A high-content image-based method for quantitatively studying context-dependent cell population dynamics

NASA Astrophysics Data System (ADS)

Garvey, Colleen M.; Spiller, Erin; Lindsay, Danika; Chiang, Chun-Te; Choi, Nathan C.; Agus, David B.; Mallick, Parag; Foo, Jasmine; Mumenthaler, Shannon M.

2016-07-01

Tumor progression results from a complex interplay between cellular heterogeneity, treatment response, microenvironment and heterocellular interactions. Existing approaches to characterize this interplay suffer from an inability to distinguish between multiple cell types, often lack environmental context, and are unable to perform multiplex phenotypic profiling of cell populations. Here we present a high-throughput platform for characterizing, with single-cell resolution, the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations both during standard growth and in response to multiple, co-occurring selective pressures. The speed of this platform enables a thorough investigation of the impacts of diverse selective pressures including genetic alterations, therapeutic interventions, heterocellular components and microenvironmental factors. The platform has been applied to both 2D and 3D culture systems and readily distinguishes between (1) cytotoxic versus cytostatic cellular responses; and (2) changes in morphological features over time and in response to perturbation. These important features can directly influence tumor evolution and clinical outcome. Our image-based approach provides a deeper insight into the cellular dynamics and heterogeneity of tumors (or other complex systems), with reduced reagents and time, offering advantages over traditional biological assays.

Label-free imaging to study phenotypic behavioural traits of cells in complex co-cultures

NASA Astrophysics Data System (ADS)

Suman, Rakesh; Smith, Gabrielle; Hazel, Kathryn E. A.; Kasprowicz, Richard; Coles, Mark; O'Toole, Peter; Chawla, Sangeeta

2016-02-01

Time-lapse imaging is a fundamental tool for studying cellular behaviours, however studies of primary cells in complex co-culture environments often requires fluorescent labelling and significant light exposure that can perturb their natural function over time. Here, we describe ptychographic phase imaging that permits prolonged label-free time-lapse imaging of microglia in the presence of neurons and astrocytes, which better resembles in vivo microenvironments. We demonstrate the use of ptychography as an assay to study the phenotypic behaviour of microglial cells in primary neuronal co-cultures through the addition of cyclosporine A, a potent immune-modulator.

Cellular complexity captured in durable silica biocomposites

PubMed Central

Kaehr, Bryan; Townson, Jason L.; Kalinich, Robin M.; Awad, Yasmine H.; Swartzentruber, B. S.; Dunphy, Darren R.; Brinker, C. Jeffrey

2012-01-01

Tissue-derived cultured cells exhibit a remarkable range of morphological features in vitro, depending on phenotypic expression and environmental interactions. Translation of these cellular architectures into inorganic materials would provide routes to generate hierarchical nanomaterials with stabilized structures and functions. Here, we describe the fabrication of cell/silica composites (CSCs) and their conversion to silica replicas using mammalian cells as scaffolds to direct complex structure formation. Under mildly acidic solution conditions, silica deposition is restricted to the molecularly crowded cellular template. Inter- and intracellular heterogeneity from the nano- to macroscale is captured and dimensionally preserved in CSCs following drying and subjection to extreme temperatures allowing, for instance, size and shape preserving pyrolysis of cellular architectures to form conductive carbon replicas. The structural and behavioral malleability of the starting material (cultured cells) provides opportunities to develop robust and economical biocomposites with programmed structures and functions. PMID:23045634

Stapled peptide inhibitors of RAB25 target context-specific phenotypes in cancer | Office of Cancer Genomics

Cancer.gov

Recent evidence has established a role for the small GTPase RAB25, as well as related effector proteins, in enacting both pro-oncogenic and anti-oncogenic phenotypes in specific cellular contexts. Here we report the development of all-hydrocarbon stabilized peptides derived from the RAB-binding FIP-family of proteins to target RAB25. Relative to unmodified peptides, optimized stapled peptides exhibit increased structural stability, binding affinity, cell permeability, and inhibition of RAB25:FIP complex formation.

Connecting synthetic chemistry decisions to cell and genome biology using small-molecule phenotypic profiling

PubMed Central

Wagner, Bridget K.; Clemons, Paul A.

2009-01-01

Discovering small-molecule modulators for thousands of gene products requires multiple stages of biological testing, specificity evaluation, and chemical optimization. Many cellular profiling methods, including cellular sensitivity, gene-expression, and cellular imaging, have emerged as methods to assess the functional consequences of biological perturbations. Cellular profiling methods applied to small-molecule science provide opportunities to use complex phenotypic information to prioritize and optimize small-molecule structures simultaneously against multiple biological endpoints. As throughput increases and cost decreases for such technologies, we see an emerging paradigm of using more information earlier in probe- and drug-discovery efforts. Moreover, increasing access to public datasets makes possible the construction of “virtual” profiles of small-molecule performance, even when multiplexed measurements were not performed or when multidimensional profiling was not the original intent. We review some key conceptual advances in small-molecule phenotypic profiling, emphasizing connections to other information, such as protein-binding measurements, genetic perturbations, and cell states. We argue that to maximally leverage these measurements in probe and drug discovery requires a fundamental connection to synthetic chemistry, allowing the consequences of synthetic decisions to be described in terms of changes in small-molecule profiles. Mining such data in the context of chemical structure and synthesis strategies can inform decisions about chemistry procurement and library development, leading to optimal small-molecule screening collections. PMID:19825513

Norovirus P particle efficiently elicits innate, humoral and cellular immunity.

PubMed

Fang, Hao; Tan, Ming; Xia, Ming; Wang, Leyi; Jiang, Xi

2013-01-01

Norovirus (NoV) P domain complexes, the 24 mer P particles and the P dimers, induced effective humoral immunity, but their role in the cellular immune responses remained unclear. We reported here a study on cellular immune responses of the two P domain complexes in comparison with the virus-like particle (VLP) of a GII.4 NoV (VA387) in mice. The P domain complexes induced significant central memory CD4(+) T cell phenotypes (CD4(+) CD44(+) CD62L(+) CCR7(+)) and activated polyclonal CD4(+) T cells as shown by production of Interleukin (IL)-2, Interferon (IFN)-γ, and Tumor Necrosis Factor (TNF)-α. Most importantly, VA387-specific CD4(+) T cell epitope induced a production of IFN-γ, indicating an antigen-specific CD4(+) T cell response in P domain complex-immunized mice. Furthermore, P domain complexes efficiently induced bone marrow-derived dendritic cell (BMDC) maturation, evidenced by up-regulation of co-stimulatory and MHC class II molecules, as well as production of IL-12 and IL-1β. Finally, P domain complex-induced mature dendritic cells (DCs) elicited proliferation of specific CD4(+) T cells targeting VA387 P domain. Overall, we conclude that the NoV P domain complexes are efficiently presented by DCs to elicit not only humoral but also cellular immune responses against NoVs. Since the P particle is highly effective for both humoral and cellular immune responses and easily produced in Escherichia coli (E. coli), it is a good choice of vaccine against NoVs and a vaccine platform against other diseases.

Neurophysiology of Drosophila Models of Parkinson's Disease

PubMed Central

West, Ryan J. H.; Furmston, Rebecca; Williams, Charles A. C.; Elliott, Christopher J. H.

2015-01-01

We provide an insight into the role Drosophila has played in elucidating neurophysiological perturbations associated with Parkinson's disease- (PD-) related genes. Synaptic signalling deficits are observed in motor, central, and sensory systems. Given the neurological impact of disease causing mutations within these same genes in humans the phenotypes observed in fly are of significant interest. As such we observe four unique opportunities provided by fly nervous system models of Parkinson's disease. Firstly, Drosophila models are instrumental in exploring the mechanisms of neurodegeneration, with several PD-related mutations eliciting related phenotypes including sensitivity to energy supply and vesicular deformities. These are leading to the identification of plausible cellular mechanisms, which may be specific to (dopaminergic) neurons and synapses rather than general cellular phenotypes. Secondly, models show noncell autonomous signalling within the nervous system, offering the opportunity to develop our understanding of the way pathogenic signalling propagates, resembling Braak's scheme of spreading pathology in PD. Thirdly, the models link physiological deficits to changes in synaptic structure. While the structure-function relationship is complex, the genetic tractability of Drosophila offers the chance to separate fundamental changes from downstream consequences. Finally, the strong neuronal phenotypes permit relevant first in vivo drug testing. PMID:25960916

Not just black and white: pigment pattern development and evolution in vertebrates

PubMed Central

Mills, Margaret G.; Patterson, Larissa B.

2009-01-01

Animals display diverse colors and patterns that vary within and between species. Similar phenotypes appear in both closely related and widely divergent taxa. Pigment patterns thus provide an opportunity to explore how development is altered to produce differences in form and whether similar phenotypes share a common genetic basis. Understanding the development and evolution of pigment patterns requires knowledge of the cellular interactions and signaling pathways that produce those patterns. These complex traits provide unparalleled opportunities for integrating studies from ecology and behavior to molecular biology and biophysics. PMID:19073271

The extraction of simple relationships in growth factor-specific multiple-input and multiple-output systems in cell-fate decisions by backward elimination PLS regression.

PubMed

Akimoto, Yuki; Yugi, Katsuyuki; Uda, Shinsuke; Kudo, Takamasa; Komori, Yasunori; Kubota, Hiroyuki; Kuroda, Shinya

2013-01-01

Cells use common signaling molecules for the selective control of downstream gene expression and cell-fate decisions. The relationship between signaling molecules and downstream gene expression and cellular phenotypes is a multiple-input and multiple-output (MIMO) system and is difficult to understand due to its complexity. For example, it has been reported that, in PC12 cells, different types of growth factors activate MAP kinases (MAPKs) including ERK, JNK, and p38, and CREB, for selective protein expression of immediate early genes (IEGs) such as c-FOS, c-JUN, EGR1, JUNB, and FOSB, leading to cell differentiation, proliferation and cell death; however, how multiple-inputs such as MAPKs and CREB regulate multiple-outputs such as expression of the IEGs and cellular phenotypes remains unclear. To address this issue, we employed a statistical method called partial least squares (PLS) regression, which involves a reduction of the dimensionality of the inputs and outputs into latent variables and a linear regression between these latent variables. We measured 1,200 data points for MAPKs and CREB as the inputs and 1,900 data points for IEGs and cellular phenotypes as the outputs, and we constructed the PLS model from these data. The PLS model highlighted the complexity of the MIMO system and growth factor-specific input-output relationships of cell-fate decisions in PC12 cells. Furthermore, to reduce the complexity, we applied a backward elimination method to the PLS regression, in which 60 input variables were reduced to 5 variables, including the phosphorylation of ERK at 10 min, CREB at 5 min and 60 min, AKT at 5 min and JNK at 30 min. The simple PLS model with only 5 input variables demonstrated a predictive ability comparable to that of the full PLS model. The 5 input variables effectively extracted the growth factor-specific simple relationships within the MIMO system in cell-fate decisions in PC12 cells.

Reduction of protein translation and activation of autophagy protect against PINK1 pathogenesis in Drosophila melanogaster.

PubMed

Liu, Song; Lu, Bingwei

2010-12-09

Mutations in PINK1 and Parkin cause familial, early onset Parkinson's disease. In Drosophila melanogaster, PINK1 and Parkin mutants show similar phenotypes, such as swollen and dysfunctional mitochondria, muscle degeneration, energy depletion, and dopaminergic (DA) neuron loss. We previously showed that PINK1 and Parkin genetically interact with the mitochondrial fusion/fission pathway, and PINK1 and Parkin were recently proposed to form a mitochondrial quality control system that involves mitophagy. However, the in vivo relationships among PINK1/Parkin function, mitochondrial fission/fusion, and autophagy remain unclear; and other cellular events critical for PINK1 pathogenesis remain to be identified. Here we show that PINK1 genetically interacted with the protein translation pathway. Enhanced translation through S6K activation significantly exacerbated PINK1 mutant phenotypes, whereas reduction of translation showed suppression. Induction of autophagy by Atg1 overexpression also rescued PINK1 mutant phenotypes, even in the presence of activated S6K. Downregulation of translation and activation of autophagy were already manifested in PINK1 mutant, suggesting that they represent compensatory cellular responses to mitochondrial dysfunction caused by PINK1 inactivation, presumably serving to conserve energy. Interestingly, the enhanced PINK1 mutant phenotype in the presence of activated S6K could be fully rescued by Parkin, apparently in an autophagy-independent manner. Our results reveal complex cellular responses to PINK1 inactivation and suggest novel therapeutic strategies through manipulation of the compensatory responses.

Reduction of Protein Translation and Activation of Autophagy Protect against PINK1 Pathogenesis in Drosophila melanogaster

PubMed Central

Liu, Song; Lu, Bingwei

2010-01-01

Mutations in PINK1 and Parkin cause familial, early onset Parkinson's disease. In Drosophila melanogaster, PINK1 and Parkin mutants show similar phenotypes, such as swollen and dysfunctional mitochondria, muscle degeneration, energy depletion, and dopaminergic (DA) neuron loss. We previously showed that PINK1 and Parkin genetically interact with the mitochondrial fusion/fission pathway, and PINK1 and Parkin were recently proposed to form a mitochondrial quality control system that involves mitophagy. However, the in vivo relationships among PINK1/Parkin function, mitochondrial fission/fusion, and autophagy remain unclear; and other cellular events critical for PINK1 pathogenesis remain to be identified. Here we show that PINK1 genetically interacted with the protein translation pathway. Enhanced translation through S6K activation significantly exacerbated PINK1 mutant phenotypes, whereas reduction of translation showed suppression. Induction of autophagy by Atg1 overexpression also rescued PINK1 mutant phenotypes, even in the presence of activated S6K. Downregulation of translation and activation of autophagy were already manifested in PINK1 mutant, suggesting that they represent compensatory cellular responses to mitochondrial dysfunction caused by PINK1 inactivation, presumably serving to conserve energy. Interestingly, the enhanced PINK1 mutant phenotype in the presence of activated S6K could be fully rescued by Parkin, apparently in an autophagy-independent manner. Our results reveal complex cellular responses to PINK1 inactivation and suggest novel therapeutic strategies through manipulation of the compensatory responses. PMID:21151574

Mammalian synthetic biology for studying the cell

PubMed Central

Mathur, Melina; Xiang, Joy S.

2017-01-01

Synthetic biology is advancing the design of genetic devices that enable the study of cellular and molecular biology in mammalian cells. These genetic devices use diverse regulatory mechanisms to both examine cellular processes and achieve precise and dynamic control of cellular phenotype. Synthetic biology tools provide novel functionality to complement the examination of natural cell systems, including engineered molecules with specific activities and model systems that mimic complex regulatory processes. Continued development of quantitative standards and computational tools will expand capacities to probe cellular mechanisms with genetic devices to achieve a more comprehensive understanding of the cell. In this study, we review synthetic biology tools that are being applied to effectively investigate diverse cellular processes, regulatory networks, and multicellular interactions. We also discuss current challenges and future developments in the field that may transform the types of investigation possible in cell biology. PMID:27932576

Living Organisms Author Their Read-Write Genomes in Evolution

PubMed Central

2017-01-01

Evolutionary variations generating phenotypic adaptations and novel taxa resulted from complex cellular activities altering genome content and expression: (i) Symbiogenetic cell mergers producing the mitochondrion-bearing ancestor of eukaryotes and chloroplast-bearing ancestors of photosynthetic eukaryotes; (ii) interspecific hybridizations and genome doublings generating new species and adaptive radiations of higher plants and animals; and, (iii) interspecific horizontal DNA transfer encoding virtually all of the cellular functions between organisms and their viruses in all domains of life. Consequently, assuming that evolutionary processes occur in isolated genomes of individual species has become an unrealistic abstraction. Adaptive variations also involved natural genetic engineering of mobile DNA elements to rewire regulatory networks. In the most highly evolved organisms, biological complexity scales with “non-coding” DNA content more closely than with protein-coding capacity. Coincidentally, we have learned how so-called “non-coding” RNAs that are rich in repetitive mobile DNA sequences are key regulators of complex phenotypes. Both biotic and abiotic ecological challenges serve as triggers for episodes of elevated genome change. The intersections of cell activities, biosphere interactions, horizontal DNA transfers, and non-random Read-Write genome modifications by natural genetic engineering provide a rich molecular and biological foundation for understanding how ecological disruptions can stimulate productive, often abrupt, evolutionary transformations. PMID:29211049

Living Organisms Author Their Read-Write Genomes in Evolution.

PubMed

Shapiro, James A

2017-12-06

Evolutionary variations generating phenotypic adaptations and novel taxa resulted from complex cellular activities altering genome content and expression: (i) Symbiogenetic cell mergers producing the mitochondrion-bearing ancestor of eukaryotes and chloroplast-bearing ancestors of photosynthetic eukaryotes; (ii) interspecific hybridizations and genome doublings generating new species and adaptive radiations of higher plants and animals; and, (iii) interspecific horizontal DNA transfer encoding virtually all of the cellular functions between organisms and their viruses in all domains of life. Consequently, assuming that evolutionary processes occur in isolated genomes of individual species has become an unrealistic abstraction. Adaptive variations also involved natural genetic engineering of mobile DNA elements to rewire regulatory networks. In the most highly evolved organisms, biological complexity scales with "non-coding" DNA content more closely than with protein-coding capacity. Coincidentally, we have learned how so-called "non-coding" RNAs that are rich in repetitive mobile DNA sequences are key regulators of complex phenotypes. Both biotic and abiotic ecological challenges serve as triggers for episodes of elevated genome change. The intersections of cell activities, biosphere interactions, horizontal DNA transfers, and non-random Read-Write genome modifications by natural genetic engineering provide a rich molecular and biological foundation for understanding how ecological disruptions can stimulate productive, often abrupt, evolutionary transformations.

Asymmetric Facial Bone Fragmentation Mirrors Asymmetric Distribution of Cranial Neuromasts in Blind Mexican Cavefish.

PubMed

Gross, Joshua B; Gangidine, Andrew; Powers, Amanda K

2016-11-01

Craniofacial asymmetry is a convergent trait widely distributed across animals that colonize the extreme cave environment. Although craniofacial asymmetry can be discerned easily, other complex phenotypes (such as sensory organ position and numerical variation) are challenging to score and compare. Certain bones of the craniofacial complex demonstrate substantial asymmetry, and co-localize to regions harboring dramatically expanded numbers of mechanosensory neuromasts. To determine if a relationship exists between this expansion and bone fragmentation in cavefish, we developed a quantitative measure of positional symmetry across the left-right axis. We found that three different cave-dwelling populations were significantly more asymmetric compared to surface-dwelling fish. Moreover, cave populations did not differ in the degree of neuromast asymmetry. This work establishes a method for quantifying symmetry of a complex phenotype, and demonstrates that facial bone fragmentation mirrors the asymmetric distribution of neuromasts in different cavefish populations. Further developmental studies will provide a clearer picture of the developmental and cellular changes that accompany this extreme phenotype, and help illuminate the genetic basis for facial asymmetry in vertebrates.

A knowledge based approach to matching human neurodegenerative disease and animal models

PubMed Central

Maynard, Sarah M.; Mungall, Christopher J.; Lewis, Suzanna E.; Imam, Fahim T.; Martone, Maryann E.

2013-01-01

Neurodegenerative diseases present a wide and complex range of biological and clinical features. Animal models are key to translational research, yet typically only exhibit a subset of disease features rather than being precise replicas of the disease. Consequently, connecting animal to human conditions using direct data-mining strategies has proven challenging, particularly for diseases of the nervous system, with its complicated anatomy and physiology. To address this challenge we have explored the use of ontologies to create formal descriptions of structural phenotypes across scales that are machine processable and amenable to logical inference. As proof of concept, we built a Neurodegenerative Disease Phenotype Ontology (NDPO) and an associated Phenotype Knowledge Base (PKB) using an entity-quality model that incorporates descriptions for both human disease phenotypes and those of animal models. Entities are drawn from community ontologies made available through the Neuroscience Information Framework (NIF) and qualities are drawn from the Phenotype and Trait Ontology (PATO). We generated ~1200 structured phenotype statements describing structural alterations at the subcellular, cellular and gross anatomical levels observed in 11 human neurodegenerative conditions and associated animal models. PhenoSim, an open source tool for comparing phenotypes, was used to issue a series of competency questions to compare individual phenotypes among organisms and to determine which animal models recapitulate phenotypic aspects of the human disease in aggregate. Overall, the system was able to use relationships within the ontology to bridge phenotypes across scales, returning non-trivial matches based on common subsumers that were meaningful to a neuroscientist with an advanced knowledge of neuroanatomy. The system can be used both to compare individual phenotypes and also phenotypes in aggregate. This proof of concept suggests that expressing complex phenotypes using formal ontologies provides considerable benefit for comparing phenotypes across scales and species. PMID:23717278

Mammalian synthetic biology for studying the cell.

PubMed

Mathur, Melina; Xiang, Joy S; Smolke, Christina D

2017-01-02

Synthetic biology is advancing the design of genetic devices that enable the study of cellular and molecular biology in mammalian cells. These genetic devices use diverse regulatory mechanisms to both examine cellular processes and achieve precise and dynamic control of cellular phenotype. Synthetic biology tools provide novel functionality to complement the examination of natural cell systems, including engineered molecules with specific activities and model systems that mimic complex regulatory processes. Continued development of quantitative standards and computational tools will expand capacities to probe cellular mechanisms with genetic devices to achieve a more comprehensive understanding of the cell. In this study, we review synthetic biology tools that are being applied to effectively investigate diverse cellular processes, regulatory networks, and multicellular interactions. We also discuss current challenges and future developments in the field that may transform the types of investigation possible in cell biology. © 2017 Mathur et al.

Engineering yeast transcription machinery for improved ethanol tolerance and production.

PubMed

Alper, Hal; Moxley, Joel; Nevoigt, Elke; Fink, Gerald R; Stephanopoulos, Gregory

2006-12-08

Global transcription machinery engineering (gTME) is an approach for reprogramming gene transcription to elicit cellular phenotypes important for technological applications. Here we show the application of gTME to Saccharomyces cerevisiae for improved glucose/ethanol tolerance, a key trait for many biofuels programs. Mutagenesis of the transcription factor Spt15p and selection led to dominant mutations that conferred increased tolerance and more efficient glucose conversion to ethanol. The desired phenotype results from the combined effect of three separate mutations in the SPT15 gene [serine substituted for phenylalanine (Phe(177)Ser) and, similarly, Tyr(195)His, and Lys(218)Arg]. Thus, gTME can provide a route to complex phenotypes that are not readily accessible by traditional methods.

Senescence and the pro-tumorigenic stroma.

PubMed

Alspach, Elise; Fu, Yujie; Stewart, Sheila A

2013-01-01

Hayflick and Moorhead first described senescence in the late 1960's as a permanent growth arrest that primary cells underwent after a defined number of cellular divisions in culture. This observation gave rise to the hypothesis that cells contained an internal counting mechanism that limited cellular division and that this limit was an important barrier to cellular transformation. What began as an in vitro observation has led to an immense body of work that reaches into all fields of biology and is of particular interest in the areas of aging, tissue regeneration, and tumorigenesis. The initially simplistic view that senescence limits cellular division and contributes to aging while stymying tumorigenesis has now evolved into an important and complex biological process that has numerous caveats and often opposing effects on tumorigenesis. In this review, we limit our discussion to the complex role senescence plays in tumorigenesis. Throughout the review we attempt to draw many parallels to other systems including the role senescent cells play in the tumor microenvironment and their significant molecular and phenotypic similarities to cancer associated fibroblasts (CAFs).

Retinal dystrophies, genomic applications in diagnosis and prospects for therapy

PubMed Central

Nash, Benjamin M.; Wright, Dale C.; Grigg, John R.; Bennetts, Bruce

2015-01-01

Retinal dystrophies (RDs) are degenerative diseases of the retina which have marked clinical and genetic heterogeneity. Common presentations among these disorders include night or colour blindness, tunnel vision and subsequent progression to complete blindness. The known causative disease genes have a variety of developmental and functional roles with mutations in more than 120 genes shown to be responsible for the phenotypes. In addition, mutations within the same gene have been shown to cause different disease phenotypes, even amongst affected individuals within the same family highlighting further levels of complexity. The known disease genes encode proteins involved in retinal cellular structures, phototransduction, the visual cycle, and photoreceptor structure or gene regulation. This review aims to demonstrate the high degree of genetic complexity in both the causative disease genes and their associated phenotypes, highlighting the more common clinical manifestation of retinitis pigmentosa (RP). The review also provides insight to recent advances in genomic molecular diagnosis and gene and cell-based therapies for the RDs. PMID:26835369

Cationic liposome/DNA complexes: from structure to interactions with cellular membranes.

PubMed

Caracciolo, Giulio; Amenitsch, Heinz

2012-10-01

Gene-based therapeutic approaches are based upon the concept that, if a disease is caused by a mutation in a gene, then adding back the wild-type gene should restore regular function and attenuate the disease phenotype. To deliver the gene of interest, both viral and nonviral vectors are used. Viruses are efficient, but their application is impeded by detrimental side-effects. Among nonviral vectors, cationic liposomes are the most promising candidates for gene delivery. They form stable complexes with polyanionic DNA (lipoplexes). Despite several advantages over viral vectors, the transfection efficiency (TE) of lipoplexes is too low compared with those of engineered viral vectors. This is due to lack of knowledge about the interactions between complexes and cellular components. Rational design of efficient lipoplexes therefore requires deeper comprehension of the interactions between the vector and the DNA as well as the cellular pathways and mechanisms involved. The importance of the lipoplex structure in biological function is revealed in the application of synchrotron small-angle X-ray scattering in combination with functional TE measurements. According to current understanding, the structure of lipoplexes can change upon interaction with cellular membranes and such changes affect the delivery efficiency. Recently, a correlation between the mechanism of gene release from complexes, the structure, and the physical and chemical parameters of the complexes has been established. Studies aimed at correlating structure and activity of lipoplexes are reviewed herein. This is a fundamental step towards rational design of highly efficient lipid gene vectors.

TM4SF5-Mediated Roles in the Development of Fibrotic Phenotypes

PubMed Central

Ryu, Jihye

2017-01-01

Transmembrane 4 L six family member 5 (TM4SF5) can form tetraspanin-enriched microdomains (TERMs) on the cell's surface. TERMs contain protein-protein complexes comprised of tetraspanins, growth factor receptors, and integrins. These complexes regulate communication between extracellular and intracellular spaces to control diverse cellular functions. TM4SF5 influences the epithelial-mesenchymal transition (EMT), aberrant multilayer cellular growth, drug resistance, enhanced migration and invasion, circulation through the bloodstream, tumor-initiation property, metastasis, and muscle development in zebrafish. Here, current data on TM4SF5's roles in the development of fibrotic phenotypes are reviewed. TM4SF5 is induced by transforming growth factor β1 (TGFβ1) signaling via a collaboration with epidermal growth factor receptor (EGFR) activation. TM4SF5, by itself or in concert with other receptors, transduces signals intracellularly. In hepatocytes, TM4SF5 expression regulates cell cycle progression, migration, and expression of extracellular matrix components. In CCl4-treated mice, TM4SF5, α-smooth muscle actin (α-SMA), and collagen I expression are observed together along the fibrotic septa regions of the liver. These fibrotic phenotypes are diminished by anti-TM4SF5 reagents, such as a specific small compound [TSAHC, 4′-(p-toluenesulfonylamido)-4-hydroxychalcone] or a chimeric antibody. This review discusses the antifibrotic strategies that target TM4SF5 and its associated protein networks that regulate the intracellular signaling necessary for fibrotic functions of hepatocytes. PMID:28458469

Mutation of the Diamond-Blackfan Anemia Gene Rps7 in Mouse Results in Morphological and Neuroanatomical Phenotypes

PubMed Central

Watkins-Chow, Dawn E.; Cooke, Joanna; Pidsley, Ruth; Edwards, Andrew; Slotkin, Rebecca; Leeds, Karen E.; Mullen, Raymond; Baxter, Laura L.; Campbell, Thomas G.; Salzer, Marion C.; Biondini, Laura; Gibney, Gretchen; Phan Dinh Tuy, Françoise; Chelly, Jamel; Morris, H. Douglas; Riegler, Johannes; Lythgoe, Mark F.; Arkell, Ruth M.; Loreni, Fabrizio; Flint, Jonathan

2013-01-01

The ribosome is an evolutionarily conserved organelle essential for cellular function. Ribosome construction requires assembly of approximately 80 different ribosomal proteins (RPs) and four different species of rRNA. As RPs co-assemble into one multi-subunit complex, mutation of the genes that encode RPs might be expected to give rise to phenocopies, in which the same phenotype is associated with loss-of-function of each individual gene. However, a more complex picture is emerging in which, in addition to a group of shared phenotypes, diverse RP gene-specific phenotypes are observed. Here we report the first two mouse mutations (Rps7Mtu and Rps7Zma) of ribosomal protein S7 (Rps7), a gene that has been implicated in Diamond-Blackfan anemia. Rps7 disruption results in decreased body size, abnormal skeletal morphology, mid-ventral white spotting, and eye malformations. These phenotypes are reported in other murine RP mutants and, as demonstrated for some other RP mutations, are ameliorated by Trp53 deficiency. Interestingly, Rps7 mutants have additional overt malformations of the developing central nervous system and deficits in working memory, phenotypes that are not reported in murine or human RP gene mutants. Conversely, Rps7 mouse mutants show no anemia or hyperpigmentation, phenotypes associated with mutation of human RPS7 and other murine RPs, respectively. We provide two novel RP mouse models and expand the repertoire of potential phenotypes that should be examined in RP mutants to further explore the concept of RP gene-specific phenotypes. PMID:23382688

A method to integrate patterned electrospun fibers with microfluidic systems to generate complex microenvironments for cell culture applications

PubMed Central

Wallin, Patric; Zandén, Carl; Carlberg, Björn; Hellström Erkenstam, Nina; Liu, Johan; Gold, Julie

2012-01-01

The properties of a cell’s microenvironment are one of the main driving forces in cellular fate processes and phenotype expression invivo. The ability to create controlled cell microenvironments invitro becomes increasingly important for studying or controlling phenotype expression in tissue engineering and drug discovery applications. This includes the capability to modify material surface properties within well-defined liquid environments in cell culture systems. One successful approach to mimic extra cellular matrix is with porous electrospun polymer fiber scaffolds, while microfluidic networks have been shown to efficiently generate spatially and temporally defined liquid microenvironments. Here, a method to integrate electrospun fibers with microfluidic networks was developed in order to form complex cell microenvironments with the capability to vary relevant parameters. Spatially defined regions of electrospun fibers of both aligned and random orientation were patterned on glass substrates that were irreversibly bonded to microfluidic networks produced in poly-dimethyl-siloxane. Concentration gradients obtained in the fiber containing channels were characterized experimentally and compared with values obtained by computational fluid dynamic simulations. Velocity and shear stress profiles, as well as vortex formation, were calculated to evaluate the influence of fiber pads on fluidic properties. The suitability of the system to support cell attachment and growth was demonstrated with a fibroblast cell line. The potential of the platform was further verified by a functional investigation of neural stem cell alignment in response to orientation of electrospun fibers versus a microfluidic generated chemoattractant gradient of stromal cell-derived factor 1 alpha. The described method is a competitive strategy to create complex microenvironments invitro that allow detailed studies on the interplay of topography, substrate surface properties, and soluble microenvironment on cellular fate processes. PMID:23781291

High content screening in neurodegenerative diseases.

PubMed

Jain, Shushant; van Kesteren, Ronald E; Heutink, Peter

2012-01-06

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention. High-throughput screening (HTS) is an ideal methodology to address these issues. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS). HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. HCS has many advantages over HTS, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism. Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets. Copyright © 2012 Creative Commons Attribution License

Mitochondrial respiratory chain Complex I defects in Fanconi anemia complementation group A.

PubMed

Ravera, Silvia; Vaccaro, Daniele; Cuccarolo, Paola; Columbaro, Marta; Capanni, Cristina; Bartolucci, Martina; Panfoli, Isabella; Morelli, Alessandro; Dufour, Carlo; Cappelli, Enrico; Degan, Paolo

2013-10-01

Fanconi anemia (FA) is a rare and complex inherited blood disorder of the child. At least 15 genes are associated with the disease. The highest frequency of mutations belongs to groups A, C and G. Genetic instability and cytokine hypersensitivity support the selection of leukemic over non-leukemic stem cells. FA cellular phenotype is characterized by alterations in red-ox state, mitochondrial functionality and energy metabolism as reported in the past however a clear picture of the altered biochemical phenotype in FA is still elusive and the final biochemical defect(s) still unknown. Here we report an analysis of the respiratory fluxes in FANCA primary fibroblasts, lymphocytes and lymphoblasts. FANCA mutants show defective respiration through Complex I, diminished ATP production and metabolic sufferance with an increased AMP/ATP ratio. Respiration in FANCC mutants is normal. Treatment with N-acetyl-cysteine (NAC) restores oxygen consumption to normal level. Defective respiration in FANCA mutants appear correlated with the FA pro-oxidative phenotype which is consistent with the altered morphology of FANCA mitochondria. Electron microscopy measures indeed show profound alterations in mitochondrial ultrastructure and shape. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

An in vivo multiplexed small molecule screening platform

PubMed Central

Yang, Dian; Ogasawara, Daisuke; Dix, Melissa M.; Rogers, Zoë N.; Chuang, Chen-Hua; McFarland, Christopher D.; Chiou, Shin-Heng; Brown, J. Mark; Cravatt, Benjamin F.; Bogyo, Matthew; Winslow, Monte M.

2016-01-01

Phenotype-based small molecule screening is a powerful method to identify regulators of cellular function. However, such screens are generally performed in vitro using conditions that do not necessarily model complex physiological conditions or disease states. Here, we use molecular cell barcoding to enable direct in vivo phenotypic screening of libraries of small molecules. The multiplexed nature of this approach allows rapid in vivo analysis of hundreds to thousands of compounds. Using this platform, we screened >700 covalent inhibitors directed towards hydrolases for their effect on pancreatic cancer metastatic seeding. We identified multiple hits and confirmed the relevant target of one compound as the lipase ABHD6. Pharmacological and genetic studies confirmed the role of this enzyme as a regulator of metastatic fitness. Our results highlight the applicability of this multiplexed screening platform for investigating complex processes in vivo. PMID:27617390

Modulation of Cellular Stress Response via the Erythropoietin/CD131 Heteroreceptor Complex in Mouse Mesenchymal-Derived Cells

PubMed Central

Bohr, Stefan; Patel, Suraj J; Vasko, Radovan; Shen, Keyue; Iracheta-Vellve, Arvin; Lee, Jungwoo; Bale, Shyam Sundhar; Chakraborty, Nilay; Brines, Michael; Cerami, Anthony; Berthiaume, Francois; Yarmush, Martin L

2014-01-01

Tissue protective properties of erythropoietin (EPO) have let to the discovery of an alternative EPO-signaling via an EPO-R/CD131 receptor complex which can now be specifically targeted through pharmaceutically designed short sequence peptides such as ARA290. However, little is still known about specific functions of alternative EPO-signaling in defined cell populations. In this study we investigated effects of signaling through EPO-R/CD131 complex on cellular stress responses and pro-inflammatory activation in different mesenchymal-derived phenotypes. We show that anti-apoptotic, anti-inflammatory effects of ARA290 and EPO coincide with the externalization of CD131 receptor component as an immediate response to cellular stress. In addition, alternative EPO-signaling strongly modulated transcriptional, translational or metabolic responses after stressor removal. Specifically, we saw that ARA290 was able overcome a TNFα-mediated inhibition of transcription factor activation related to cell stress responses, most notably of serum response factor (SRF), heat shock transcription factor protein 1 (HSF1) and activator protein 1 (AP1). We conclude that alternative EPO-signaling acts as a modulator of pro-inflammatory signaling pathways and likely plays a role in restoring tissue homeostasis. PMID:25373867

Metabotropic glutamate receptor 5 couples cellular prion protein to intracellular signalling in Alzheimer’s disease

PubMed Central

Haas, Laura T.; Salazar, Santiago V.; Kostylev, Mikhail A.; Um, Ji Won; Kaufman, Adam C.

2016-01-01

Alzheimer’s disease-related phenotypes in mice can be rescued by blockade of either cellular prion protein or metabotropic glutamate receptor 5. We sought genetic and biochemical evidence that these proteins function cooperatively as an obligate complex in the brain. We show that cellular prion protein associates via transmembrane metabotropic glutamate receptor 5 with the intracellular protein mediators Homer1b/c, calcium/calmodulin-dependent protein kinase II, and the Alzheimer’s disease risk gene product protein tyrosine kinase 2 beta. Coupling of cellular prion protein to these intracellular proteins is modified by soluble amyloid-β oligomers, by mouse brain Alzheimer’s disease transgenes or by human Alzheimer’s disease pathology. Amyloid-β oligomer-triggered phosphorylation of intracellular protein mediators and impairment of synaptic plasticity in vitro requires Prnp–Grm5 genetic interaction, being absent in transheterozygous loss-of-function, but present in either single heterozygote. Importantly, genetic coupling between Prnp and Grm5 is also responsible for signalling, for survival and for synapse loss in Alzheimer’s disease transgenic model mice. Thus, the interaction between metabotropic glutamate receptor 5 and cellular prion protein has a central role in Alzheimer’s disease pathogenesis, and the complex is a potential target for disease-modifying intervention. PMID:26667279

Label-free cell-cycle analysis by high-throughput quantitative phase time-stretch imaging flow cytometry

NASA Astrophysics Data System (ADS)

Mok, Aaron T. Y.; Lee, Kelvin C. M.; Wong, Kenneth K. Y.; Tsia, Kevin K.

2018-02-01

Biophysical properties of cells could complement and correlate biochemical markers to characterize a multitude of cellular states. Changes in cell size, dry mass and subcellular morphology, for instance, are relevant to cell-cycle progression which is prevalently evaluated by DNA-targeted fluorescence measurements. Quantitative-phase microscopy (QPM) is among the effective biophysical phenotyping tools that can quantify cell sizes and sub-cellular dry mass density distribution of single cells at high spatial resolution. However, limited camera frame rate and thus imaging throughput makes QPM incompatible with high-throughput flow cytometry - a gold standard in multiparametric cell-based assay. Here we present a high-throughput approach for label-free analysis of cell cycle based on quantitative-phase time-stretch imaging flow cytometry at a throughput of > 10,000 cells/s. Our time-stretch QPM system enables sub-cellular resolution even at high speed, allowing us to extract a multitude (at least 24) of single-cell biophysical phenotypes (from both amplitude and phase images). Those phenotypes can be combined to track cell-cycle progression based on a t-distributed stochastic neighbor embedding (t-SNE) algorithm. Using multivariate analysis of variance (MANOVA) discriminant analysis, cell-cycle phases can also be predicted label-free with high accuracy at >90% in G1 and G2 phase, and >80% in S phase. We anticipate that high throughput label-free cell cycle characterization could open new approaches for large-scale single-cell analysis, bringing new mechanistic insights into complex biological processes including diseases pathogenesis.

Clinical Impact and Cellular Mechanisms of Iron Overload-Associated Bone Loss

PubMed Central

Jeney, Viktória

2017-01-01

Diseases/conditions with diverse etiology, such as hemoglobinopathies, hereditary hemochromatosis and menopause, could lead to chronic iron accumulation. This condition is frequently associated with a bone phenotype; characterized by low bone mass, osteoporosis/osteopenia, altered microarchitecture and biomechanics, and increased incidence of fractures. Osteoporotic bone phenotype constitutes a major complication in patients with iron overload. The purpose of this review is to summarize what we have learnt about iron overload-associated bone loss from clinical studies and animal models. Bone is a metabolically active tissue that undergoes continuous remodeling with the involvement of osteoclasts that resorb mineralized bone, and osteoblasts that form new bone. Growing evidence suggests that both increased bone resorption and decreased bone formation are involved in the pathological bone-loss in iron overload conditions. We will discuss the cellular and molecular mechanisms that are involved in this detrimental process. Fuller understanding of this complex mechanism may lead to the development of improved therapeutics meant to interrupt the pathologic effects of excess iron on bone. PMID:28270766

Dynamic Interactions Between Cancer Stem Cells And Their Stromal Partners.

PubMed

Park, Tea Soon; Donnenberg, Vera S; Donnenberg, Albert D; Zambidis, Elias T; Zimmerlin, Ludovic

2014-03-01

The cancer stem cell (CSC) paradigm presumes the existence of self-renewing cancer cells capable of regenerating all tumor compartments and exhibiting stem cell-associated phenotypes. Recent interpretations of the CSC hypothesis envision stemness as a dynamic trait of tumor-initiating cells rather than a defined and unique cell type. Bidirectional crosstalk between the tumor microenvironment and the cancer bulk is well described in the literature and the tumor-associated stroma, vasculature and immune infiltrate have all been implicated as direct contributors to tumor development. These non-neoplastic cell types have also been shown to organize specific niches within the tumor bulk where they can control the intra-tumor CSC content and alter the fate of CSCs and tumor progenitors during tumorigenesis to acquire phenotypic features for invasion, metastasis and dormancy. Despite the complexity of the tumor-stroma interactome, novel therapeutic approaches envision combining tumor-ablative treatment with manipulation of the tumor microenvironment. We will review the currently available literature that provides clues about the complex cellular network that regulate the CSC phenotype and its niches during tumor progression.

T Cell Adaptive Immunity Proceeds through Environment-Induced Adaptation from the Exposure of Cryptic Genetic Variation

PubMed Central

Whitacre, James M.; Lin, Joseph; Harding, Angus

2011-01-01

Evolution is often characterized as a process involving incremental genetic changes that are slowly discovered and fixed in a population through genetic drift and selection. However, a growing body of evidence is finding that changes in the environment frequently induce adaptations that are much too rapid to occur by an incremental genetic search process. Rapid evolution is hypothesized to be facilitated by mutations present within the population that are silent or “cryptic” within the first environment but are co-opted or “exapted” to the new environment, providing a selective advantage once revealed. Although cryptic mutations have recently been shown to facilitate evolution in RNA enzymes, their role in the evolution of complex phenotypes has not been proven. In support of this wider role, this paper describes an unambiguous relationship between cryptic genetic variation and complex phenotypic responses within the immune system. By reviewing the biology of the adaptive immune system through the lens of evolution, we show that T cell adaptive immunity constitutes an exemplary model system where cryptic alleles drive rapid adaptation of complex traits. In naive T cells, normally cryptic differences in T cell receptor reveal diversity in activation responses when the cellular population is presented with a novel environment during infection. We summarize how the adaptive immune response presents a well studied and appropriate experimental system that can be used to confirm and expand upon theoretical evolutionary models describing how seemingly small and innocuous mutations can drive rapid cellular evolution. PMID:22363338

CellCognition: time-resolved phenotype annotation in high-throughput live cell imaging.

PubMed

Held, Michael; Schmitz, Michael H A; Fischer, Bernd; Walter, Thomas; Neumann, Beate; Olma, Michael H; Peter, Matthias; Ellenberg, Jan; Gerlich, Daniel W

2010-09-01

Fluorescence time-lapse imaging has become a powerful tool to investigate complex dynamic processes such as cell division or intracellular trafficking. Automated microscopes generate time-resolved imaging data at high throughput, yet tools for quantification of large-scale movie data are largely missing. Here we present CellCognition, a computational framework to annotate complex cellular dynamics. We developed a machine-learning method that combines state-of-the-art classification with hidden Markov modeling for annotation of the progression through morphologically distinct biological states. Incorporation of time information into the annotation scheme was essential to suppress classification noise at state transitions and confusion between different functional states with similar morphology. We demonstrate generic applicability in different assays and perturbation conditions, including a candidate-based RNA interference screen for regulators of mitotic exit in human cells. CellCognition is published as open source software, enabling live-cell imaging-based screening with assays that directly score cellular dynamics.

Network Medicine: From Cellular Networks to the Human Diseasome

NASA Astrophysics Data System (ADS)

Barabasi, Albert-Laszlo

2014-03-01

Given the functional interdependencies between the molecular components in a human cell, a disease is rarely a consequence of an abnormality in a single gene, but reflects the perturbations of the complex intracellular network. The tools of network science offer a platform to explore systematically not only the molecular complexity of a particular disease, leading to the identification of disease modules and pathways, but also the molecular relationships between apparently distinct (patho)phenotypes. Advances in this direction not only enrich our understanding of complex systems, but are also essential to identify new disease genes, to uncover the biological significance of disease-associated mutations identified by genome-wide association studies and full genome sequencing, and to identify drug targets and biomarkers for complex diseases.

Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis

PubMed Central

Dragovic, Rebecca A.; Gardiner, Christopher; Brooks, Alexandra S.; Tannetta, Dionne S.; Ferguson, David J.P.; Hole, Patrick; Carr, Bob; Redman, Christopher W.G.; Harris, Adrian L.; Dobson, Peter J.; Harrison, Paul; Sargent, Ian L.

2011-01-01

Cellular microvesicles and nanovesicles (exosomes) are involved in many disease processes and have major potential as biomarkers. However, developments in this area are constrained by limitations in the technology available for their measurement. Here we report on the use of fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles. In this system vesicles are visualized by light scattering using a light microscope. A video is taken, and the NTA software tracks the brownian motion of individual vesicles and calculates their size and total concentration. Using human placental vesicles and plasma, we have demonstrated that NTA can measure cellular vesicles as small as ∼50 nm and is far more sensitive than conventional flow cytometry (lower limit ∼300 nm). By combining NTA with fluorescence measurement we have demonstrated that vesicles can be labeled with specific antibody-conjugated quantum dots, allowing their phenotype to be determined. From the Clinical Editor The authors of this study utilized fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles, demonstrating that NTA is far more sensitive than conventional flow cytometry. PMID:21601655

Resolving phylogenetic incongruence to articulate homology and phenotypic evolution: a case study from Nematoda

PubMed Central

Ragsdale, Erik J.; Baldwin, James G.

2010-01-01

Modern morphology-based systematics, including questions of incongruence with molecular data, emphasizes analysis over similarity criteria to assess homology. Yet detailed examination of a few key characters, using new tools and processes such as computerized, three-dimensional ultrastructural reconstruction of cell complexes, can resolve apparent incongruence by re-examining primary homologies. In nematodes of Tylenchomorpha, a parasitic feeding phenotype is thus reconciled with immediate free-living outgroups. Closer inspection of morphology reveals phenotypes congruent with molecular-based phylogeny and points to a new locus of homology in mouthparts. In nematode models, the study of individually homologous cells reveals a conserved modality of evolution among dissimilar feeding apparati adapted to divergent lifestyles. Conservatism of cellular components, consistent with that of other body systems, allows meaningful comparative morphology in difficult groups of microscopic organisms. The advent of phylogenomics is synergistic with morphology in systematics, providing an honest test of homology in the evolution of phenotype. PMID:20106846

Large-scale image-based profiling of single-cell phenotypes in arrayed CRISPR-Cas9 gene perturbation screens.

PubMed

de Groot, Reinoud; Lüthi, Joel; Lindsay, Helen; Holtackers, René; Pelkmans, Lucas

2018-01-23

High-content imaging using automated microscopy and computer vision allows multivariate profiling of single-cell phenotypes. Here, we present methods for the application of the CISPR-Cas9 system in large-scale, image-based, gene perturbation experiments. We show that CRISPR-Cas9-mediated gene perturbation can be achieved in human tissue culture cells in a timeframe that is compatible with image-based phenotyping. We developed a pipeline to construct a large-scale arrayed library of 2,281 sequence-verified CRISPR-Cas9 targeting plasmids and profiled this library for genes affecting cellular morphology and the subcellular localization of components of the nuclear pore complex (NPC). We conceived a machine-learning method that harnesses genetic heterogeneity to score gene perturbations and identify phenotypically perturbed cells for in-depth characterization of gene perturbation effects. This approach enables genome-scale image-based multivariate gene perturbation profiling using CRISPR-Cas9. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

[Phenotypic heterogeneity of chronic obstructive pulmonary disease].

PubMed

Garcia-Aymerich, Judith; Agustí, Alvar; Barberà, Joan A; Belda, José; Farrero, Eva; Ferrer, Antoni; Ferrer, Jaume; Gáldiz, Juan B; Gea, Joaquim; Gómez, Federico P; Monsó, Eduard; Morera, Josep; Roca, Josep; Sauleda, Jaume; Antó, Josep M

2009-03-01

A functional definition of chronic obstructive pulmonary disease (COPD) based on airflow limitation has largely dominated the field. However, a view has emerged that COPD involves a complex array of cellular, organic, functional, and clinical events, with a growing interest in disentangling the phenotypic heterogeneity of COPD. The present review is based on the opinion of the authors, who have extensive research experience in several aspects of COPD. The starting assumption of the review is that current knowledge on the pathophysiology and clinical features of COPD allows us to classify phenotypic information in terms of the following dimensions: respiratory symptoms and health status, acute exacerbations, lung function, structural changes, local and systemic inflammation, and systemic effects. Twenty-six phenotypic traits were identified and assigned to one of the 6 dimensions. For each dimension, a summary is provided of the best evidence on the relationships among phenotypic traits, in particular among those corresponding to different dimensions, and on the relationship between these traits and relevant events in the natural history of COPD. The information has been organized graphically into a phenotypic matrix where each cell representing a pair of phenotypic traits is linked to relevant references. The information provided has the potential to increase our understanding of the heterogeneity of COPD phenotypes and help us plan future studies on aspects that are as yet unexplored.

Prenatal alcohol exposure and cellular differentiation: a role for Polycomb and Trithorax group proteins in FAS phenotypes?

PubMed

Veazey, Kylee J; Muller, Daria; Golding, Michael C

2013-01-01

Exposure to alcohol significantly alters the developmental trajectory of progenitor cells and fundamentally compromises tissue formation (i.e., histogenesis). Emerging research suggests that ethanol can impair mammalian development by interfering with the execution of molecular programs governing differentiation. For example, ethanol exposure disrupts cellular migration, changes cell-cell interactions, and alters growth factor signaling pathways. Additionally, ethanol can alter epigenetic mechanisms controlling gene expression. Normally, lineage-specific regulatory factors (i.e., transcription factors) establish the transcriptional networks of each new cell type; the cell's identity then is maintained through epigenetic alterations in the way in which the DNA encoding each gene becomes packaged within the chromatin. Ethanol exposure can induce epigenetic changes that do not induce genetic mutations but nonetheless alter the course of fetal development and result in a large array of patterning defects. Two crucial enzyme complexes--the Polycomb and Trithorax proteins--are central to the epigenetic programs controlling the intricate balance between self-renewal and the execution of cellular differentiation, with diametrically opposed functions. Prenatal ethanol exposure may disrupt the functions of these two enzyme complexes, altering a crucial aspect of mammalian differentiation. Characterizing the involvement of Polycomb and Trithorax group complexes in the etiology of fetal alcohol spectrum disorders will undoubtedly enhance understanding of the role that epigenetic programming plays in this complex disorder.

Chronic innate immune activation of TBK1 suppresses mTORC1 activity and dysregulates cellular metabolism.

PubMed

Hasan, Maroof; Gonugunta, Vijay K; Dobbs, Nicole; Ali, Aktar; Palchik, Guillermo; Calvaruso, Maria A; DeBerardinis, Ralph J; Yan, Nan

2017-01-24

Three-prime repair exonuclease 1 knockout (Trex1 -/- ) mice suffer from systemic inflammation caused largely by chronic activation of the cyclic GMP-AMP synthase-stimulator of interferon genes-TANK-binding kinase-interferon regulatory factor 3 (cGAS-STING-TBK1-IRF3) signaling pathway. We showed previously that Trex1-deficient cells have reduced mammalian target of rapamycin complex 1 (mTORC1) activity, although the underlying mechanism is unclear. Here, we performed detailed metabolic analysis in Trex1 -/- mice and cells that revealed both cellular and systemic metabolic defects, including reduced mitochondrial respiration and increased glycolysis, energy expenditure, and fat metabolism. We also genetically separated the inflammatory and metabolic phenotypes by showing that Sting deficiency rescued both inflammatory and metabolic phenotypes, whereas Irf3 deficiency only rescued inflammation on the Trex1 -/- background, and many metabolic defects persist in Trex1 -/- Irf3 -/- cells and mice. We also showed that Leptin deficiency (ob/ob) increased lipogenesis and prolonged survival of Trex1 -/- mice without dampening inflammation. Mechanistically, we identified TBK1 as a key regulator of mTORC1 activity in Trex1 -/- cells. Together, our data demonstrate that chronic innate immune activation of TBK1 suppresses mTORC1 activity, leading to dysregulated cellular metabolism.

Phenotypic screening for developmental neurotoxicity ...

EPA Pesticide Factsheets

There are large numbers of environmental chemicals with little or no available information on their toxicity, including developmental neurotoxicity. Because of the resource-intensive nature of traditional animal tests, high-throughput (HTP) methods that can rapidly evaluate chemicals for the potential to affect the developing brain are being explored. Typically, HTP screening uses biochemical and molecular assays to detect the interaction of a chemical with a known target or molecular initiating event (e.g., the mechanism of action). For developmental neurotoxicity, however, the mechanism(s) is often unknown. Thus, we have developed assays for detecting chemical effects on the key events of neurodevelopment at the cellular level (e.g., proliferation, differentiation, neurite growth, synaptogenesis, network formation). Cell-based assays provide a test system at a level of biological complexity that encompasses many potential neurotoxic mechanisms. For example, phenotypic assessment of neurite outgrowth at the cellular level can detect chemicals that target kinases, ion channels, or esterases at the molecular level. The results from cell-based assays can be placed in a conceptual framework using an Adverse Outcome Pathway (AOP) which links molecular, cellular, and organ level effects with apical measures of developmental neurotoxicity. Testing a wide range of concentrations allows for the distinction between selective effects on neurodevelopmental and non-specific

Molecular Mechanisms Modulating the Phenotype of Macrophages and Microglia

PubMed Central

Amici, Stephanie A.; Dong, Joycelyn; Guerau-de-Arellano, Mireia

2017-01-01

Macrophages and microglia play crucial roles during central nervous system development, homeostasis and acute events such as infection or injury. The diverse functions of tissue macrophages and microglia are mirrored by equally diverse phenotypes. A model of inflammatory/M1 versus a resolution phase/M2 macrophages has been widely used. However, the complexity of macrophage function can only be achieved by the existence of varied, plastic and tridimensional macrophage phenotypes. Understanding how tissue macrophages integrate environmental signals via molecular programs to define pathogen/injury inflammatory responses provides an opportunity to better understand the multilayered nature of macrophages, as well as target and modulate cellular programs to control excessive inflammation. This is particularly important in MS and other neuroinflammatory diseases, where chronic inflammatory macrophage and microglial responses may contribute to pathology. Here, we perform a comprehensive review of our current understanding of how molecular pathways modulate tissue macrophage phenotype, covering both classic pathways and the emerging role of microRNAs, receptor-tyrosine kinases and metabolism in macrophage phenotype. In addition, we discuss pathway parallels in microglia, novel markers helpful in the identification of peripheral macrophages versus microglia and markers linked to their phenotype. PMID:29176977

Identifying niche-mediated regulatory factors of stem cell phenotypic state: a systems biology approach.

PubMed

Ravichandran, Srikanth; Del Sol, Antonio

2017-02-01

Understanding how the cellular niche controls the stem cell phenotype is often hampered due to the complexity of variegated niche composition, its dynamics, and nonlinear stem cell-niche interactions. Here, we propose a systems biology view that considers stem cell-niche interactions as a many-body problem amenable to simplification by the concept of mean field approximation. This enables approximation of the niche effect on stem cells as a constant field that induces sustained activation/inhibition of specific stem cell signaling pathways in all stem cells within heterogeneous populations exhibiting the same phenotype (niche determinants). This view offers a new basis for the development of single cell-based computational approaches for identifying niche determinants, which has potential applications in regenerative medicine and tissue engineering. © 2017 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Pleiotropic and Epistatic Network-Based Discovery: Integrated Networks for Target Gene Discovery

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

Weighill, Deborah; Jones, Piet; Shah, Manesh

Biological organisms are complex systems that are composed of functional networks of interacting molecules and macro-molecules. Complex phenotypes are the result of orchestrated, hierarchical, heterogeneous collections of expressed genomic variants. However, the effects of these variants are the result of historic selective pressure and current environmental and epigenetic signals, and, as such, their co-occurrence can be seen as genome-wide correlations in a number of different manners. Biomass recalcitrance (i.e., the resistance of plants to degradation or deconstruction, which ultimately enables access to a plant's sugars) is a complex polygenic phenotype of high importance to biofuels initiatives. This study makes usemore » of data derived from the re-sequenced genomes from over 800 different Populus trichocarpa genotypes in combination with metabolomic and pyMBMS data across this population, as well as co-expression and co-methylation networks in order to better understand the molecular interactions involved in recalcitrance, and identify target genes involved in lignin biosynthesis/degradation. A Lines Of Evidence (LOE) scoring system is developed to integrate the information in the different layers and quantify the number of lines of evidence linking genes to target functions. This new scoring system was applied to quantify the lines of evidence linking genes to lignin-related genes and phenotypes across the network layers, and allowed for the generation of new hypotheses surrounding potential new candidate genes involved in lignin biosynthesis in P. trichocarpa, including various AGAMOUS-LIKE genes. Lastly, the resulting Genome Wide Association Study networks, integrated with Single Nucleotide Polymorphism (SNP) correlation, co-methylation, and co-expression networks through the LOE scores are proving to be a powerful approach to determine the pleiotropic and epistatic relationships underlying cellular functions and, as such, the molecular basis for complex phenotypes, such as recalcitrance.« less

Pleiotropic and Epistatic Network-Based Discovery: Integrated Networks for Target Gene Discovery

DOE PAGES

Weighill, Deborah; Jones, Piet; Shah, Manesh; ...

2018-05-11

Biological organisms are complex systems that are composed of functional networks of interacting molecules and macro-molecules. Complex phenotypes are the result of orchestrated, hierarchical, heterogeneous collections of expressed genomic variants. However, the effects of these variants are the result of historic selective pressure and current environmental and epigenetic signals, and, as such, their co-occurrence can be seen as genome-wide correlations in a number of different manners. Biomass recalcitrance (i.e., the resistance of plants to degradation or deconstruction, which ultimately enables access to a plant's sugars) is a complex polygenic phenotype of high importance to biofuels initiatives. This study makes usemore » of data derived from the re-sequenced genomes from over 800 different Populus trichocarpa genotypes in combination with metabolomic and pyMBMS data across this population, as well as co-expression and co-methylation networks in order to better understand the molecular interactions involved in recalcitrance, and identify target genes involved in lignin biosynthesis/degradation. A Lines Of Evidence (LOE) scoring system is developed to integrate the information in the different layers and quantify the number of lines of evidence linking genes to target functions. This new scoring system was applied to quantify the lines of evidence linking genes to lignin-related genes and phenotypes across the network layers, and allowed for the generation of new hypotheses surrounding potential new candidate genes involved in lignin biosynthesis in P. trichocarpa, including various AGAMOUS-LIKE genes. Lastly, the resulting Genome Wide Association Study networks, integrated with Single Nucleotide Polymorphism (SNP) correlation, co-methylation, and co-expression networks through the LOE scores are proving to be a powerful approach to determine the pleiotropic and epistatic relationships underlying cellular functions and, as such, the molecular basis for complex phenotypes, such as recalcitrance.« less

eIF4E and eIF4GI have distinct and differential imprints on multiple myeloma's proteome and signaling

PubMed Central

Attar-Schneider, Oshrat; Pasmanik-Chor, Metsada; Tartakover-Matalon, Shelly

2015-01-01

Accumulating data indicate translation plays a role in cancer biology, particularly its rate limiting stage of initiation. Despite this evolving recognition, the function and importance of specific translation initiation factors is unresolved. The eukaryotic translation initiation complex eIF4F consists of eIF4E and eIF4G at a 1:1 ratio. Although it is expected that they display interdependent functions, several publications suggest independent mechanisms. This study is the first to directly assess the relative contribution of eIF4F components to the expressed cellular proteome, transcription factors, microRNAs, and phenotype in a malignancy known for extensive protein synthesis-multiple myeloma (MM). Previously, we have shown that eIF4E/eIF4GI attenuation (siRNA/Avastin) deleteriously affected MM cells' fate and reduced levels of eIF4E/eIF4GI established targets. Here, we demonstrated that eIF4E/eIF4GI indeed have individual influences on cell proteome. We used an objective, high throughput assay of mRNA microarrays to examine the significance of eIF4E/eIF4GI silencing to several cellular facets such as transcription factors, microRNAs and phenotype. We showed different imprints for eIF4E and eIF4GI in all assayed aspects. These results promote our understanding of the relative contribution and importance of eIF4E and eIF4GI to the malignant phenotype and shed light on their function in eIF4F translation initiation complex. PMID:25717031

Phenotypic Spectrum in Osteogenesis Imperfecta Due to Mutations in TMEM38B: Unraveling a Complex Cellular Defect.

PubMed

Webb, Emma A; Balasubramanian, Meena; Fratzl-Zelman, Nadja; Cabral, Wayne A; Titheradge, Hannah; Alsaedi, Atif; Saraff, Vrinda; Vogt, Julie; Cole, Trevor; Stewart, Susan; Crabtree, Nicola J; Sargent, Brandi M; Gamsjaeger, Sonja; Paschalis, Eleftherios P; Roschger, Paul; Klaushofer, Klaus; Shaw, Nick J; Marini, Joan C; Högler, Wolfgang

2017-06-01

Recessive mutations in TMEM38B cause type XIV osteogenesis imperfecta (OI) by dysregulating intracellular calcium flux. Clinical and bone material phenotype description and osteoblast differentiation studies. Natural history study in pediatric research centers. Eight patients with type XIV OI. Clinical examinations included bone mineral density, radiographs, echocardiography, and muscle biopsy. Bone biopsy samples (n = 3) were analyzed using histomorphometry, quantitative backscattered electron microscopy, and Raman microspectroscopy. Cellular differentiation studies were performed on proband and control osteoblasts and normal murine osteoclasts. Type XIV OI clinical phenotype ranges from asymptomatic to severe. Previously unreported features include vertebral fractures, periosteal cloaking, coxa vara, and extraskeletal features (muscular hypotonia, cardiac abnormalities). Proband lumbar spine bone density z score was reduced [median -3.3 (range -4.77 to +0.1; n = 7)] and increased by +1.7 (1.17 to 3.0; n = 3) following bisphosphonate therapy. TMEM38B mutant bone has reduced trabecular bone volume, osteoblast, and particularly osteoclast numbers, with >80% reduction in bone resorption. Bone matrix mineralization is normal and nanoporosity low. We demonstrate a complex osteoblast differentiation defect with decreased expression of early markers and increased expression of late and mineralization-related markers. Predominance of trimeric intracellular cation channel type B over type A expression in murine osteoclasts supports an intrinsic osteoclast defect underlying low bone turnover. OI type XIV has a bone histology, matrix mineralization, and osteoblast differentiation pattern that is distinct from OI with collagen defects. Probands are responsive to bisphosphonates and some show muscular and cardiovascular features possibly related to intracellular calcium flux abnormalities. Copyright © 2017 Endocrine Society

Interactome Networks and Human Disease

PubMed Central

Vidal, Marc; Cusick, Michael E.; Barabási, Albert-László

2011-01-01

Complex biological systems and cellular networks may underlie most genotype to phenotype relationships. Here we review basic concepts in network biology, discussing different types of interactome networks and the insights that can come from analyzing them. We elaborate on why interactome networks are important to consider in biology, how they can be mapped and integrated with each other, what global properties are starting to emerge from interactome network models, and how these properties may relate to human disease. PMID:21414488

A targeted genetic modifier screen links the SWI2/SNF2 protein domino to growth and autophagy genes in Drosophila melanogaster.

PubMed

Kwon, Matt Hyoung; Callaway, Heather; Zhong, Jim; Yedvobnick, Barry

2013-05-20

Targeted genetic studies can facilitate phenotypic analyses and provide important insights into development and other complex processes. The SWI2/SNF2 DNA-dependent ATPase Domino (Dom) of Drosophila melanogaster, a component of the Tip60 acetyltransferase complex, has been associated with a wide spectrum of cellular processes at multiple developmental stages. These include hematopoiesis, cell proliferation, homeotic gene regulation, histone exchange during DNA repair, and Notch signaling. To explore the wider gene network associated with Dom action, we used RNAi directed against domino (dom) to mediate loss-of-function at the wing margin, a tissue that is readily scored for phenotypic changes. Dom RNAi driven through GAL4-UAS elicited dominant wing nicking that responded phenotypically to the dose of dom and other loci known to function with dom. We screened for phenotypic modifiers of this wing phenotype among 2500 transpositions of the EP P element and found both enhancers and suppressors. Several classes of modifier were obtained, including those encoding transcription factors, RNA regulatory proteins, and factors that regulate cell growth, proliferation and autophagy, a lysosomal degradation pathway that affects cell growth under conditions of starvation and stress. Our analysis is consistent with prior studies, suggesting that Dom acts pleiotropically as a positive effector of Notch signaling and a repressor of proliferation. This genetic system should facilitate screens for additional loci associated with Dom function, and complement biochemical approaches to their regulatory activity.

Detection of dysregulated protein-association networks by high-throughput proteomics predicts cancer vulnerabilities.

PubMed

Lapek, John D; Greninger, Patricia; Morris, Robert; Amzallag, Arnaud; Pruteanu-Malinici, Iulian; Benes, Cyril H; Haas, Wilhelm

2017-10-01

The formation of protein complexes and the co-regulation of the cellular concentrations of proteins are essential mechanisms for cellular signaling and for maintaining homeostasis. Here we use isobaric-labeling multiplexed proteomics to analyze protein co-regulation and show that this allows the identification of protein-protein associations with high accuracy. We apply this 'interactome mapping by high-throughput quantitative proteome analysis' (IMAHP) method to a panel of 41 breast cancer cell lines and show that deviations of the observed protein co-regulations in specific cell lines from the consensus network affects cellular fitness. Furthermore, these aberrant interactions serve as biomarkers that predict the drug sensitivity of cell lines in screens across 195 drugs. We expect that IMAHP can be broadly used to gain insight into how changing landscapes of protein-protein associations affect the phenotype of biological systems.

Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein

PubMed Central

Viktorova, Ekaterina G.; Nchoutmboube, Jules; Ford-Siltz, Lauren A.

2015-01-01

ABSTRACT It is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses. IMPORTANCE Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can easily generate mutants resistant to practically any compounds targeting viral proteins. An alternative approach is to target stable cellular factors recruited for the virus-specific functions. In the present study, we analyzed the factors permitting and restricting the establishment of the resistance of poliovirus, a small (+)RNA virus, to brefeldin A (BFA), a drug targeting a cellular component of the viral replication complex. We found that the emergence and replication potential of resistant mutants is cell type dependent and that BFA resistance reduces virus fitness. Our data provide a rational approach to the development of antiviral therapeutics targeting host factors. PMID:25653442

Next Generation Tissue Engineering of Orthopedic Soft Tissue-to-Bone Interfaces.

PubMed

Boys, Alexander J; McCorry, Mary Clare; Rodeo, Scott; Bonassar, Lawrence J; Estroff, Lara A

2017-09-01

Soft tissue-to-bone interfaces are complex structures that consist of gradients of extracellular matrix materials, cell phenotypes, and biochemical signals. These interfaces, called entheses for ligaments, tendons, and the meniscus, are crucial to joint function, transferring mechanical loads and stabilizing orthopedic joints. When injuries occur to connected soft tissue, the enthesis must be re-established to restore function, but due to structural complexity, repair has proven challenging. Tissue engineering offers a promising solution for regenerating these tissues. This prospective review discusses methodologies for tissue engineering the enthesis, outlined in three key design inputs: materials processing methods, cellular contributions, and biochemical factors.

Next Generation Tissue Engineering of Orthopedic Soft Tissue-to-Bone Interfaces

PubMed Central

Boys, Alexander J.; McCorry, Mary Clare; Rodeo, Scott; Bonassar, Lawrence J.; Estroff, Lara A.

2017-01-01

Soft tissue-to-bone interfaces are complex structures that consist of gradients of extracellular matrix materials, cell phenotypes, and biochemical signals. These interfaces, called entheses for ligaments, tendons, and the meniscus, are crucial to joint function, transferring mechanical loads and stabilizing orthopedic joints. When injuries occur to connected soft tissue, the enthesis must be re-established to restore function, but due to structural complexity, repair has proven challenging. Tissue engineering offers a promising solution for regenerating these tissues. This prospective review discusses methodologies for tissue engineering the enthesis, outlined in three key design inputs: materials processing methods, cellular contributions, and biochemical factors. PMID:29333332

Wild yeast harbor a variety of distinct amyloid structures with strong prion-inducing capabilities

PubMed Central

Westergard, Laura; True, Heather L.

2014-01-01

Summary Variation in amyloid structures profoundly influences a wide array of pathological phenotypes in mammalian protein conformation disorders and dominantly inherited phenotypes in yeast. Here, we describe, for the first time, naturally occurring, self-propagating, structural variants of a prion protein isolated from wild strains of the yeast Saccharomyces cerevisiae. Variants of the [RNQ+] prion propagating in a variety of wild yeast differ biochemically, in their intracellular distributions, and in their ability to promote formation of the [PSI+] prion. [PSI+] is an epigenetic regulator of cellular phenotype and adaptability. Strikingly, we find that most natural [RNQ+] variants induced [PSI+] at high frequencies and the majority of [PSI+] variants elicited strong cellular phenotypes. We hypothesize that the presence of an efficient [RNQ+] template primes the cell for [PSI+] formation in order to induce [PSI+] in conditions where it would be advantageous. These studies utilize naturally occurring structural variants to expand our understanding of the consequences of diverse prion conformations on cellular phenotypes. PMID:24673812

Current status and perspectives in atomic force microscopy-based identification of cellular transformation

PubMed Central

Dong, Chenbo; Hu, Xiao; Dinu, Cerasela Zoica

2016-01-01

Understanding the complex interplay between cells and their biomechanics and how the interplay is influenced by the extracellular microenvironment, as well as how the transforming potential of a tissue from a benign to a cancerous one is related to the dynamics of both the cell and its surroundings, holds promise for the development of targeted translational therapies. This review provides a comprehensive overview of atomic force microscopy-based technology and its applications for identification of cellular progression to a cancerous phenotype. The review also offers insights into the advancements that are required for the next user-controlled tool to allow for the identification of early cell transformation and thus potentially lead to improved therapeutic outcomes. PMID:27274238

Myopathology of Adult and Paediatric Mitochondrial Diseases

PubMed Central

Phadke, Rahul

2017-01-01

Mitochondria are dynamic organelles ubiquitously present in nucleated eukaryotic cells, subserving multiple metabolic functions, including cellular ATP generation by oxidative phosphorylation (OXPHOS). The OXPHOS machinery comprises five transmembrane respiratory chain enzyme complexes (RC). Defective OXPHOS gives rise to mitochondrial diseases (mtD). The incredible phenotypic and genetic diversity of mtD can be attributed at least in part to the RC dual genetic control (nuclear DNA (nDNA) and mitochondrial DNA (mtDNA)) and the complex interaction between the two genomes. Despite the increasing use of next-generation-sequencing (NGS) and various omics platforms in unravelling novel mtD genes and pathomechanisms, current clinical practice for investigating mtD essentially involves a multipronged approach including clinical assessment, metabolic screening, imaging, pathological, biochemical and functional testing to guide molecular genetic analysis. This review addresses the broad muscle pathology landscape including genotype–phenotype correlations in adult and paediatric mtD, the role of immunodiagnostics in understanding some of the pathomechanisms underpinning the canonical features of mtD, and recent diagnostic advances in the field. PMID:28677615

Altered Neuronal and Circuit Excitability in Fragile X Syndrome.

PubMed

Contractor, Anis; Klyachko, Vitaly A; Portera-Cailliau, Carlos

2015-08-19

Fragile X syndrome (FXS) results from a genetic mutation in a single gene yet produces a phenotypically complex disorder with a range of neurological and psychiatric problems. Efforts to decipher how perturbations in signaling pathways lead to the myriad alterations in synaptic and cellular functions have provided insights into the molecular underpinnings of this disorder. From this large body of data, the theme of circuit hyperexcitability has emerged as a potential explanation for many of the neurological and psychiatric symptoms in FXS. The mechanisms for hyperexcitability range from alterations in the expression or activity of ion channels to changes in neurotransmitters and receptors. Contributions of these processes are often brain region and cell type specific, resulting in complex effects on circuit function that manifest as altered excitability. Here, we review the current state of knowledge of the molecular, synaptic, and circuit-level mechanisms underlying hyperexcitability and their contributions to the FXS phenotypes. Copyright © 2015 Elsevier Inc. All rights reserved.

The cellular and molecular etiology of the craniofacial defects in the avian ciliopathic mutant talpid2

USDA-ARS?s Scientific Manuscript database

talpid2 is an avian autosomal recessive mutant with a myriad of congenital malformations, including polydactyly and facial clefting. Although phenotypically similar to talpid3, talpid2 has a distinct facial phenotype and an unknown cellular, molecular and genetic basis. We set out to determine the e...

Epigenetic Influences on Brain Development and Plasticity

PubMed Central

Fagiolini, Michela; Jensen, Catherine L.; Champagne, Frances A.

2009-01-01

A fine interplay exists between sensory experience and innate genetic programs leading to the sculpting of neuronal circuits during early brain development. Recent evidence suggests that the dynamic regulation of gene expression through epigenetic mechanisms is at the interface between environmental stimuli and long-lasting molecular, cellular and complex behavioral phenotypes acquired during periods of developmental plasticity. Understanding these mechanisms may give insight into the formation of critical periods and provide new strategies for increasing plasticity and adaptive change in adulthood. PMID:19545993

In situ macrophage phenotypic transition is affected by altered cellular composition prior to acute sterile muscle injury.

PubMed

Patsalos, Andreas; Pap, Attila; Varga, Tamas; Trencsenyi, Gyorgy; Contreras, Gerardo Alvarado; Garai, Ildiko; Papp, Zoltan; Dezso, Balazs; Pintye, Eva; Nagy, Laszlo

2017-09-01

The in situ phenotypic switch of macrophages is delayed in acute injury following irradiation. The combination of bone marrow transplantation and local muscle radiation protection allows for the identification of a myeloid cell contribution to tissue repair. PET-MRI allows monitoring of myeloid cell invasion and metabolism. Altered cellular composition prior to acute sterile injury affects the in situ phenotypic transition of invading myeloid cells to repair macrophages. There is reciprocal intercellular communication between local muscle cell compartments, such as PAX7 positive cells, and recruited macrophages during skeletal muscle regeneration. Skeletal muscle regeneration is a complex interplay between various cell types including invading macrophages. Their recruitment to damaged tissues upon acute sterile injuries is necessary for clearance of necrotic debris and for coordination of tissue regeneration. This highly dynamic process is characterized by an in situ transition of infiltrating monocytes from an inflammatory (Ly6C high ) to a repair (Ly6C low ) macrophage phenotype. The importance of the macrophage phenotypic shift and the cross-talk of the local muscle tissue with the infiltrating macrophages during tissue regeneration upon injury are not fully understood and their study lacks adequate methodology. Here, using an acute sterile skeletal muscle injury model combined with irradiation, bone marrow transplantation and in vivo imaging, we show that preserved muscle integrity and cell composition prior to the injury is necessary for the repair macrophage phenotypic transition and subsequently for proper and complete tissue regeneration. Importantly, by using a model of in vivo ablation of PAX7 positive cells, we show that this radiosensitive skeletal muscle progenitor pool contributes to macrophage phenotypic transition following acute sterile muscle injury. In addition, local muscle tissue radioprotection by lead shielding during irradiation preserves normal macrophage transition dynamics and subsequently muscle tissue regeneration. Taken together, our data suggest the existence of a more extensive and reciprocal cross-talk between muscle tissue compartments, including satellite cells, and infiltrating myeloid cells upon tissue damage. These interactions shape the macrophage in situ phenotypic shift, which is indispensable for normal muscle tissue repair dynamics. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

PDT-based combinations in overcoming chemoresistance from stromal and heterotypic cellular communication (Conference Presentation)

NASA Astrophysics Data System (ADS)

Rizvi, Imran; Bulin, Anne-Laure; Anbil, Sriram R.; Briars, Emma A.; Vecchio, Daniela; Celli, Jonathan P.; Broekgaarden, Mans; Hasan, Tayyaba

2017-02-01

Targeting the molecular and cellular cues that influence treatment resistance in tumors is critical to effectively treating unresponsive populations of stubborn disease. The informed design of mechanism-based combinations is emerging as increasingly important to targeting resistance and improving the efficacy of conventional treatments, while minimizing toxicity. Photodynamic therapy (PDT) has been shown to synergize with conventional agents and to overcome the evasion pathways that cause resistance. Increasing evidence shows that PDT-based combinations cooperate mechanistically with, and improve the therapeutic index of, traditional chemotherapies. These and other findings emphasize the importance of including PDT as part of comprehensive treatment plans for cancer, particularly in complex disease sites. Identifying effective combinations requires a multi-faceted approach that includes the development of bioengineered cancer models and corresponding image analysis tools. The molecular and phenotypic basis of verteporfin-mediated PDT-based enhancement of chemotherapeutic efficacy and predictability in complex 3D models for ovarian cancer will be presented.

A Journey Through a Leaf: Phenomics Analysis of Leaf Growth in Arabidopsis thaliana

PubMed Central

Vanhaeren, Hannes; Gonzalez, Nathalie; Inzé, Dirk

2015-01-01

In Arabidopsis, leaves contribute to the largest part of the aboveground biomass. In these organs, light is captured and converted into chemical energy, which plants use to grow and complete their life cycle. Leaves emerge as a small pool of cells at the vegetative shoot apical meristem and develop into planar, complex organs through different interconnected cellular events. Over the last decade, numerous phenotyping techniques have been developed to visualize and quantify leaf size and growth, leading to the identification of numerous genes that contribute to the final size of leaves. In this review, we will start at the Arabidopsis rosette level and gradually zoom in from a macroscopic view on leaf growth to a microscopic and molecular view. Along this journey, we describe different techniques that have been key to identify important events during leaf development and discuss approaches that will further help unraveling the complex cellular and molecular mechanisms that underlie leaf growth. PMID:26217168

Cellular stress responses to chronic heat shock and shell damage in temperate Mya truncata.

PubMed

Sleight, Victoria A; Peck, Lloyd S; Dyrynda, Elisabeth A; Smith, Valerie J; Clark, Melody S

2018-05-12

Acclimation, via phenotypic flexibility, is a potential means for a fast response to climate change. Understanding the molecular mechanisms underpinning phenotypic flexibility can provide a fine-scale cellular understanding of how organisms acclimate. In the last 30 years, Mya truncata populations around the UK have faced an average increase in sea surface temperature of 0.7 °C and further warming of between 1.5 and 4 °C, in all marine regions adjacent to the UK, is predicted by the end of the century. Hence, data are required on the ability of M. truncata to acclimate to physiological stresses, and most notably, chronic increases in temperature. Animals in the present study were exposed to chronic heat-stress for 2 months prior to shell damage and subsequently, only 3, out of 20 damaged individuals, were able to repair their shells within 2 weeks. Differentially expressed genes (between control and damaged animals) were functionally enriched with processes relating to cellular stress, the immune response and biomineralisation. Comparative transcriptomics highlighted genes, and more broadly molecular mechanisms, that are likely to be pivotal in this lack of acclimation. This study demonstrates that discovery-led transcriptomic profiling of animals during stress-response experiments can shed light on the complexity of biological processes and changes within organisms that can be more difficult to detect at higher levels of biological organisation.

Optogenetic Approaches to Drug Discovery in Neuroscience and Beyond.

PubMed

Zhang, Hongkang; Cohen, Adam E

2017-07-01

Recent advances in optogenetics have opened new routes to drug discovery, particularly in neuroscience. Physiological cellular assays probe functional phenotypes that connect genomic data to patient health. Optogenetic tools, in particular tools for all-optical electrophysiology, now provide a means to probe cellular disease models with unprecedented throughput and information content. These techniques promise to identify functional phenotypes associated with disease states and to identify compounds that improve cellular function regardless of whether the compound acts directly on a target or through a bypass mechanism. This review discusses opportunities and unresolved challenges in applying optogenetic techniques throughout the discovery pipeline - from target identification and validation, to target-based and phenotypic screens, to clinical trials. Copyright © 2017 Elsevier Ltd. All rights reserved.

Complex Determinants of Epithelial: Mesenchymal Phenotypic Plasticity in Ovarian Cancer

PubMed Central

Klymenko, Yuliya; Kim, Oleg; Stack, M. Sharon

2017-01-01

Unlike most epithelial malignancies which metastasize hematogenously, metastasis of epithelial ovarian cancer (EOC) occurs primarily via transcoelomic dissemination, characterized by exfoliation of cells from the primary tumor, avoidance of detachment-induced cell death (anoikis), movement throughout the peritoneal cavity as individual cells and multi-cellular aggregates (MCAs), adhesion to and disruption of the mesothelial lining of the peritoneum, and submesothelial matrix anchoring and proliferation to generate widely disseminated metastases. This exceptional microenvironment is highly permissive for phenotypic plasticity, enabling mesenchymal-to-epithelial (MET) and epithelial-to-mesenchymal (EMT) transitions. In this review, we summarize current knowledge on EOC heterogeneity in an EMT context, outline major regulators of EMT in ovarian cancer, address controversies in EMT and EOC chemoresistance, and highlight computational modeling approaches toward understanding EMT/MET in EOC. PMID:28792442

Are There Roles for Brain Cell Senescence in Aging and Neurodegenerative Disorders?

PubMed Central

Tan, Florence C. C.; Hutchison, Emmette R.; Eitan, Erez; Mattson, Mark P.

2014-01-01

The term cellular senescence was introduced more than five decades ago to describe the state of growth arrest observed in aging cells. Since this initial discovery, the phenotypes associated with cellular senescence have expanded beyond growth arrest to include alterations in cellular metabolism, secreted cytokines, epigenetic regulation and protein expression. Recently, senescence has been shown to play an important role in vivo not only in relation to aging, but also during embryonic development. Thus, cellular senescence serves different purposes and comprises a wide range of distinct phenotypes across multiple cell types. Whether all cell types, including post-mitotic neurons, are capable of entering into a senescent state remains unclear. In this review we examine recent data that suggest that cellular senescence plays a role in brain aging and, notably, may not be limited to glia but also neurons. We suggest that there is a high level of similarity between some of the pathological changes that occur in the brain in Alzheimer’s and Parkinson’s diseases and those phenotypes observed in cellular senescence, leading us to propose that neurons and glia can exhibit hallmarks of senescence previously documented in peripheral tissues. PMID:25305051

Are there roles for brain cell senescence in aging and neurodegenerative disorders?

PubMed

Tan, Florence C C; Hutchison, Emmette R; Eitan, Erez; Mattson, Mark P

2014-12-01

The term cellular senescence was introduced more than five decades ago to describe the state of growth arrest observed in aging cells. Since this initial discovery, the phenotypes associated with cellular senescence have expanded beyond growth arrest to include alterations in cellular metabolism, secreted cytokines, epigenetic regulation and protein expression. Recently, senescence has been shown to play an important role in vivo not only in relation to aging, but also during embryonic development. Thus, cellular senescence serves different purposes and comprises a wide range of distinct phenotypes across multiple cell types. Whether all cell types, including post-mitotic neurons, are capable of entering into a senescent state remains unclear. In this review we examine recent data that suggest that cellular senescence plays a role in brain aging and, notably, may not be limited to glia but also neurons. We suggest that there is a high level of similarity between some of the pathological changes that occur in the brain in Alzheimer's and Parkinson's diseases and those phenotypes observed in cellular senescence, leading us to propose that neurons and glia can exhibit hallmarks of senescence previously documented in peripheral tissues.

Evidence for complete epistasis of null mutations in murine Fanconi anemia genes Fanca and Fancg.

PubMed

van de Vrugt, Henri J; Koomen, Mireille; Bakker, Sietske; Berns, Mariska A D; Cheng, Ngan Ching; van der Valk, Martin A; de Vries, Yne; Rooimans, Martin A; Oostra, Anneke B; Hoatlin, Maureen E; Te Riele, Hein; Joenje, Hans; Arwert, Fré

2011-12-10

Fanconi anemia (FA) is a heritable disease characterized by bone marrow failure, congenital abnormalities, and cancer predisposition. The 15 identified FA genes operate in a molecular pathway to preserve genomic integrity. Within this pathway the FA core complex operates as an ubiquitin ligase that activates the complex of FANCD2 and FANCI to coordinate DNA repair. The FA core complex is formed by at least 12 proteins. However, only the FANCL subunit displays ubiquitin ligase activity. FANCA and FANCG are members of the FA core complex for which no other functions have been described than to participate in protein interactions. In this study we generated mice with combined null alleles for Fanca and Fancg to identify extended functions for these genes by characterizing the double mutant mice and cells. Double mutant a(-/-)/g(-/-) mice were born at near Mendelian frequencies without apparent developmental abnormalities. Histological analysis of a(-/-)/g(-/-) mice revealed a Leydig cell hyperplasia and frequent vacuolization of Sertoli cells in testes, while ovaries were depleted from developing follicles and displayed an interstitial cell hyperplasia. These gonadal aberrations were associated with a compromised fertility of a(-/-)/g(-/-) males and females. During the first year of life a(-/-)/g(-/-) did not develop malignancies or bone marrow failure. At the cellular level a(-/-)/g(-/-), Fanca(-/-), and Fancg(-/-) cells proved equally compromised in DNA crosslink and homology-directed repair. Overall the phenotype of a(-/-)/g(-/-) double knockout mice and cells appeared highly similar to the phenotype of Fanca or Fancg single knockouts. The lack of an augmented phenotype suggest that null mutations in Fanca or Fancg are fully epistatic, making additional important functions outside of the FA core complex highly unlikely. 2011 Elsevier B.V. All rights reserved.

WAVE2 regulates epithelial morphology and cadherin isoform switching through regulation of Twist and Abl.

PubMed

Bryce, Nicole S; Reynolds, Albert B; Koleske, Anthony J; Weaver, Alissa M

2013-01-01

Epithelial morphogenesis is a dynamic process that involves coordination of signaling and actin cytoskeletal rearrangements. We analyzed the contribution of the branched actin regulator WAVE2 in the development of 3-dimensional (3D) epithelial structures. WAVE2-knockdown (WAVE2-KD) cells formed large multi-lobular acini that continued to proliferate at an abnormally late stage compared to control acini. Immunostaining of the cell-cell junctions of WAVE2-KD acini revealed weak and heterogeneous E-cadherin staining despite little change in actin filament localization to the same junctions. Analysis of cadherin expression demonstrated a decrease in E-cadherin and an increase in N-cadherin protein and mRNA abundance in total cell lysates. In addition, WAVE2-KD cells exhibited an increase in the mRNA levels of the epithelial-mesenchymal transition (EMT)-associated transcription factor Twist1. KD of Twist1 expression in WAVE2-KD cells reversed the cadherin switching and completely rescued the aberrant 3D morphological phenotype. Activity of the WAVE2 complex binding partner Abl kinase was also increased in WAVE2-KD cells, as assessed by tyrosine phosphorylation of the Abl substrate CrkL. Inhibition of Abl with STI571 rescued the multi-lobular WAVE2-KD 3D phenotype whereas overexpression of Abl kinase phenocopied the WAVE2-KD phenotype. The WAVE2 complex regulates breast epithelial morphology by a complex mechanism involving repression of Twist1 expression and Abl kinase activity. These data reveal a critical role for WAVE2 complex in regulation of cellular signaling and epithelial morphogenesis.

TWIST1-WDR5-Hottip regulates Hoxa9 chromatin to facilitate prostate cancer metastasis

PubMed Central

Malek, Reem; Gajula, Rajendra P.; Williams, Russell D.; Nghiem, Belinda; Simons, Brian W.; Nugent, Katriana; Wang, Hailun; Taparra, Kekoa; Lemtiri-Chlieh, Ghali; Yoon, Arum R.; True, Lawrence; An, Steven S.; DeWeese, Theodore L.; Ross, Ashley E.; Schaeffer, Edward M.; Pienta, Kenneth J.; Hurley, Paula J.; Morrissey, Colm; Tran, Phuoc T.

2017-01-01

TWIST1 is a transcription factor critical for development which can promote prostate cancer metastasis. During embryonic development, TWIST1 and HOXA9 are co-expressed in mouse prostate and then silenced post-natally. Here we report that TWIST1 and HOXA9 co-expression are re-activated in mouse and human primary prostate tumors and are further enriched in human metastases, correlating with survival. TWIST1 formed a complex with WDR5 and the lncRNA Hottip/HOTTIP, members of the MLL/COMPASS-like H3K4 methylases, which regulate chromatin in the Hox/HOX cluster during development. TWIST1 overexpression led to co-enrichment of TWIST1 and WDR5 as well increased H3K4me3 chromatin at the Hoxa9/HOXA9 promoter which was dependent on WDR5. Expression of WDR5 and Hottip/HOTTIP was also required for TWIST1-induced upregulation of HOXA9 and aggressive cellular phenotypes such as invasion and migration. Pharmacological inhibition of HOXA9 prevented TWIST1-induced aggressive prostate cancer cellular phenotypes in vitro and metastasis in vivo. This study demonstrates a novel mechanism by which TWIST1 regulates chromatin and gene expression by cooperating with the COMPASS-like complex to increase H3K4 trimethylation at target gene promoters. Our findings highlight a TWIST1-HOXA9 embryonic prostate developmental program that is reactivated during prostate cancer metastasis and is therapeutically targetable. PMID:28484075

The topology and dynamics of complex networks

NASA Astrophysics Data System (ADS)

Dezso, Zoltan

We start with a brief introduction about the topological properties of real networks. Most real networks are scale-free, being characterized by a power-law degree distribution. The scale-free nature of real networks leads to unexpected properties such as the vanishing epidemic threshold. Traditional methods aiming to reduce the spreading rate of viruses cannot succeed on eradicating the epidemic on a scale-free network. We demonstrate that policies that discriminate between the nodes, curing mostly the highly connected nodes, can restore a finite epidemic threshold and potentially eradicate the virus. We find that the more biased a policy is towards the hubs, the more chance it has to bring the epidemic threshold above the virus' spreading rate. We continue by studying a large Web portal as a model system for a rapidly evolving network. We find that the visitation pattern of a news document decays as a power law, in contrast with the exponential prediction provided by simple models of site visitation. This is rooted in the inhomogeneous nature of the browsing pattern characterizing individual users: the time interval between consecutive visits by the same user to the site follows a power law distribution, in contrast with the exponential expected for Poisson processes. We show that the exponent characterizing the individual user's browsing patterns determines the power-law decay in a document's visitation. Finally, we turn our attention to biological networks and demonstrate quantitatively that protein complexes in the yeast, Saccharomyces cerevisiae, are comprised of a core in which subunits are highly coexpressed, display the same deletion phenotype (essential or non-essential) and share identical functional classification and cellular localization. The results allow us to define the deletion phenotype and cellular task of most known complexes, and to identify with high confidence the biochemical role of hundreds of proteins with yet unassigned functionality.

A cadmium-sensitive, glutathione-deficient mutant of Arabidopsis thaliana.

PubMed Central

Howden, R; Andersen, C R; Goldsbrough, P B; Cobbett, C S

1995-01-01

The roots of the cadmium-sensitive mutant of Arabidopsis thaliana, cad1-1, become brown in the presence of cadmium. A new cadmium-sensitive mutant affected at a second locus, cad2, has been identified using this phenotype. Genetic analysis has grown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus. Assays of cadmium accumulation by intact plants indicated that the mutant is deficient in its ability to sequester cadmium. Undifferentiated callus tissue was also cadmium sensitive, suggesting that the mutant phenotype is expressed at the cellular level. The level of cadmium-binding complexes formed in vivo was decreased compared with the wild type and accumulation of phytochelatins was about 10% of that in the wild type. The level of glutathione, the substrate for phytochelatin biosynthesis, in tissues of the mutant was decreased to about 15 to 30% of that in the wild type. Thus, the deficiency in phytochelatin biosynthesis can be explained by a deficiency in glutathione. PMID:7770518

Gene signature critical to cancer phenotype as a paradigm for anti-cancer drug discovery

PubMed Central

Sampson, Erik R.; McMurray, Helene R.; Hassane, Duane C.; Newman, Laurel; Salzman, Peter; Jordan, Craig T.; Land, Hartmut

2013-01-01

Malignant cell transformation commonly results in the deregulation of thousands of cellular genes, an observation that suggests a complex biological process and an inherently challenging scenario for the development of effective cancer interventions. To better define the genes/pathways essential to regulating the malignant phenotype, we recently described a novel strategy based on the cooperative nature of carcinogenesis that focuses on genes synergistically deregulated in response to cooperating oncogenic mutations. These so-called “cooperation response genes” (CRGs) are highly enriched for genes critical for the cancer phenotype, thereby suggesting their causal role in the malignant state. Here we show that CRGs play an essential role in drug-mediated anti-cancer activity and that anti-cancer agents can be identified through their ability to antagonize the CRG expression profile. These findings provide proof-of-concept for the use of the CRG signature as a novel means of drug discovery with relevance to underlying anti-cancer drug mechanisms. PMID:22964631

Disease signatures for schizophrenia and bipolar disorder using patient-derived induced pluripotent stem cells.

PubMed

Watmuff, Bradley; Berkovitch, Shaunna S; Huang, Joanne H; Iaconelli, Jonathan; Toffel, Steven; Karmacharya, Rakesh

2016-06-01

Schizophrenia and bipolar disorder are complex psychiatric disorders that present unique challenges in the study of disease biology. There are no objective biological phenotypes for these disorders, which are characterized by complex genetics and prominent roles for gene-environment interactions. The study of the neurobiology underlying these severe psychiatric disorders has been hindered by the lack of access to the tissue of interest - neurons from patients. The advent of reprogramming methods that enable generation of induced pluripotent stem cells (iPSCs) from patient fibroblasts and peripheral blood mononuclear cells has opened possibilities for new approaches to study relevant disease biology using iPSC-derived neurons. While early studies with patient iPSCs have led to promising and intriguing leads, significant hurdles remain in our attempts to capture the complexity of these disorders in vitro. We present here an overview of studies to date of schizophrenia and bipolar disorder using iPSC-derived neuronal cells and discuss potential future directions that can result in the identification of robust and valid cellular phenotypes that in turn can lay the groundwork for meaningful clinical advances. Copyright © 2016 Elsevier Inc. All rights reserved.

Fast and accurate automated cell boundary determination for fluorescence microscopy

NASA Astrophysics Data System (ADS)

Arce, Stephen Hugo; Wu, Pei-Hsun; Tseng, Yiider

2013-07-01

Detailed measurement of cell phenotype information from digital fluorescence images has the potential to greatly advance biomedicine in various disciplines such as patient diagnostics or drug screening. Yet, the complexity of cell conformations presents a major barrier preventing effective determination of cell boundaries, and introduces measurement error that propagates throughout subsequent assessment of cellular parameters and statistical analysis. State-of-the-art image segmentation techniques that require user-interaction, prolonged computation time and specialized training cannot adequately provide the support for high content platforms, which often sacrifice resolution to foster the speedy collection of massive amounts of cellular data. This work introduces a strategy that allows us to rapidly obtain accurate cell boundaries from digital fluorescent images in an automated format. Hence, this new method has broad applicability to promote biotechnology.

Air Pollution Stress and the Aging Phenotype: The Telomere Connection.

PubMed

Martens, Dries S; Nawrot, Tim S

2016-09-01

Aging is a complex physiological phenomenon. The question why some subjects grow old while remaining free from disease whereas others prematurely die remains largely unanswered. We focus here on the role of air pollution in biological aging. Hallmarks of aging can be grouped into three main categories: genomic instability, telomere attrition, and epigenetic alterations leading to altered mitochondrial function and cellular senescence. At birth, the initial telomere length of a person is largely determined by environmental factors. Telomere length shortens with each cell division and exposure to air pollution as well as low residential greens space exposure is associated with shorter telomere length. Recent studies show that the estimated effects of particulate air pollution exposure on the telomere mitochondrial axis of aging may play an important role in chronic health effects of air pollution. The exposome encompasses all exposures over an entire life. As telomeres can be considered as the cellular memories of exposure to oxidative stress and inflammation, telomere maintenance may be a proxy for assessing the "exposome". If telomeres are causally related to the aging phenotype and environmental air pollution is an important determinant of telomere length, this might provide new avenues for future preventive strategies.

Systems metabolic engineering: genome-scale models and beyond.

PubMed

Blazeck, John; Alper, Hal

2010-07-01

The advent of high throughput genome-scale bioinformatics has led to an exponential increase in available cellular system data. Systems metabolic engineering attempts to use data-driven approaches--based on the data collected with high throughput technologies--to identify gene targets and optimize phenotypical properties on a systems level. Current systems metabolic engineering tools are limited for predicting and defining complex phenotypes such as chemical tolerances and other global, multigenic traits. The most pragmatic systems-based tool for metabolic engineering to arise is the in silico genome-scale metabolic reconstruction. This tool has seen wide adoption for modeling cell growth and predicting beneficial gene knockouts, and we examine here how this approach can be expanded for novel organisms. This review will highlight advances of the systems metabolic engineering approach with a focus on de novo development and use of genome-scale metabolic reconstructions for metabolic engineering applications. We will then discuss the challenges and prospects for this emerging field to enable model-based metabolic engineering. Specifically, we argue that current state-of-the-art systems metabolic engineering techniques represent a viable first step for improving product yield that still must be followed by combinatorial techniques or random strain mutagenesis to achieve optimal cellular systems.

Bistability, epigenetics, and bet-hedging in bacteria.

PubMed

Veening, Jan-Willem; Smits, Wiep Klaas; Kuipers, Oscar P

2008-01-01

Clonal populations of microbial cells often show a high degree of phenotypic variability under homogeneous conditions. Stochastic fluctuations in the cellular components that determine cellular states can cause two distinct subpopulations, a property called bistability. Phenotypic heterogeneity can be readily obtained by interlinking multiple gene regulatory pathways, effectively resulting in a genetic logic-AND gate. Although switching between states can occur within the cells' lifetime, cells can also pass their cellular state over to the next generation by a mechanism known as epigenetic inheritance and thus perpetuate the phenotypic state. Importantly, heterogeneous populations can demonstrate increased fitness compared with homogeneous populations. This suggests that microbial cells employ bet-hedging strategies to maximize survival. Here, we discuss the possible roles of interlinked bistable networks, epigenetic inheritance, and bet-hedging in bacteria.

Detecting phenotype-driven transitions in regulatory network structure.

PubMed

Padi, Megha; Quackenbush, John

2018-01-01

Complex traits and diseases like human height or cancer are often not caused by a single mutation or genetic variant, but instead arise from functional changes in the underlying molecular network. Biological networks are known to be highly modular and contain dense "communities" of genes that carry out cellular processes, but these structures change between tissues, during development, and in disease. While many methods exist for inferring networks and analyzing their topologies separately, there is a lack of robust methods for quantifying differences in network structure. Here, we describe ALPACA (ALtered Partitions Across Community Architectures), a method for comparing two genome-scale networks derived from different phenotypic states to identify condition-specific modules. In simulations, ALPACA leads to more nuanced, sensitive, and robust module discovery than currently available network comparison methods. As an application, we use ALPACA to compare transcriptional networks in three contexts: angiogenic and non-angiogenic subtypes of ovarian cancer, human fibroblasts expressing transforming viral oncogenes, and sexual dimorphism in human breast tissue. In each case, ALPACA identifies modules enriched for processes relevant to the phenotype. For example, modules specific to angiogenic ovarian tumors are enriched for genes associated with blood vessel development, and modules found in female breast tissue are enriched for genes involved in estrogen receptor and ERK signaling. The functional relevance of these new modules suggests that not only can ALPACA identify structural changes in complex networks, but also that these changes may be relevant for characterizing biological phenotypes.

Contribution of high-content imaging technologies to the development of anti-infective drugs.

PubMed

Ang, Michelle Lay Teng; Pethe, Kevin

2016-08-01

Originally developed to study fundamental aspects of cellular biology, high-content imaging (HCI) was rapidly adapted to study host-pathogen interactions at the cellular level and adopted as a technology of choice to unravel disease biology. HCI platforms allow for the visualization and quantification of discrete phenotypes that cannot be captured using classical screening approaches. A key advantage of high-content screening technologies lies in the possibility to develop and interrogate physiologically significant, predictive ex vivo disease models that reproduce complex conditions relevant for infection. Here we review and discuss recent advances in HCI technologies and chemical biology approaches that are contributing to an increased understanding of the intricate host-pathogen interrelationship on the cellular level, and which will foster the development of novel therapeutic approaches for the treatment of human bacterial and protozoan infections. © 2016 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC. © 2016 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.

A novel microduplication of ARID1B: Clinical, genetic, and proteomic findings.

PubMed

Seabra, Catarina M; Szoko, Nicholas; Erdin, Serkan; Ragavendran, Ashok; Stortchevoi, Alexei; Maciel, Patrícia; Lundberg, Kathleen; Schlatzer, Daniela; Smith, Janice; Talkowski, Michael E; Gusella, James F; Natowicz, Marvin R

2017-09-01

Genetic alterations of ARID1B have been recently recognized as one of the most common mendelian causes of intellectual disability and are associated with both syndromic and non-syndromic phenotypes. The ARID1B protein, a subunit of the chromatin remodeling complex SWI/SNF-A, is involved in the regulation of transcription and multiple downstream cellular processes. We report here the clinical, genetic, and proteomic phenotypes of an individual with a unique apparent de novo mutation of ARID1B due to an intragenic duplication. His neurodevelopmental phenotype includes a severe speech/language disorder with full scale IQ scores 78-98 and scattered academic skill levels, expanding the phenotypic spectrum of ARID1B mutations. Haploinsufficiency of ARID1B was determined both by RNA sequencing and quantitative RT-PCR. Fluorescence in situ hybridization analysis supported an intragenic localization of the ARID1B copy number gain. Principal component analysis revealed marked differentiation of the subject's lymphoblast proteome from that of controls. Of 3426 proteins quantified, 1014 were significantly up- or down-regulated compared to controls (q < 0.01). Pathway analysis revealed highly significant enrichment for canonical pathways of EIF2 and EIF4 signaling, protein ubiquitination, tRNA charging and chromosomal replication, among others. Network analyses revealed down-regulation of: (1) intracellular components involved in organization of membranes, organelles, and vesicles; (2) aspects of cell cycle control, signal transduction, and nuclear protein export; (3) ubiquitination and proteosomal function; and (4) aspects of mRNA synthesis/splicing. Further studies are needed to determine the detailed molecular and cellular mechanisms by which constitutional haploinsufficiency of ARID1B causes syndromic and non-syndromic developmental disabilities. © 2017 Wiley Periodicals, Inc.

Elementary Mode Analysis: A Useful Metabolic Pathway Analysis Tool for Characterizing Cellular Metabolism

PubMed Central

Trinh, Cong T.; Wlaschin, Aaron; Srienc, Friedrich

2010-01-01

Elementary Mode Analysis is a useful Metabolic Pathway Analysis tool to identify the structure of a metabolic network that links the cellular phenotype to the corresponding genotype. The analysis can decompose the intricate metabolic network comprised of highly interconnected reactions into uniquely organized pathways. These pathways consisting of a minimal set of enzymes that can support steady state operation of cellular metabolism represent independent cellular physiological states. Such pathway definition provides a rigorous basis to systematically characterize cellular phenotypes, metabolic network regulation, robustness, and fragility that facilitate understanding of cell physiology and implementation of metabolic engineering strategies. This mini-review aims to overview the development and application of elementary mode analysis as a metabolic pathway analysis tool in studying cell physiology and as a basis of metabolic engineering. PMID:19015845

RAPTOR controls developmental growth transitions by altering the hormonal and metabolic balance.

PubMed

Salem, Mohamed A; Li, Yan; Bajdzienko, Krzysztof; Fisahn, Joachim; Watanabe, Mutsumi; Hoefgen, Rainer; Schöttler, Mark Aurel; Giavalisco, Patrick

2018-04-23

Vegetative growth requires the systemic coordination of numerous cellular processes, which are controlled by regulatory proteins that monitor extra- and intra-cellular cues and translate them into growth decisions. In eukaryotes, one of the central factors regulating growth is the Ser/Thr protein kinase Target of Rapamycin (TOR), which forms complexes with regulatory proteins. To understand the function of one such regulatory protein, Regulatory-Associated Protein of TOR 1B (RAPTOR1B) in plants, we analyzed the effect of raptor1b mutations on growth and physiology in Arabidopsis thaliana by detailed phenotyping, metabolomic, lipidomic, and proteomic analysis. Mutation of RAPTR1B resulted in a strong reduction of TOR kinase activity, leading to massive changes in central carbon and nitrogen metabolism, accumulation of excess starch, and induction of autophagy. These shifts led to a significant, reduction of plant growth that occurred non-linearly during developmental stage transtions.. This phenotype was accompanied by changes in cell morphology and tissue anatomy. In contrast to previous studies in rice, we found that the Arabidopsis raptor1b mutation did not affect chloroplast development or photosynthetic electron transport efficiency; however, it resulted in decreased CO2 assimilation rate and increased stomatal conductance. The raptor1b mutants also had reduced abscisic acid levels. Surprisingly, ABA feeding experiments resulted in partial complementation of the growth phenotypes, indicating the tight interaction between TOR function and hormone synthesis and signaling in plants. {copyright, serif} 2018 American Society of Plant Biologists. All rights reserved.

Accelerating glioblastoma drug discovery: Convergence of patient-derived models, genome editing and phenotypic screening.

PubMed

O'Duibhir, Eoghan; Carragher, Neil O; Pollard, Steven M

2017-04-01

Patients diagnosed with glioblastoma (GBM) continue to face a bleak prognosis. It is critical that new effective therapeutic strategies are developed. GBM stem cells have molecular hallmarks of neural stem and progenitor cells and it is possible to propagate both non-transformed normal neural stem cells and GBM stem cells, in defined, feeder-free, adherent culture. These primary stem cell lines provide an experimental model that is ideally suited to cell-based drug discovery or genetic screens in order to identify tumour-specific vulnerabilities. For many solid tumours, including GBM, the genetic disruptions that drive tumour initiation and growth have now been catalogued. CRISPR/Cas-based genome editing technologies have recently emerged, transforming our ability to functionally annotate the human genome. Genome editing opens prospects for engineering precise genetic changes in normal and GBM-derived neural stem cells, which will provide more defined and reliable genetic models, with critical matched pairs of isogenic cell lines. Generation of more complex alleles such as knock in tags or fluorescent reporters is also now possible. These new cellular models can be deployed in cell-based phenotypic drug discovery (PDD). Here we discuss the convergence of these advanced technologies (iPS cells, neural stem cell culture, genome editing and high content phenotypic screening) and how they herald a new era in human cellular genetics that should have a major impact in accelerating glioblastoma drug discovery. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Quantitative Assessment of Eye Phenotypes for Functional Genetic Studies Using Drosophila melanogaster

PubMed Central

Iyer, Janani; Wang, Qingyu; Le, Thanh; Pizzo, Lucilla; Grönke, Sebastian; Ambegaokar, Surendra S.; Imai, Yuzuru; Srivastava, Ashutosh; Troisí, Beatriz Llamusí; Mardon, Graeme; Artero, Ruben; Jackson, George R.; Isaacs, Adrian M.; Partridge, Linda; Lu, Bingwei; Kumar, Justin P.; Girirajan, Santhosh

2016-01-01

About two-thirds of the vital genes in the Drosophila genome are involved in eye development, making the fly eye an excellent genetic system to study cellular function and development, neurodevelopment/degeneration, and complex diseases such as cancer and diabetes. We developed a novel computational method, implemented as Flynotyper software (http://flynotyper.sourceforge.net), to quantitatively assess the morphological defects in the Drosophila eye resulting from genetic alterations affecting basic cellular and developmental processes. Flynotyper utilizes a series of image processing operations to automatically detect the fly eye and the individual ommatidium, and calculates a phenotypic score as a measure of the disorderliness of ommatidial arrangement in the fly eye. As a proof of principle, we tested our method by analyzing the defects due to eye-specific knockdown of Drosophila orthologs of 12 neurodevelopmental genes to accurately document differential sensitivities of these genes to dosage alteration. We also evaluated eye images from six independent studies assessing the effect of overexpression of repeats, candidates from peptide library screens, and modifiers of neurotoxicity and developmental processes on eye morphology, and show strong concordance with the original assessment. We further demonstrate the utility of this method by analyzing 16 modifiers of sine oculis obtained from two genome-wide deficiency screens of Drosophila and accurately quantifying the effect of its enhancers and suppressors during eye development. Our method will complement existing assays for eye phenotypes, and increase the accuracy of studies that use fly eyes for functional evaluation of genes and genetic interactions. PMID:26994292

Metabolomics, Standards, and Metabolic Modeling for Synthetic Biology in Plants

PubMed Central

Hill, Camilla Beate; Czauderna, Tobias; Klapperstück, Matthias; Roessner, Ute; Schreiber, Falk

2015-01-01

Life on earth depends on dynamic chemical transformations that enable cellular functions, including electron transfer reactions, as well as synthesis and degradation of biomolecules. Biochemical reactions are coordinated in metabolic pathways that interact in a complex way to allow adequate regulation. Biotechnology, food, biofuel, agricultural, and pharmaceutical industries are highly interested in metabolic engineering as an enabling technology of synthetic biology to exploit cells for the controlled production of metabolites of interest. These approaches have only recently been extended to plants due to their greater metabolic complexity (such as primary and secondary metabolism) and highly compartmentalized cellular structures and functions (including plant-specific organelles) compared with bacteria and other microorganisms. Technological advances in analytical instrumentation in combination with advances in data analysis and modeling have opened up new approaches to engineer plant metabolic pathways and allow the impact of modifications to be predicted more accurately. In this article, we review challenges in the integration and analysis of large-scale metabolic data, present an overview of current bioinformatics methods for the modeling and visualization of metabolic networks, and discuss approaches for interfacing bioinformatics approaches with metabolic models of cellular processes and flux distributions in order to predict phenotypes derived from specific genetic modifications or subjected to different environmental conditions. PMID:26557642

Effects of helminths and Mycobacterium tuberculosis infection on HIV-1: a cellular immunological perspective.

PubMed

Mouser, Emily E I M; Pollakis, Georgios; Paxton, William A

2012-05-01

In many regions of the world, a high prevalence of HIV-1, helminthic and Mycobacterium tuberculosis (Mtb) infections can be found. Here, we summarize the types of immune responses induced and/or modulated by these pathogens and the consequences for HIV-1 disease. Helminths predominantly induce strong T helper (Th) 2 cellular responses which are downregulated in chronic disease. The anatomical niche populated by helminths plays a key factor in the effect these parasites have on HIV-1 transmission and subsequent replication. Gut-associated helminths have been found to increase HIV-1 transmission via the lesions they provide. In spite of this, the many immune modulatory molecules secreted by the parasites may inhibit or slow HIV-1 infection. In contrast, Mtb is mainly restricted to the lung and the Mtb-specific Th cells induced are highly susceptible to HIV-1 infection and replication. Antigens from both pathogens have immunomodulatory activity that can skew cellular immune responses in specific directions. The effect of helminths and Mtb on modulating immune responses is varied and complex with both their location and phenotype potentially influencing HIV-1 disease. These pathogens have evolved a complex array of molecules which have the capacity to modulate immunity and preserve pathogen survival.

Discovering novel phenotypes with automatically inferred dynamic models: a partial melanocyte conversion in Xenopus

NASA Astrophysics Data System (ADS)

Lobo, Daniel; Lobikin, Maria; Levin, Michael

2017-01-01

Progress in regenerative medicine requires reverse-engineering cellular control networks to infer perturbations with desired systems-level outcomes. Such dynamic models allow phenotypic predictions for novel perturbations to be rapidly assessed in silico. Here, we analyzed a Xenopus model of conversion of melanocytes to a metastatic-like phenotype only previously observed in an all-or-none manner. Prior in vivo genetic and pharmacological experiments showed that individual animals either fully convert or remain normal, at some characteristic frequency after a given perturbation. We developed a Machine Learning method which inferred a model explaining this complex, stochastic all-or-none dataset. We then used this model to ask how a new phenotype could be generated: animals in which only some of the melanocytes converted. Systematically performing in silico perturbations, the model predicted that a combination of altanserin (5HTR2 inhibitor), reserpine (VMAT inhibitor), and VP16-XlCreb1 (constitutively active CREB) would break the all-or-none concordance. Remarkably, applying the predicted combination of three reagents in vivo revealed precisely the expected novel outcome, resulting in partial conversion of melanocytes within individuals. This work demonstrates the capability of automated analysis of dynamic models of signaling networks to discover novel phenotypes and predictively identify specific manipulations that can reach them.

Adrenal cortex expression quantitative trait loci in a German Holstein × Charolais cross.

PubMed

Brand, Bodo; Scheinhardt, Markus O; Friedrich, Juliane; Zimmer, Daisy; Reinsch, Norbert; Ponsuksili, Siriluck; Schwerin, Manfred; Ziegler, Andreas

2016-10-06

The importance of the adrenal gland in regard to lactation and reproduction in cattle has been recognized early. Caused by interest in animal welfare and the impact of stress on economically important traits in farm animals the adrenal gland and its function within the stress response is of increasing interest. However, the molecular mechanisms and pathways involved in stress-related effects on economically important traits in farm animals are not fully understood. Gene expression is an important mechanism underlying complex traits, and genetic variants affecting the transcript abundance are thought to influence the manifestation of an expressed phenotype. We therefore investigated the genetic background of adrenocortical gene expression by applying an adaptive linear rank test to identify genome-wide expression quantitative trait loci (eQTL) for adrenal cortex transcripts in cattle. A total of 10,986 adrenal cortex transcripts and 37,204 single nucleotide polymorphisms (SNPs) were analysed in 145 F2 cows of a Charolais × German Holstein cross. We identified 505 SNPs that were associated with the abundance of 129 transcripts, comprising 482 cis effects and 17 trans effects. These SNPs were located on all chromosomes but X, 16, 24 and 28. Associated genes are mainly involved in molecular and cellular functions comprising free radical scavenging, cellular compromise, cell morphology and lipid metabolism, including genes such as CYP27A1 and LHCGR that have been shown to affect economically important traits in cattle. In this study we showed that adrenocortical eQTL affect the expression of genes known to contribute to the phenotypic manifestation in cattle. Furthermore, some of the identified genes and related molecular pathways were previously shown to contribute to the phenotypic variation of behaviour, temperament and growth at the onset of puberty in the same population investigated here. We conclude that eQTL analysis appears to be a useful approach providing insight into the molecular and genetic background of complex traits in cattle and will help to understand molecular networks involved.

Chemical Fluxes in Cellular Steady States Measured by Fluorescence Correlation Spectroscopy

NASA Astrophysics Data System (ADS)

Qian, Hong; Elson, Elliot L.

Genetically, identical cells adopt phenotypes that have different structures, functions, and metabolic properties. In multi-cellular organisms, for example, tissue-specific phenotypes distinguish muscle cells, liver cells, fibroblasts, and blood cells that differ in biochemical functions, geometric forms, and interactions with extracellular environments. Tissue-specific cells usually have different metabolic functions such as synthesis of distinct spectra of secreted proteins, e.g., by liver or pancreatic cells, or of structural proteins, e.g., muscle vs. epithelial cells. But more importantly, a phenotype should include a dynamic aspect: different phenotypes can have distinctly different dynamic functions such as contraction of muscle cells and locomotion of leukocytes. The phenotypes of differentiated tissue cells are typically stable, but they can respond to changes in external conditions, e.g., as in the hypertrophy of muscle cells in response to extra load [1] or the phenotypic shift of fibroblasts to myofibroblasts as part of the wound healing response [2]. Cells pass through sequences of phenotypes during development and also undergo malignant phenotypic transformations as occur in cancer and heart disease.

Differential network entropy reveals cancer system hallmarks

PubMed Central

West, James; Bianconi, Ginestra; Severini, Simone; Teschendorff, Andrew E.

2012-01-01

The cellular phenotype is described by a complex network of molecular interactions. Elucidating network properties that distinguish disease from the healthy cellular state is therefore of critical importance for gaining systems-level insights into disease mechanisms and ultimately for developing improved therapies. By integrating gene expression data with a protein interaction network we here demonstrate that cancer cells are characterised by an increase in network entropy. In addition, we formally demonstrate that gene expression differences between normal and cancer tissue are anticorrelated with local network entropy changes, thus providing a systemic link between gene expression changes at the nodes and their local correlation patterns. In particular, we find that genes which drive cell-proliferation in cancer cells and which often encode oncogenes are associated with reductions in network entropy. These findings may have potential implications for identifying novel drug targets. PMID:23150773

WAVE2 Regulates Epithelial Morphology and Cadherin Isoform Switching through Regulation of Twist and Abl

PubMed Central

Bryce, Nicole S.; Reynolds, Albert B.; Koleske, Anthony J.; Weaver, Alissa M.

2013-01-01

Background Epithelial morphogenesis is a dynamic process that involves coordination of signaling and actin cytoskeletal rearrangements. Principal Findings We analyzed the contribution of the branched actin regulator WAVE2 in the development of 3-dimensional (3D) epithelial structures. WAVE2-knockdown (WAVE2-KD) cells formed large multi-lobular acini that continued to proliferate at an abnormally late stage compared to control acini. Immunostaining of the cell-cell junctions of WAVE2-KD acini revealed weak and heterogeneous E-cadherin staining despite little change in actin filament localization to the same junctions. Analysis of cadherin expression demonstrated a decrease in E-cadherin and an increase in N-cadherin protein and mRNA abundance in total cell lysates. In addition, WAVE2-KD cells exhibited an increase in the mRNA levels of the epithelial-mesenchymal transition (EMT)-associated transcription factor Twist1. KD of Twist1 expression in WAVE2-KD cells reversed the cadherin switching and completely rescued the aberrant 3D morphological phenotype. Activity of the WAVE2 complex binding partner Abl kinase was also increased in WAVE2-KD cells, as assessed by tyrosine phosphorylation of the Abl substrate CrkL. Inhibition of Abl with STI571 rescued the multi-lobular WAVE2-KD 3D phenotype whereas overexpression of Abl kinase phenocopied the WAVE2-KD phenotype. Conclusions The WAVE2 complex regulates breast epithelial morphology by a complex mechanism involving repression of Twist1 expression and Abl kinase activity. These data reveal a critical role for WAVE2 complex in regulation of cellular signaling and epithelial morphogenesis. PMID:23691243

Solving Immunology?

PubMed Central

Vodovotz, Yoram; Xia, Ashley; Read, Elizabeth L.; Bassaganya-Riera, Josep; Hafler, David A.; Sontag, Eduardo; Wang, Jin; Tsang, John S.; Day, Judy D.; Kleinstein, Steven; Butte, Atul J.; Altman, Matthew C; Hammond, Ross; Sealfon, Stuart C.

2016-01-01

Emergent responses of the immune system result from integration of molecular and cellular networks over time and across multiple organs. High-content and high-throughput analysis technologies, concomitantly with data-driven and mechanistic modeling, hold promise for systematic interrogation of these complex pathways. However, connecting genetic variation and molecular mechanisms to individual phenotypes and health outcomes has proven elusive. Gaps remain in data, and disagreements persist about the value of mechanistic modeling for immunology. Here, we present the perspectives that emerged from the NIAID workshop “Complex Systems Science, Modeling and Immunity” and subsequent discussions regarding the potential synergy of high-throughput data acquisition, data-driven modeling and mechanistic modeling to define new mechanisms of immunological disease and to accelerate the translation of these insights into therapies. PMID:27986392

Loss of AP-5 results in accumulation of aberrant endolysosomes: defining a new type of lysosomal storage disease

PubMed Central

Hirst, Jennifer; Edgar, James R.; Esteves, Typhaine; Darios, Frédéric; Madeo, Marianna; Chang, Jaerak; Roda, Ricardo H.; Dürr, Alexandra; Anheim, Mathieu; Gellera, Cinzia; Li, Jun; Züchner, Stephan; Mariotti, Caterina; Stevanin, Giovanni; Blackstone, Craig; Kruer, Michael C.; Robinson, Margaret S.

2015-01-01

Adaptor proteins (AP 1–5) are heterotetrameric complexes that facilitate specialized cargo sorting in vesicular-mediated trafficking. Mutations in AP5Z1, encoding a subunit of the AP-5 complex, have been reported to cause hereditary spastic paraplegia (HSP), although their impact at the cellular level has not been assessed. Here we characterize three independent fibroblast lines derived from skin biopsies of patients harbouring nonsense mutations in AP5Z1 and presenting with spastic paraplegia accompanied by neuropathy, parkinsonism and/or cognitive impairment. In all three patient-derived lines, we show that there is complete loss of AP-5 ζ protein and a reduction in the associated AP-5 µ5 protein. Using ultrastructural analysis, we show that these patient-derived lines consistently exhibit abundant multilamellar structures that are positive for markers of endolysosomes and are filled with aberrant storage material organized as exaggerated multilamellar whorls, striated belts and ‘fingerprint bodies’. This phenotype can be replicated in a HeLa cell culture model by siRNA knockdown of AP-5 ζ. The cellular phenotype bears striking resemblance to features described in a number of lysosomal storage diseases (LSDs). Collectively, these findings reveal an emerging picture of the role of AP-5 in endosomal and lysosomal homeostasis, illuminates a potential pathomechanism that is relevant to the role of AP-5 in neurons and expands the understanding of recessive HSPs. Moreover, the resulting accumulation of storage material in endolysosomes leads us to propose that AP-5 deficiency represents a new type of LSDs. PMID:26085577

TWIST1-WDR5-Hottip Regulates Hoxa9 Chromatin to Facilitate Prostate Cancer Metastasis.

PubMed

Malek, Reem; Gajula, Rajendra P; Williams, Russell D; Nghiem, Belinda; Simons, Brian W; Nugent, Katriana; Wang, Hailun; Taparra, Kekoa; Lemtiri-Chlieh, Ghali; Yoon, Arum R; True, Lawrence; An, Steven S; DeWeese, Theodore L; Ross, Ashley E; Schaeffer, Edward M; Pienta, Kenneth J; Hurley, Paula J; Morrissey, Colm; Tran, Phuoc T

2017-06-15

TWIST1 is a transcription factor critical for development that can promote prostate cancer metastasis. During embryonic development, TWIST1 and HOXA9 are coexpressed in mouse prostate and then silenced postnatally. Here we report that TWIST1 and HOXA9 coexpression are reactivated in mouse and human primary prostate tumors and are further enriched in human metastases, correlating with survival. TWIST1 formed a complex with WDR5 and the lncRNA Hottip/HOTTIP, members of the MLL/COMPASS-like H3K4 methylases, which regulate chromatin in the Hox/HOX cluster during development. TWIST1 overexpression led to coenrichment of TWIST1 and WDR5 as well as increased H3K4me3 chromatin at the Hoxa9/HOXA9 promoter, which was dependent on WDR5. Expression of WDR5 and Hottip/HOTTIP was also required for TWIST1-induced upregulation of HOXA9 and aggressive cellular phenotypes such as invasion and migration. Pharmacologic inhibition of HOXA9 prevented TWIST1-induced aggressive prostate cancer cellular phenotypes in vitro and metastasis in vivo This study demonstrates a novel mechanism by which TWIST1 regulates chromatin and gene expression by cooperating with the COMPASS-like complex to increase H3K4 trimethylation at target gene promoters. Our findings highlight a TWIST1-HOXA9 embryonic prostate developmental program that is reactivated during prostate cancer metastasis and is therapeutically targetable. Cancer Res; 77(12); 3181-93. ©2017 AACR . ©2017 American Association for Cancer Research.

Human mesenchymal stem cells cultured on silk hydrogels with variable stiffness and growth factor differentiate into mature smooth muscle cell phenotype.

PubMed

Floren, Michael; Bonani, Walter; Dharmarajan, Anirudh; Motta, Antonella; Migliaresi, Claudio; Tan, Wei

2016-02-01

Cell-matrix and cell-biomolecule interactions play critical roles in a diversity of biological events including cell adhesion, growth, differentiation, and apoptosis. Evidence suggests that a concise crosstalk of these environmental factors may be required to direct stem cell differentiation toward matured cell type and function. However, the culmination of these complex interactions to direct stem cells into highly specific phenotypes in vitro is still widely unknown, particularly in the context of implantable biomaterials. In this study, we utilized tunable hydrogels based on a simple high pressure CO2 method and silk fibroin (SF) the structural protein of Bombyx mori silk fibers. Modification of SF protein starting water solution concentration results in hydrogels of variable stiffness while retaining key structural parameters such as matrix pore size and β-sheet crystallinity. To further resolve the complex crosstalk of chemical signals with matrix properties, we chose to investigate the role of 3D hydrogel stiffness and transforming growth factor (TGF-β1), with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells. Our data revealed the potential to upregulate matured vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72h). Overall, our observations suggest that chemical and physical stimuli within the cellular microenvironment are tightly coupled systems involved in the fate decisions of hMSCs. The production of tunable scaffold materials that are biocompatible and further specialized to mimic tissue-specific niche environments will be of considerable value to future tissue engineering platforms. This article investigates the role of silk fibroin hydrogel stiffness and transforming growth factor (TGF-β1), with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells. Specifically, we demonstrate the upregulation of mature vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72h). Moreover, we demonstrate the potential to direct specialized hMSC differentiation by modulating stiffness and growth factor using silk fibroin, a well-tolerated and -defined biomaterial with an impressive portfolio of tissue engineering applications. Altogether, our study reinforce the fact that complex differentiation protocols may be simplified by engineering the cellular microenvironment on multiple scales, i.e. matrix stiffness with growth factor. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Why do bacteria divide?

PubMed Central

Norris, Vic

2015-01-01

The problem of not only how but also why cells divide can be tackled using recent ideas. One idea from the origins of life – Life as independent of its constituents – is that a living entity like a cell is a particular pattern of connectivity between its constituents. This means that if the growing cell were just to get bigger the average connectivity between its constituents per unit mass – its cellular connectivity – would decrease and the cell would lose its identity. The solution is division which restores connectivity. The corollary is that the cell senses decreasing cellular connectivity and uses this information to trigger division. A second idea from phenotypic diversity – Life on the Scales of Equilibria – is that a bacterium must find strategies that allow it to both survive and grow. This means that it has learnt to reconcile the opposing constraints that these strategies impose. The solution is that the cell cycle generates daughter cells with different phenotypes based on sufficiently complex equilibrium (E) and non-equilibrium (NE) cellular compounds and structures appropriate for survival and growth, respectively, alias ‘hyperstructures.’ The corollary is that the cell senses both the quantity of E material and the intensity of use of NE material and then uses this information to trigger the cell cycle. A third idea from artificial intelligence – Competitive Coherence – is that a cell selects the active subset of elements that actively determine its phenotype from a much larger set of available elements. This means that the selection of an active subset of a specific size and composition must be done so as to generate both a coherent cell state, in which the cell’s contents work together harmoniously, and a coherent sequence of cell states, each coherent with respect to itself and to an unpredictable environment. The solution is the use of a range of mechanisms ranging from hyperstructure dynamics to the cell cycle itself. PMID:25932025

Type IV Collagens and Basement Membrane Diseases: Cell Biology and Pathogenic Mechanisms.

PubMed

Mao, Mao; Alavi, Marcel V; Labelle-Dumais, Cassandre; Gould, Douglas B

2015-01-01

Basement membranes are highly specialized extracellular matrices. Once considered inert scaffolds, basement membranes are now viewed as dynamic and versatile environments that modulate cellular behaviors to regulate tissue development, function, and repair. Increasing evidence suggests that, in addition to providing structural support to neighboring cells, basement membranes serve as reservoirs of growth factors that direct and fine-tune cellular functions. Type IV collagens are a major component of all basement membranes. They evolved along with the earliest multicellular organisms and have been integrated into diverse fundamental biological processes as time and evolution shaped the animal kingdom. The roles of basement membranes in humans are as complex and diverse as their distributions and molecular composition. As a result, basement membrane defects result in multisystem disorders with ambiguous and overlapping boundaries that likely reflect the simultaneous interplay and integration of multiple cellular pathways and processes. Consequently, there will be no single treatment for basement membrane disorders, and therapies are likely to be as varied as the phenotypes. Understanding tissue-specific pathology and the underlying molecular mechanism is the present challenge; personalized medicine will rely upon understanding how a given mutation impacts diverse cellular functions. Copyright © 2015 Elsevier Inc. All rights reserved.

Global Analysis of Yeast Endosomal Transport Identifies the Vps55/68 Sorting Complex

PubMed Central

Schluter, Cayetana; Lam, Karen K.Y.; Brumm, Jochen; Wu, Bella W.; Saunders, Matthew; Stevens, Tom H.

2008-01-01

Endosomal transport is critical for cellular processes ranging from receptor down-regulation and retroviral budding to the immune response. A full understanding of endosome sorting requires a comprehensive picture of the multiprotein complexes that orchestrate vesicle formation and fusion. Here, we use unsupervised, large-scale phenotypic analysis and a novel computational approach for the global identification of endosomal transport factors. This technique effectively identifies components of known and novel protein assemblies. We report the characterization of a previously undescribed endosome sorting complex that contains two well-conserved proteins with four predicted membrane-spanning domains. Vps55p and Vps68p form a complex that acts with or downstream of ESCRT function to regulate endosomal trafficking. Loss of Vps68p disrupts recycling to the TGN as well as onward trafficking to the vacuole without preventing the formation of lumenal vesicles within the MVB. Our results suggest the Vps55/68 complex mediates a novel, conserved step in the endosomal maturation process. PMID:18216282

Platform for combined analysis of functional and biomolecular phenotypes of the same cell.

PubMed

Kelbauskas, L; Ashili, S; Zeng, J; Rezaie, A; Lee, K; Derkach, D; Ueberroth, B; Gao, W; Paulson, T; Wang, H; Tian, Y; Smith, D; Reid, B; Meldrum, Deirdre R

2017-03-16

Functional and molecular cell-to-cell variability is pivotal at the cellular, tissue and whole-organism levels. Yet, the ultimate goal of directly correlating the function of the individual cell with its biomolecular profile remains elusive. We present a platform for integrated analysis of functional and transcriptional phenotypes in the same single cells. We investigated changes in the cellular respiration and gene expression diversity resulting from adaptation to repeated episodes of acute hypoxia in a premalignant progression model. We find differential, progression stage-specific alterations in phenotypic heterogeneity and identify cells with aberrant phenotypes. To our knowledge, this study is the first demonstration of an integrated approach to elucidate how heterogeneity at the transcriptional level manifests in the physiologic profile of individual cells in the context of disease progression.

Poly-Omic Prediction of Complex Traits: OmicKriging

PubMed Central

Wheeler, Heather E.; Aquino-Michaels, Keston; Gamazon, Eric R.; Trubetskoy, Vassily V.; Dolan, M. Eileen; Huang, R. Stephanie; Cox, Nancy J.; Im, Hae Kyung

2014-01-01

High-confidence prediction of complex traits such as disease risk or drug response is an ultimate goal of personalized medicine. Although genome-wide association studies have discovered thousands of well-replicated polymorphisms associated with a broad spectrum of complex traits, the combined predictive power of these associations for any given trait is generally too low to be of clinical relevance. We propose a novel systems approach to complex trait prediction, which leverages and integrates similarity in genetic, transcriptomic, or other omics-level data. We translate the omic similarity into phenotypic similarity using a method called Kriging, commonly used in geostatistics and machine learning. Our method called OmicKriging emphasizes the use of a wide variety of systems-level data, such as those increasingly made available by comprehensive surveys of the genome, transcriptome, and epigenome, for complex trait prediction. Furthermore, our OmicKriging framework allows easy integration of prior information on the function of subsets of omics-level data from heterogeneous sources without the sometimes heavy computational burden of Bayesian approaches. Using seven disease datasets from the Wellcome Trust Case Control Consortium (WTCCC), we show that OmicKriging allows simple integration of sparse and highly polygenic components yielding comparable performance at a fraction of the computing time of a recently published Bayesian sparse linear mixed model method. Using a cellular growth phenotype, we show that integrating mRNA and microRNA expression data substantially increases performance over either dataset alone. Using clinical statin response, we show improved prediction over existing methods. PMID:24799323

Dendritic Cytoskeletal Architecture Is Modulated by Combinatorial Transcriptional Regulation in Drosophila melanogaster.

PubMed

Das, Ravi; Bhattacharjee, Shatabdi; Patel, Atit A; Harris, Jenna M; Bhattacharya, Surajit; Letcher, Jamin M; Clark, Sarah G; Nanda, Sumit; Iyer, Eswar Prasad R; Ascoli, Giorgio A; Cox, Daniel N

2017-12-01

Transcription factors (TFs) have emerged as essential cell autonomous mediators of subtype specific dendritogenesis; however, the downstream effectors of these TFs remain largely unknown, as are the cellular events that TFs control to direct morphological change. As dendritic morphology is largely dictated by the organization of the actin and microtubule (MT) cytoskeletons, elucidating TF-mediated cytoskeletal regulatory programs is key to understanding molecular control of diverse dendritic morphologies. Previous studies in Drosophila melanogaster have demonstrated that the conserved TFs Cut and Knot exert combinatorial control over aspects of dendritic cytoskeleton development, promoting actin and MT-based arbor morphology, respectively. To investigate transcriptional targets of Cut and/or Knot regulation, we conducted systematic neurogenomic studies, coupled with in vivo genetic screens utilizing multi-fluor cytoskeletal and membrane marker reporters. These analyses identified a host of putative Cut and/or Knot effector molecules, and a subset of these putative TF targets converge on modulating dendritic cytoskeletal architecture, which are grouped into three major phenotypic categories, based upon neuromorphometric analyses: complexity enhancer, complexity shifter, and complexity suppressor. Complexity enhancer genes normally function to promote higher order dendritic growth and branching with variable effects on MT stabilization and F-actin organization, whereas complexity shifter and complexity suppressor genes normally function in regulating proximal-distal branching distribution or in restricting higher order branching complexity, respectively, with spatially restricted impacts on the dendritic cytoskeleton. Collectively, we implicate novel genes and cellular programs by which TFs distinctly and combinatorially govern dendritogenesis via cytoskeletal modulation. Copyright © 2017 by the Genetics Society of America.

The puzzle of immune phenotypes of childhood asthma.

PubMed

Landgraf-Rauf, Katja; Anselm, Bettina; Schaub, Bianca

2016-12-01

Asthma represents the most common chronic childhood disease worldwide. Whereas preschool children present with wheezing triggered by different factors (multitrigger and viral wheeze), clinical asthma manifestation in school children has previously been classified as allergic and non-allergic asthma. For both, the underlying immunological mechanisms are not yet understood in depth in children. Treatment is still prescribed regardless of underlying mechanisms, and children are not always treated successfully. This review summarizes recent key findings on the complex mechanisms of the development and manifestation of childhood asthma. Whereas traditional classification of childhood asthma is primarily based on clinical symptoms like wheezing and atopy, novel approaches to specify asthma phenotypes are under way and face challenges such as including the stability of phenotypes over time and transition into adulthood. Epidemiological studies enclose more information on the patient's disease history and environmental influences. Latest studies define endotypes based on molecular and cellular mechanisms, for example defining risk and protective single nucleotide polymorphisms (SNPs) and new immune phenotypes, showing promising results. Also, regulatory T cells and recently discovered T helper cell subtypes such as Th9 and Th17 cells were shown to be important for the development of asthma. Innate lymphoid cells (ILC) could play a critical role in asthma patients as they produce different cytokines associated with asthma. Epigenetic findings showed different acetylation and methylation patterns for children with allergic and non-allergic asthma. On a posttranscriptional level, miRNAs are regulating factors identified to differ between asthma patients and healthy controls and also indicate differences within asthma phenotypes. Metabolomics is another exciting chapter important for endotyping asthmatic children. Despite the development of new biomarkers and the discovery of new immunological molecules, the complex puzzle of childhood asthma is still far from being completed. Addressing the current challenges of distinct clinical asthma and wheeze phenotypes, including their stability and underlying endotypes, involves addressing the interplay of innate and adaptive immune regulatory mechanisms in large, interdisciplinary cohorts.

A Comparative Analysis of the Murine Thymic Microenvironment in Normal, Autoimmune, and Immunodeficiency States

PubMed Central

Takeoka, Yuichi; Chen, Shao-Yuan; Boyd, Richard L.; Tsuneyama, Koichi; Taguchi, Nobuhisa; Morita, Shinji; Yago, Hisashi; Suehiro, Seishi; Ansari, Aftab A.; Shultz, Leonard D.

1997-01-01

It is widely accepted that the thymic microenvironment regulates normal thymopoiesis through a highly coordinated and complex series of cellular and cytokine interactions. A direct corollary of this is that abnormalities within the microenvironment could be of etiologic significance in T-cell-based diseases. Our laboratory has developed a large panel of monoclonal antibodies (mAbs) that react specifically with epithelial or nonepithelial markers in the thymus. We have taken advantage of these reagents to characterize the thymic microenvironment of several genetic strains of mice, including BALB/cJ, C57BL/6J, NZB/BlnJ, SM/J, NOD/Ltz, NOD/Ltz-scid/sz, C57BL/6J-Hcph me/Hcph me, and ALY/NscJcl-aly/aly mice, and littermate control animals. We report herein that control mice, including strains of several backgrounds, have a very consistent phenotypic profile with this panel of monoclonal antibodies, including reactivity with thymic epithelial cells in the cortex, the medulla and the corticomedullary junction, and the extracellular matrix. In contrast, the disease-prone strains studied have unique, abnormal staining of thymic cortex and medulla at both the structural and cellular levels. These phenotypic data suggest that abnormalities in interactions between developing thymocytes and stromal cells characterize disease-prone mice. PMID:9587708

Functional Genomic Analysis of the let-7 Regulatory Network in Caenorhabditis elegans

PubMed Central

Zisoulis, Dimitrios G.; Lovci, Michael T.; Melnik-Martinez, Katya V.; Yeo, Gene W.; Pasquinelli, Amy E.

2013-01-01

The let-7 microRNA (miRNA) regulates cellular differentiation across many animal species. Loss of let-7 activity causes abnormal development in Caenorhabditis elegans and unchecked cellular proliferation in human cells, which contributes to tumorigenesis. These defects are due to improper expression of protein-coding genes normally under let-7 regulation. While some direct targets of let-7 have been identified, the genome-wide effect of let-7 insufficiency in a developing animal has not been fully investigated. Here we report the results of molecular and genetic assays aimed at determining the global network of genes regulated by let-7 in C. elegans. By screening for mis-regulated genes that also contribute to let-7 mutant phenotypes, we derived a list of physiologically relevant potential targets of let-7 regulation. Twenty new suppressors of the rupturing vulva or extra seam cell division phenotypes characteristic of let-7 mutants emerged. Three of these genes, opt-2, prmt-1, and T27D12.1, were found to associate with Argonaute in a let-7–dependent manner and are likely novel direct targets of this miRNA. Overall, a complex network of genes with various activities is subject to let-7 regulation to coordinate developmental timing across tissues during worm development. PMID:23516374

The Arabidopsis COP9 SIGNALOSOME INTERACTING F-BOX KELCH 1 protein forms an SCF ubiquitin ligase and regulates hypocotyl elongation.

PubMed

Franciosini, Anna; Lombardi, Benedetta; Iafrate, Silvia; Pecce, Valeria; Mele, Giovanni; Lupacchini, Leonardo; Rinaldi, Gianmarco; Kondou, Youichi; Gusmaroli, Giuliana; Aki, Shiori; Tsuge, Tomohiko; Deng, Xing-Wang; Matsui, Minami; Vittorioso, Paola; Costantino, Paolo; Serino, Giovanna

2013-09-01

The regulation of protein turnover by the ubiquitin proteasome system (UPS) is a major posttranslational mechanism in eukaryotes. One of the key components of the UPS, the COP9 signalosome (CSN), regulates 'cullin-ring' E3 ubiquitin ligases. In plants, CSN participates in diverse cellular and developmental processes, ranging from light signaling to cell cycle control. In this work, we isolated a new plant-specific CSN-interacting F-box protein, which we denominated CFK1 (COP9 INTERACTING F-BOX KELCH 1). We show that, in Arabidopsis thaliana, CFK1 is a component of a functional ubiquitin ligase complex. We also show that CFK1 stability is regulated by CSN and by proteasome-dependent proteolysis, and that light induces accumulation of the CFK1 transcript in the hypocotyl. Analysis of CFK1 knockdown, mutant, and overexpressing seedlings indicates that CFK1 promotes hypocotyl elongation by increasing cell size. Reduction of CSN levels enhances the short hypocotyl phenotype of CFK1-depleted seedlings, while complete loss of CSN activity suppresses the long-hypocotyl phenotype of CFK1-overexpressing seedlings. We propose that CFK1 (and its regulation by CSN) is a novel component of the cellular mechanisms controlling hypocotyl elongation.

Dynamic self-guiding analysis of Alzheimer's disease

PubMed Central

Kurakin, Alexei; Bredesen, Dale E.

2015-01-01

We applied a self-guiding evolutionary algorithm to initiate the synthesis of the Alzheimer's disease-related data and literature. A protein interaction network associated with amyloid-beta precursor protein (APP) and a seed model that treats Alzheimer's disease as progressive dysregulation of APP-associated signaling were used as dynamic “guides” and structural “filters” in the recursive search, analysis, and assimilation of data to drive the evolution of the seed model in size, detail, and complexity. Analysis of data and literature across sub-disciplines and system-scale discovery platforms suggests a key role of dynamic cytoskeletal connectivity in the stability, plasticity, and performance of multicellular networks and architectures. Chronic impairment and/or dysregulation of cell adhesions/synapses, cytoskeletal networks, and/or reversible epithelial-to-mesenchymal-like transitions, which enable and mediate the stable and coherent yet dynamic and reconfigurable multicellular architectures, may lead to the emergence and persistence of the disordered, wound-like pockets/microenvironments of chronically disconnected cells. Such wound-like microenvironments support and are supported by pro-inflammatory, pro-secretion, de-differentiated cellular phenotypes with altered metabolism and signaling. The co-evolution of wound-like microenvironments and their inhabitants may lead to the selection and stabilization of degenerated cellular phenotypes, via acquisition of epigenetic modifications and mutations, which eventually result in degenerative disorders such as cancer and Alzheimer's disease. PMID:26041885

A Stochastic Model of the Yeast Cell Cycle Reveals Roles for Feedback Regulation in Limiting Cellular Variability.

PubMed

Barik, Debashis; Ball, David A; Peccoud, Jean; Tyson, John J

2016-12-01

The cell division cycle of eukaryotes is governed by a complex network of cyclin-dependent protein kinases (CDKs) and auxiliary proteins that govern CDK activities. The control system must function reliably in the context of molecular noise that is inevitable in tiny yeast cells, because mistakes in sequencing cell cycle events are detrimental or fatal to the cell or its progeny. To assess the effects of noise on cell cycle progression requires not only extensive, quantitative, experimental measurements of cellular heterogeneity but also comprehensive, accurate, mathematical models of stochastic fluctuations in the CDK control system. In this paper we provide a stochastic model of the budding yeast cell cycle that accurately accounts for the variable phenotypes of wild-type cells and more than 20 mutant yeast strains simulated in different growth conditions. We specifically tested the role of feedback regulations mediated by G1- and SG2M-phase cyclins to minimize the noise in cell cycle progression. Details of the model are informed and tested by quantitative measurements (by fluorescence in situ hybridization) of the joint distributions of mRNA populations in yeast cells. We use the model to predict the phenotypes of ~30 mutant yeast strains that have not yet been characterized experimentally.

Exercises in Anatomy, Connectivity, and Morphology using Neuromorpho.org and the Allen Brain Atlas.

PubMed

Chu, Philip; Peck, Joshua; Brumberg, Joshua C

2015-01-01

Laboratory instruction of neuroscience is often limited by the lack of physical resources and supplies (e.g., brains specimens, dissection kits, physiological equipment). Online databases can serve as supplements to material labs by providing professionally collected images of brain specimens and their underlying cellular populations with resolution and quality that is extremely difficult to access for strictly pedagogical purposes. We describe a method using two online databases, the Neuromorpho.org and the Allen Brain Atlas (ABA), that freely provide access to data from working brain scientists that can be modified for laboratory instruction/exercises. Neuromorpho.org is the first neuronal morphology database that provides qualitative and quantitative data from reconstructed cells analyzed in published scientific reports. The Neuromorpho.org database contains cross species and multiple neuronal phenotype datasets which allows for comparative examinations. The ABA provides modules that allow students to study the anatomy of the rodent brain, as well as observe the different cellular phenotypes that exist using histochemical labeling. Using these tools in conjunction, advanced students can ask questions about qualitative and quantitative neuronal morphology, then examine the distribution of the same cell types across the entire brain to gain a full appreciation of the magnitude of the brain's complexity.

A Stochastic Model of the Yeast Cell Cycle Reveals Roles for Feedback Regulation in Limiting Cellular Variability

PubMed Central

Ball, David A.

2016-01-01

The cell division cycle of eukaryotes is governed by a complex network of cyclin-dependent protein kinases (CDKs) and auxiliary proteins that govern CDK activities. The control system must function reliably in the context of molecular noise that is inevitable in tiny yeast cells, because mistakes in sequencing cell cycle events are detrimental or fatal to the cell or its progeny. To assess the effects of noise on cell cycle progression requires not only extensive, quantitative, experimental measurements of cellular heterogeneity but also comprehensive, accurate, mathematical models of stochastic fluctuations in the CDK control system. In this paper we provide a stochastic model of the budding yeast cell cycle that accurately accounts for the variable phenotypes of wild-type cells and more than 20 mutant yeast strains simulated in different growth conditions. We specifically tested the role of feedback regulations mediated by G1- and SG2M-phase cyclins to minimize the noise in cell cycle progression. Details of the model are informed and tested by quantitative measurements (by fluorescence in situ hybridization) of the joint distributions of mRNA populations in yeast cells. We use the model to predict the phenotypes of ~30 mutant yeast strains that have not yet been characterized experimentally. PMID:27935947

NADPH oxidase promotes Parkinsonian phenotypes by impairing autophagic flux in an mTORC1-independent fashion in a cellular model of Parkinson’s disease

PubMed Central

Pal, Rituraj; Bajaj, Lakshya; Sharma, Jaiprakash; Palmieri, Michela; Di Ronza, Alberto; Lotfi, Parisa; Chaudhury, Arindam; Neilson, Joel; Sardiello, Marco; Rodney, George G.

2016-01-01

Oxidative stress and aberrant accumulation of misfolded proteins in the cytosol are key pathological features associated with Parkinson’s disease (PD). NADPH oxidase (Nox2) is upregulated in the pathogenesis of PD; however, the underlying mechanism(s) of Nox2-mediated oxidative stress in PD pathogenesis are still unknown. Using a rotenone-inducible cellular model of PD, we observed that a short exposure to rotenone (0.5 μM) resulted in impaired autophagic flux through activation of a Nox2 dependent Src/PI3K/Akt axis, with a consequent disruption of a Beclin1-VPS34 interaction that was independent of mTORC1 activity. Sustained exposure to rotenone at a higher dose (10 μM) decreased mTORC1 activity; however, autophagic flux was still impaired due to dysregulation of lysosomal activity with subsequent induction of the apoptotic machinery. Cumulatively, our results highlight a complex pathogenic mechanism for PD where short- and long-term oxidative stress alters different signaling pathways, ultimately resulting in anomalous autophagic activity and disease phenotype. Inhibition of Nox2-dependent oxidative stress attenuated the impaired autophagy and cell death, highlighting the importance and therapeutic potential of these pathways for treating patients with PD. PMID:26960433

HSP90 Inhibition and Cellular Stress Elicits Phenotypic Plasticity in Hematopoietic Differentiation

PubMed Central

Lawag, Abdalla A.; Napper, Jennifer M.; Hunter, Caroline A.; Bacon, Nickolas A.; Deskins, Seth; El-hamdani, Manaf; Govender, Sarah-Leigh; Koc, Emine C.

2017-01-01

Abstract Cancer cells exist in a state of Darwinian selection using mechanisms that produce changes in gene expression through genetic and epigenetic alteration to facilitate their survival. Cellular plasticity, or the ability to alter cellular phenotype, can assist in survival of premalignant cells as they progress to full malignancy by providing another mechanism of adaptation. The connection between cellular stress and the progression of cancer has been established, although the details of the mechanisms have yet to be fully elucidated. The molecular chaperone HSP90 is often upregulated in cancers as they progress, presumably to allow cancer cells to deal with misfolded proteins and cellular stress associated with transformation. The objective of this work is to test the hypothesis that inhibition of HSP90 results in increased cell plasticity in mammalian systems that can confer a greater adaptability to selective pressures. The approach used is a murine in vitro model system of hematopoietic differentiation that utilizes a murine hematopoietic stem cell line, erythroid myeloid lymphoid (EML) clone 1, during their maturation from stem cells to granulocytic progenitors. During the differentiation protocol, 80%–90% of the cells die when placed in medium where the major growth factor is granulocyte–macrophage-colony stimulating factor. Using this selection point model, EML cells exhibit increases in cellular plasticity when they are better able to adapt to this medium and survive. Increases in cellular plasticity were found to occur upon exposure to geldanamycin to inhibit HSP90, when subjected to various forms of cellular stress, or inhibition of histone acetylation. Furthermore, we provide evidence that the cellular plasticity associated with inhibition of HSP90 in this model involves epigenetic mechanisms and is dependent upon high levels of stem cell factor signaling. This work provides evidence for a role of HSP90 and cellular stress in inducing phenotypic plasticity in mammalian systems that has new implications for cellular stress in progression and evolution of cancer. PMID:28910138

HSP90 Inhibition and Cellular Stress Elicits Phenotypic Plasticity in Hematopoietic Differentiation.

PubMed

Lawag, Abdalla A; Napper, Jennifer M; Hunter, Caroline A; Bacon, Nickolas A; Deskins, Seth; El-Hamdani, Manaf; Govender, Sarah-Leigh; Koc, Emine C; Sollars, Vincent E

2017-10-01

Cancer cells exist in a state of Darwinian selection using mechanisms that produce changes in gene expression through genetic and epigenetic alteration to facilitate their survival. Cellular plasticity, or the ability to alter cellular phenotype, can assist in survival of premalignant cells as they progress to full malignancy by providing another mechanism of adaptation. The connection between cellular stress and the progression of cancer has been established, although the details of the mechanisms have yet to be fully elucidated. The molecular chaperone HSP90 is often upregulated in cancers as they progress, presumably to allow cancer cells to deal with misfolded proteins and cellular stress associated with transformation. The objective of this work is to test the hypothesis that inhibition of HSP90 results in increased cell plasticity in mammalian systems that can confer a greater adaptability to selective pressures. The approach used is a murine in vitro model system of hematopoietic differentiation that utilizes a murine hematopoietic stem cell line, erythroid myeloid lymphoid (EML) clone 1, during their maturation from stem cells to granulocytic progenitors. During the differentiation protocol, 80%-90% of the cells die when placed in medium where the major growth factor is granulocyte-macrophage-colony stimulating factor. Using this selection point model, EML cells exhibit increases in cellular plasticity when they are better able to adapt to this medium and survive. Increases in cellular plasticity were found to occur upon exposure to geldanamycin to inhibit HSP90, when subjected to various forms of cellular stress, or inhibition of histone acetylation. Furthermore, we provide evidence that the cellular plasticity associated with inhibition of HSP90 in this model involves epigenetic mechanisms and is dependent upon high levels of stem cell factor signaling. This work provides evidence for a role of HSP90 and cellular stress in inducing phenotypic plasticity in mammalian systems that has new implications for cellular stress in progression and evolution of cancer.

Cellular FLIP can substitute for the herpes simplex virus type 1 latency-associated transcript gene to support a wild-type virus reactivation phenotype in mice

PubMed Central

Jin, Ling; Carpenter, Dale; Moerdyk-Schauwecker, Megan; Vanarsdall, Adam L; Osorio, Nelson; Hsiang, Chinhui; Jones, Clinton; Wechsler, Steven L

2010-01-01

Latency-associated transcript (LAT) deletion mutants of herpes simplex virus type 1 (HSV-1) have reduced reactivation phenotypes. Thus, LAT plays an essential role in the latency-reactivation cycle of HSV-1. We have shown that LAT has antiapoptosis activity and demonstrated that the chimeric virus, dLAT-cpIAP, resulting from replacing LAT with the baculovirus antiapoptosis gene cpIAP, has a wild-type HSV-1 reactivation phenotype in mice and rabbits. Thus, LAT can be replaced by an alternative antiapoptosis gene, confirming that LAT’s antiapoptosis activity plays an important role in the mechanism by which LAT enhances the virus’ reactivation phenotype. However, because cpIAP interferes with both of the major apoptosis pathways, these studies did not address whether LAT’s proreactivation phenotype function was due to blocking the extrinsic (Fas-ligand–, caspase-8–, or caspase-10–dependent pathway) or the intrinsic (mitochondria-, caspase-9–dependent pathway) pathway, or whether both pathways must be blocked. Here we constructed an HSV-1 LAT(−) mutant that expresses cellular FLIP (cellular FLICE-like inhibitory protein) under control of the LAT promoter and in place of LAT nucleotides 76 to 1667. Mice were ocularly infected with this mutant, designated dLAT-FLIP, and the reactivation phenotype was determined using the trigeminal ganglia explant model. dLAT-FLIP had a reactivation phenotype similar to wild-type virus and significantly higher than the LAT(−) mutant dLAT2903. Thus, the LAT function responsible for enhancing the reactivation phenotype could be replaced with an antiapoptosis gene that primarily blocks the extrinsic signaling apoptosis pathway. PMID:18989818

Extracting microtubule networks from superresolution single-molecule localization microscopy data

PubMed Central

Zhang, Zhen; Nishimura, Yukako; Kanchanawong, Pakorn

2017-01-01

Microtubule filaments form ubiquitous networks that specify spatial organization in cells. However, quantitative analysis of microtubule networks is hampered by their complex architecture, limiting insights into the interplay between their organization and cellular functions. Although superresolution microscopy has greatly facilitated high-resolution imaging of microtubule filaments, extraction of complete filament networks from such data sets is challenging. Here we describe a computational tool for automated retrieval of microtubule filaments from single-molecule-localization–based superresolution microscopy images. We present a user-friendly, graphically interfaced implementation and a quantitative analysis of microtubule network architecture phenotypes in fibroblasts. PMID:27852898

[Epigenetics, interface between environment and genes: role in complex diseases].

PubMed

Scheen, A J; Junien, C

2012-01-01

Epigenetics is the study of heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence. Epigenetics is one of the major mechanisms explaining the "Developmental Origin of Health and Diseases" (DOHaD). Besides genetic background inherited from parents, which confers susceptibility to certain pathologies, epigenetic changes constitute the memory of previous events, either positive or negative, along the life cycle, including at the in utero stage. The later exposition to hostile environment may reveal such susceptibility, with the development of various pathologies, among them numerous chronic complex diseases. The demonstration of such a sequence of events has been shown for metabolic diseases as obesity, metabolic syndrome and type 2 diabetes, cardiovascular disease and cancer. In contrast to genetic predisposition, which is irreversible, epigenetic changes are potentially reversible, thus giving targets not only for prevention, but possibly also for the treatment of certain complex diseases.

Determining Regulatory Networks Governing the Differentiation of Embryonic Stem Cells to Pancreatic Lineage

NASA Astrophysics Data System (ADS)

Banerjee, Ipsita

2009-03-01

Knowledge of pathways governing cellular differentiation to specific phenotype will enable generation of desired cell fates by careful alteration of the governing network by adequate manipulation of the cellular environment. With this aim, we have developed a novel method to reconstruct the underlying regulatory architecture of a differentiating cell population from discrete temporal gene expression data. We utilize an inherent feature of biological networks, that of sparsity, in formulating the network reconstruction problem as a bi-level mixed-integer programming problem. The formulation optimizes the network topology at the upper level and the network connectivity strength at the lower level. The method is first validated by in-silico data, before applying it to the complex system of embryonic stem (ES) cell differentiation. This formulation enables efficient identification of the underlying network topology which could accurately predict steps necessary for directing differentiation to subsequent stages. Concurrent experimental verification demonstrated excellent agreement with model prediction.

Variations in brain defects result from cellular mosaicism in the activation of heat shock signalling.

PubMed

Ishii, Seiji; Torii, Masaaki; Son, Alexander I; Rajendraprasad, Meenu; Morozov, Yury M; Kawasawa, Yuka Imamura; Salzberg, Anna C; Fujimoto, Mitsuaki; Brennand, Kristen; Nakai, Akira; Mezger, Valerie; Gage, Fred H; Rakic, Pasko; Hashimoto-Torii, Kazue

2017-05-02

Repetitive prenatal exposure to identical or similar doses of harmful agents results in highly variable and unpredictable negative effects on fetal brain development ranging in severity from high to little or none. However, the molecular and cellular basis of this variability is not well understood. This study reports that exposure of mouse and human embryonic brain tissues to equal doses of harmful chemicals, such as ethanol, activates the primary stress response transcription factor heat shock factor 1 (Hsf1) in a highly variable and stochastic manner. While Hsf1 is essential for protecting the embryonic brain from environmental stress, excessive activation impairs critical developmental events such as neuronal migration. Our results suggest that mosaic activation of Hsf1 within the embryonic brain in response to prenatal environmental stress exposure may contribute to the resulting generation of phenotypic variations observed in complex congenital brain disorders.

Multiscale Systems Analysis of Root Growth and Development: Modeling Beyond the Network and Cellular Scales

PubMed Central

Band, Leah R.; Fozard, John A.; Godin, Christophe; Jensen, Oliver E.; Pridmore, Tony; Bennett, Malcolm J.; King, John R.

2012-01-01

Over recent decades, we have gained detailed knowledge of many processes involved in root growth and development. However, with this knowledge come increasing complexity and an increasing need for mechanistic modeling to understand how those individual processes interact. One major challenge is in relating genotypes to phenotypes, requiring us to move beyond the network and cellular scales, to use multiscale modeling to predict emergent dynamics at the tissue and organ levels. In this review, we highlight recent developments in multiscale modeling, illustrating how these are generating new mechanistic insights into the regulation of root growth and development. We consider how these models are motivating new biological data analysis and explore directions for future research. This modeling progress will be crucial as we move from a qualitative to an increasingly quantitative understanding of root biology, generating predictive tools that accelerate the development of improved crop varieties. PMID:23110897

A comparison of oncogene-induced senescence and replicative senescence: implications for tumor suppression and aging.

PubMed

Nelson, David M; McBryan, Tony; Jeyapalan, Jessie C; Sedivy, John M; Adams, Peter D

2014-06-01

Cellular senescence is a stable proliferation arrest associated with an altered secretory pathway, the senescence-associated secretory phenotype. However, cellular senescence is initiated by diverse molecular triggers, such as activated oncogenes and shortened telomeres, and is associated with varied and complex physiological endpoints, such as tumor suppression and tissue aging. The extent to which distinct triggers activate divergent modes of senescence that might be associated with different physiological endpoints is largely unknown. To begin to address this, we performed gene expression profiling to compare the senescence programs associated with two different modes of senescence, oncogene-induced senescence (OIS) and replicative senescence (RS [in part caused by shortened telomeres]). While both OIS and RS are associated with many common changes in gene expression compared to control proliferating cells, they also exhibit substantial differences. These results are discussed in light of potential physiological consequences, tumor suppression and aging.

Compensatory functions and interdependency of the DNA-binding domain of BRCA2 with the BRCA1-PALB2-BRCA2 complex.

PubMed

Al Abo, Muthana; Dejsuphong, Donniphat; Hirota, Kouji; Yonetani, Yasukazu; Yamazoe, Mitsuyoshi; Kurumizaka, Hitoshi; Takeda, Shunichi

2014-02-01

BRCA1, BRCA2, and PALB2 are key players in cellular tolerance to chemotherapeutic agents, including camptothecin, cisplatin, and PARP inhibitor. The N-terminal segment of BRCA2 interacts with PALB2, thus contributing to the formation of the BRCA1-PALB2-BRCA2 complex. To understand the role played by BRCA2 in this complex, we deleted its N-terminal segment and generated BRCA2(Δ)(N) mutant cells. Although previous studies have suggested that BRCA1-PALB2 plays a role in the recruitment of BRCA2 to DNA-damage sites, BRCA2(Δ)(N) mutant cells displayed a considerably milder phenotype than did BRCA2(-/-) null-deficient cells. We hypothesized that the DNA-binding domain (DBD) of BRCA2 might compensate for a defect in BRCA2(ΔN) that prevented stable interaction with PALB2. To test this hypothesis, we disrupted the DBD of BRCA2 in wild-type and BRCA2(Δ)(N) cells. Remarkably, although the resulting BRCA2(Δ)(DBD) cells displayed a moderate phenotype, the BRCA2(Δ)(N+ΔDBD) cells displayed a very severe phenotype, as did the BRCA2(-/-) cells, suggesting that the N-terminal segment and the DBD play a substantially overlapping role in the functionality of BRCA2. We also showed that the formation of both the BRCA1-PALB2-BRCA2 complex and the DBD is required for efficient recruitment of BRCA2 to DNA-damage sites. Our study revealed the essential role played by both the BRCA1-PALB2-BRCA2 complex and the DBD in the functionality of BRCA2, as each can compensate for the other in the recruitment of BRCA2 to DNA-damage sites. This knowledge adds to our ability to accurately predict the efficacy of antimalignant therapies for patients carrying mutations in the BRCA2 gene.

Platform for combined analysis of functional and biomolecular phenotypes of the same cell

PubMed Central

Kelbauskas, L.; Ashili, S.; Zeng, J.; Rezaie, A.; Lee, K.; Derkach, D.; Ueberroth, B.; Gao, W.; Paulson, T.; Wang, H.; Tian, Y.; Smith, D.; Reid, B.; Meldrum, Deirdre R.

2017-01-01

Functional and molecular cell-to-cell variability is pivotal at the cellular, tissue and whole-organism levels. Yet, the ultimate goal of directly correlating the function of the individual cell with its biomolecular profile remains elusive. We present a platform for integrated analysis of functional and transcriptional phenotypes in the same single cells. We investigated changes in the cellular respiration and gene expression diversity resulting from adaptation to repeated episodes of acute hypoxia in a premalignant progression model. We find differential, progression stage-specific alterations in phenotypic heterogeneity and identify cells with aberrant phenotypes. To our knowledge, this study is the first demonstration of an integrated approach to elucidate how heterogeneity at the transcriptional level manifests in the physiologic profile of individual cells in the context of disease progression. PMID:28300162

Melanoma cells revive an embryonic transcriptional network to dictate phenotypic heterogeneity.

PubMed

Vandamme, Niels; Berx, Geert

2014-01-01

Compared to the overwhelming amount of literature describing how epithelial-to-mesenchymal transition (EMT)-inducing transcription factors orchestrate cellular plasticity in embryogenesis and epithelial cells, the functions of these factors in non-epithelial contexts, such as melanoma, are less clear. Melanoma is an aggressive tumor arising from melanocytes, endowed with unique features of cellular plasticity. The reversible phenotype-switching between differentiated and invasive phenotypes is increasingly appreciated as a mechanism accounting for heterogeneity in melanoma and is driven by oncogenic signaling and environmental cues. This phenotypic switch is coupled with an intriguing and somewhat counterintuitive signaling switch of EMT-inducing transcription factors. In contrast to carcinomas, different EMT-inducing transcription factors have antagonizing effects in melanoma. Balancing between these different EMT transcription factors is likely the key to successful metastatic spread of melanoma.

A Hybrid Cellular Automaton Model of Clonal Evolution in Cancer: The Emergence of the Glycolytic Phenotype

PubMed Central

Gerlee, P.; Anderson, A.R.A.

2009-01-01

We present a cellular automaton model of clonal evolution in cancer aimed at investigating the emergence of the glycolytic phenotype. In the model each cell is equipped with a micro-environment response network that determines the behaviour or phenotype of the cell based on the local environment. The response network is modelled using a feed-forward neural network, which is subject to mutations when the cells divide. This implies that cells might react differently to the environment and when space and nutrients are limited only the fittest cells will survive. With this model we have investigated the impact of the environment on the growth dynamics of the tumour. In particular we have analysed the influence of the tissue oxygen concentration and extra-cellular matrix density on the dynamics of the model. We found that the environment influences both the growth and evolutionary dynamics of the tumour. For low oxygen concentration we observe tumours with a fingered morphology, while increasing the matrix density gives rise to more compact tumours with wider fingers. The distribution of phenotypes in the tumour is also affected, and we observe that the glycolytic phenotype is most likely to emerge in a poorly oxygenated tissue with a high matrix density. Our results suggest that it is the combined effect of the oxygen concentration and matrix density that creates an environment where the glycolytic phenotype has a growth advantage and consequently is most likely to appear. PMID:18068192

P-TEFb, the Super Elongation Complex and Mediator Regulate a Subset of Non-paused Genes during Early Drosophila Embryo Development

PubMed Central

Dahlberg, Olle; Shilkova, Olga; Tang, Min; Holmqvist, Per-Henrik; Mannervik, Mattias

2015-01-01

Positive Transcription Elongation Factor b (P-TEFb) is a kinase consisting of Cdk9 and Cyclin T that releases RNA Polymerase II (Pol II) into active elongation. It can assemble into a larger Super Elongation Complex (SEC) consisting of additional elongation factors. Here, we use a miRNA-based approach to knock down the maternal contribution of P-TEFb and SEC components in early Drosophila embryos. P-TEFb or SEC depletion results in loss of cells from the embryo posterior and in cellularization defects. Interestingly, the expression of many patterning genes containing promoter-proximal paused Pol II is relatively normal in P-TEFb embryos. Instead, P-TEFb and SEC are required for expression of some non-paused, rapidly transcribed genes in pre-cellular embryos, including the cellularization gene Serendipity-α. We also demonstrate that another P-TEFb regulated gene, terminus, has an essential function in embryo development. Similar morphological and gene expression phenotypes were observed upon knock down of Mediator subunits, providing in vivo evidence that P-TEFb, the SEC and Mediator collaborate in transcription control. Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3’ end, despite affecting global Pol II Ser2 phosphorylation levels. Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes. We conclude that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in Drosophila embryos. PMID:25679530

HIV Nef-mediated cellular phenotypes are differentially expressed as a function of intracellular Nef concentrations.

PubMed

Liu, X; Schrager, J A; Lange, G D; Marsh, J W

2001-08-31

Nef is a regulatory protein encoded by the genome of both human and simian immunodeficiency virus. Its expression in T cells leads to CD4 and major histocompatibility complex class I modulation and either enhancement or suppression of T cell activation. How this viral protein achieves multiple and at times opposing activities has been unclear. Through direct measurements of Nef and the Nef-GFP fusion protein, we find that these events are mediated by different Nef concentrations. Relative to the intracellular concentration that down-modulates surface CD4, an order of magnitude increase in Nef-GFP expression is required for a comparable modulation of major histocompatibility complex class I, and a further 3-fold increase is necessary to suppress T cell activation.

Microparticles in the blood of patients with systemic lupus erythematosus (SLE): phenotypic characterization and clinical associations

PubMed Central

Mobarrez, Fariborz; Vikerfors, Anna; Gustafsson, Johanna T.; Gunnarsson, Iva; Zickert, Agneta; Larsson, Anders; Pisetsky, David S.; Wallén, Håkan; Svenungsson, Elisabet

2016-01-01

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by circulating autoantibodies and the formation of immune complexes. In these responses, the selecting self-antigens likely derive from the remains of dead and dying cells, as well as from disturbances in clearance. During cell death/activation, microparticles (MPs) can be released to the circulation. Previous MP studies in SLE have been limited in size and differ regarding numbers and phenotypes. Therefore, to characterize MPs more completely, we investigated 280 SLE patients and 280 individually matched controls. MPs were measured with flow cytometry and phenotyped according to phosphatidylserine expression (PS+/PS−), cellular origin and inflammatory markers. MPs, regardless of phenotype, are 2–10 times more abundant in SLE blood compared to controls. PS− MPs predominated in SLE, but not in controls (66% vs. 42%). Selectively in SLE, PS− MPs were more numerous in females and smokers. MP numbers decreased with declining renal function, but no clear association with disease activity was observed. The striking abundance of MPs, especially PS− MPs, suggests a generalized disturbance in SLE. MPs may be regarded as “liquid biopsies” to assess the production and clearance of dead, dying and activated cells, i.e. pivotal events for SLE pathogenesis. PMID:27777414

Diet and Energy-Sensing Inputs Affect TorC1-Mediated Axon Misrouting but Not TorC2-Directed Synapse Growth in a Drosophila Model of Tuberous Sclerosis

PubMed Central

Dimitroff, Brian; Lee, Hyun-Gwan; Zhao, Na; O'Connor, Michael B.; Neufeld, Thomas P.; Selleck, Scott B.

2012-01-01

The Target of Rapamycin (TOR) growth regulatory system is influenced by a number of different inputs, including growth factor signaling, nutrient availability, and cellular energy levels. While the effects of TOR on cell and organismal growth have been well characterized, this pathway also has profound effects on neural development and behavior. Hyperactivation of the TOR pathway by mutations in the upstream TOR inhibitors TSC1 (tuberous sclerosis complex 1) or TSC2 promotes benign tumors and neurological and behavioral deficits, a syndrome known as tuberous sclerosis (TS). In Drosophila, neuron-specific overexpression of Rheb, the direct downstream target inhibited by Tsc1/Tsc2, produced significant synapse overgrowth, axon misrouting, and phototaxis deficits. To understand how misregulation of Tor signaling affects neural and behavioral development, we examined the influence of growth factor, nutrient, and energy sensing inputs on these neurodevelopmental phenotypes. Neural expression of Pi3K, a principal mediator of growth factor inputs to Tor, caused synapse overgrowth similar to Rheb, but did not disrupt axon guidance or phototaxis. Dietary restriction rescued Rheb-mediated behavioral and axon guidance deficits, as did overexpression of AMPK, a component of the cellular energy sensing pathway, but neither was able to rescue synapse overgrowth. While axon guidance and behavioral phenotypes were affected by altering the function of a Tor complex 1 (TorC1) component, Raptor, or a TORC1 downstream element (S6k), synapse overgrowth was only suppressed by reducing the function of Tor complex 2 (TorC2) components (Rictor, Sin1). These findings demonstrate that different inputs to Tor signaling have distinct activities in nervous system development, and that Tor provides an important connection between nutrient-energy sensing systems and patterning of the nervous system. PMID:22319582

Measurement of Oxidative Stress: Mitochondrial Function Using the Seahorse System.

PubMed

Leung, Dilys T H; Chu, Simon

2018-01-01

The Seahorse XFp Analyzer is a powerful tool for the assessment of various parameters of cellular respiration. Here we describe the process of the Seahorse Cell Phenotype Test using the Seahorse XFp Analyzer to characterize the metabolic phenotype of live cells. The Seahorse XFp Analyzer can also be coupled with other assays to measure cellular energetics. Given that mitochondrial dysfunction is implicated in preeclampsia, the Seahorse XFp Analyzer will serve as a useful tool for the understanding of pathological metabolism in this disorder.

Organelles – understanding noise and heterogeneity in cell biology at an intermediate scale

PubMed Central

Chang, Amy Y.

2017-01-01

ABSTRACT Many studies over the years have shown that non-genetic mechanisms for producing cell-to-cell variation can lead to highly variable behaviors across genetically identical populations of cells. Most work to date has focused on gene expression noise as the primary source of phenotypic heterogeneity, yet other sources may also contribute. In this Commentary, we explore organelle-level heterogeneity as a potential secondary source of cellular ‘noise’ that contributes to phenotypic heterogeneity. We explore mechanisms for generating organelle heterogeneity and present evidence of functional links between organelle morphology and cellular behavior. Given the many instances in which molecular-level heterogeneity has been linked to phenotypic heterogeneity, we posit that organelle heterogeneity may similarly contribute to overall phenotypic heterogeneity and underline the importance of studying organelle heterogeneity to develop a more comprehensive understanding of phenotypic heterogeneity. Finally, we conclude with a discussion of the medical challenges associated with phenotypic heterogeneity and outline how improved methods for characterizing and controlling this heterogeneity may lead to improved therapeutic strategies and outcomes for patients. PMID:28183729

Inference of tumor evolution during chemotherapy by computational modeling and in situ analysis of genetic and phenotypic cellular diversity.

PubMed

Almendro, Vanessa; Cheng, Yu-Kang; Randles, Amanda; Itzkovitz, Shalev; Marusyk, Andriy; Ametller, Elisabet; Gonzalez-Farre, Xavier; Muñoz, Montse; Russnes, Hege G; Helland, Aslaug; Rye, Inga H; Borresen-Dale, Anne-Lise; Maruyama, Reo; van Oudenaarden, Alexander; Dowsett, Mitchell; Jones, Robin L; Reis-Filho, Jorge; Gascon, Pere; Gönen, Mithat; Michor, Franziska; Polyak, Kornelia

2014-02-13

Cancer therapy exerts a strong selection pressure that shapes tumor evolution, yet our knowledge of how tumors change during treatment is limited. Here, we report the analysis of cellular heterogeneity for genetic and phenotypic features and their spatial distribution in breast tumors pre- and post-neoadjuvant chemotherapy. We found that intratumor genetic diversity was tumor-subtype specific, and it did not change during treatment in tumors with partial or no response. However, lower pretreatment genetic diversity was significantly associated with pathologic complete response. In contrast, phenotypic diversity was different between pre- and posttreatment samples. We also observed significant changes in the spatial distribution of cells with distinct genetic and phenotypic features. We used these experimental data to develop a stochastic computational model to infer tumor growth patterns and evolutionary dynamics. Our results highlight the importance of integrated analysis of genotypes and phenotypes of single cells in intact tissues to predict tumor evolution. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Inference of tumor evolution during chemotherapy by computational modeling and in situ analysis of cellular diversity for genetic and phenotypic features

PubMed Central

Almendro, Vanessa; Cheng, Yu-Kang; Randles, Amanda; Itzkovitz, Shalev; Marusyk, Andriy; Ametller, Elisabet; Gonzalez-Farre, Xavier; Muñoz, Montse; Russnes, Hege G.; Helland, Åslaug; Rye, Inga H.; Borresen-Dale, Anne-Lise; Maruyama, Reo; van Oudenaarden, Alexander; Dowsett, Mitchell; Jones, Robin L.; Reis-Filho, Jorge; Gascon, Pere; Gönen, Mithat; Michor, Franziska; Polyak, Kornelia

2014-01-01

SUMMARY Cancer therapy exerts a strong selection pressure that shapes tumor evolution, yet our knowledge of how tumors change during treatment is limited. Here we report the analysis of cellular heterogeneity for genetic and phenotypic features and their spatial distribution in breast tumors pre- and post-neoadjuvant chemotherapy. We found that intratumor genetic diversity was tumor subtype-specific and it did not change during treatment in tumors with partial or no response. However, lower pre-treatment genetic diversity was significantly associated with complete pathologic response. In contrast, phenotypic diversity was different between pre- and post-treatment samples. We also observed significant changes in the spatial distribution of cells with distinct genetic and phenotypic features. We used these experimental data to develop a stochastic computational model to infer tumor growth patterns and evolutionary dynamics. Our results highlight the importance of integrated analysis of genotypes and phenotypes of single cells in intact tissues to predict tumor evolution. PMID:24462293

Inference of tumor evolution during chemotherapy by computational modeling and in situ analysis of genetic and phenotypic cellular diversity

DOE PAGES

Almendro, Vanessa; Cheng, Yu -Kang; Randles, Amanda; ...

2014-02-01

Cancer therapy exerts a strong selection pressure that shapes tumor evolution, yet our knowledge of how tumors change during treatment is limited. Here, we report the analysis of cellular heterogeneity for genetic and phenotypic features and their spatial distribution in breast tumors pre- and post-neoadjuvant chemotherapy. We found that intratumor genetic diversity was tumor-subtype specific, and it did not change during treatment in tumors with partial or no response. However, lower pretreatment genetic diversity was significantly associated with pathologic complete response. In contrast, phenotypic diversity was different between pre- and post-treatment samples. We also observed significant changes in the spatialmore » distribution of cells with distinct genetic and phenotypic features. We used these experimental data to develop a stochastic computational model to infer tumor growth patterns and evolutionary dynamics. Our results highlight the importance of integrated analysis of genotypes and phenotypes of single cells in intact tissues to predict tumor evolution.« less

Current Understanding of Usher Syndrome Type II

PubMed Central

Yang, Jun; Wang, Le; Song, Hongman; Sokolov, Maxim

2012-01-01

Usher syndrome is the most common deafness-blindness caused by genetic mutations. To date, three genes have been identified underlying the most prevalent form of Usher syndrome, the type II form (USH2). The proteins encoded by these genes are demonstrated to form a complex in vivo. This complex is localized mainly at the periciliary membrane complex in photoreceptors and the ankle-link of the stereocilia in hair cells. Many proteins have been found to interact with USH2 proteins in vitro, suggesting that they are potential additional components of this USH2 complex and that the genes encoding these proteins may be the candidate USH2 genes. However, further investigations are critical to establish their existence in the USH2 complex in vivo. Based on the predicted functional domains in USH2 proteins, their cellular localizations in photoreceptors and hair cells, the observed phenotypes in USH2 mutant mice, and the known knowledge about diseases similar to USH2, putative biological functions of the USH2 complex have been proposed. Finally, therapeutic approaches for this group of diseases are now being actively explored. PMID:22201796

Membrane-targeted WAVE mediates photoreceptor axon targeting in the absence of the WAVE complex in Drosophila

PubMed Central

Stephan, Raiko; Gohl, Christina; Fleige, Astrid; Klämbt, Christian; Bogdan, Sven

2011-01-01

A tight spatial-temporal coordination of F-actin dynamics is crucial for a large variety of cellular processes that shape cells. The Abelson interactor (Abi) has a conserved role in Arp2/3-dependent actin polymerization, regulating Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous protein (WAVE). In this paper, we report that Abi exerts nonautonomous control of photoreceptor axon targeting in the Drosophila visual system through WAVE. In abi mutants, WAVE is unstable but restored by reexpression of Abi, confirming that Abi controls the integrity of the WAVE complex in vivo. Remarkably, expression of a membrane-tethered WAVE protein rescues the axonal projection defects of abi mutants in the absence of the other subunits of the WAVE complex, whereas cytoplasmic WAVE only slightly affects the abi mutant phenotype. Thus complex formation not only stabilizes WAVE, but also provides further membrane-recruiting signals, resulting in an activation of WAVE. PMID:21900504

Poor maternal nutrition and accelerated postnatal growth induces an accelerated aging phenotype and oxidative stress in skeletal muscle of male rats

PubMed Central

Fernandez-Twinn, Denise S.; Chen, Jian Hua; Hargreaves, Iain P.; Neergheen, Viruna; Aiken, Catherine E.; Ozanne, Susan E.

2016-01-01

ABSTRACT ‘Developmental programming’, which occurs as a consequence of suboptimal in utero and early environments, can be associated with metabolic dysfunction in later life, including an increased incidence of cardiovascular disease and type 2 diabetes, and predisposition of older men to sarcopenia. However, the molecular mechanisms underpinning these associations are poorly understood. Many conditions associated with developmental programming are also known to be associated with the aging process. We therefore utilized our well-established rat model of low birth weight and accelerated postnatal catch-up growth (termed ‘recuperated’) in this study to establish the effects of suboptimal maternal nutrition on age-associated factors in skeletal muscle. We demonstrated accelerated telomere shortening (a robust marker of cellular aging) as evidenced by a reduced frequency of long telomeres (48.5-8.6 kb) and an increased frequency of short telomeres (4.2-1.3 kb) in vastus lateralis muscle from aged recuperated offspring compared to controls. This was associated with increased protein expression of the DNA-damage-repair marker 8-oxoguanine-glycosylase (OGG1) in recuperated offspring. Recuperated animals also demonstrated an oxidative stress phenotype, with decreased citrate synthase activity, increased electron-transport-complex activities of complex I, complex II-III and complex IV (all markers of functional mitochondria), and increased xanthine oxidase (XO), p67phox and nuclear-factor kappa-light-chain-enhancer of activated B-cells (NF-κB). Recuperated offspring also demonstrated increased antioxidant defense capacity, with increased protein expression of manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD), catalase and heme oxygenase-1 (HO1), all of which are known targets of NF-κB and can be upregulated as a consequence of oxidative stress. Recuperated offspring also had a pro-inflammatory phenotype, as evidenced by increased tumor necrosis factor-α (TNFα) and interleukin-1β (IL1β) protein levels. Taken together, we demonstrate, for the first time to our knowledge, an accelerated aging phenotype in skeletal muscle in the context of developmental programming. These findings may pave the way for suitable interventions in at-risk populations. PMID:27585884

Intelligent Interfaces for Mining Large-Scale RNAi-HCS Image Databases

PubMed Central

Lin, Chen; Mak, Wayne; Hong, Pengyu; Sepp, Katharine; Perrimon, Norbert

2010-01-01

Recently, High-content screening (HCS) has been combined with RNA interference (RNAi) to become an essential image-based high-throughput method for studying genes and biological networks through RNAi-induced cellular phenotype analyses. However, a genome-wide RNAi-HCS screen typically generates tens of thousands of images, most of which remain uncategorized due to the inadequacies of existing HCS image analysis tools. Until now, it still requires highly trained scientists to browse a prohibitively large RNAi-HCS image database and produce only a handful of qualitative results regarding cellular morphological phenotypes. For this reason we have developed intelligent interfaces to facilitate the application of the HCS technology in biomedical research. Our new interfaces empower biologists with computational power not only to effectively and efficiently explore large-scale RNAi-HCS image databases, but also to apply their knowledge and experience to interactive mining of cellular phenotypes using Content-Based Image Retrieval (CBIR) with Relevance Feedback (RF) techniques. PMID:21278820

Multi-target drugs to address multiple checkpoints in complex inflammatory pathologies: evolutionary cues for novel "first-in-class" anti-inflammatory drug candidates: a reviewer's perspective.

PubMed

Mathew, Geetha; Unnikrishnan, M K

2015-10-01

Inflammation is a complex, metabolically expensive process involving multiple signaling pathways and regulatory mechanisms which have evolved over evolutionary timescale. Addressing multiple targets of inflammation holistically, in moderation, is probably a more evolutionarily viable strategy, as compared to current therapy which addresses drug targets in isolation. Polypharmacology, addressing multiple targets, is commonly used in complex ailments, suggesting the superior safety and efficacy profile of multi-target (MT) drugs. Phenotypic drug discovery, which generated successful MT and first-in-class drugs in the past, is now re-emerging. A multi-pronged approach, which modulates the evolutionarily conserved, robust and pervasive cellular mechanisms of tissue repair, with AMPK at the helm, regulating the complex metabolic/immune/redox pathways underlying inflammation, is perhaps a more viable strategy than addressing single targets in isolation. Molecules that modulate multiple molecular mechanisms of inflammation in moderation (modulating TH cells toward the anti-inflammatory phenotype, activating AMPK, stimulating Nrf2 and inhibiting NFκB) might serve as a model for a novel Darwinian "first-in-class" therapeutic category that holistically addresses immune, redox and metabolic processes associated with inflammatory repair. Such a multimodal biological activity is supported by the fact that several non-calorific pleiotropic natural products with anti-inflammatory action have been incorporated into diet (chiefly guided by the adaptive development of olfacto-gustatory preferences over evolutionary timescales) rendering such molecules, endowed with evolutionarily privileged molecular scaffolds, naturally oriented toward multiple targets.

Target identification by image analysis.

PubMed

Fetz, V; Prochnow, H; Brönstrup, M; Sasse, F

2016-05-04

Covering: 1997 to the end of 2015Each biologically active compound induces phenotypic changes in target cells that are characteristic for its mode of action. These phenotypic alterations can be directly observed under the microscope or made visible by labelling structural elements or selected proteins of the cells with dyes. A comparison of the cellular phenotype induced by a compound of interest with the phenotypes of reference compounds with known cellular targets allows predicting its mode of action. While this approach has been successfully applied to the characterization of natural products based on a visual inspection of images, recent studies used automated microscopy and analysis software to increase speed and to reduce subjective interpretation. In this review, we give a general outline of the workflow for manual and automated image analysis, and we highlight natural products whose bacterial and eucaryotic targets could be identified through such approaches.

Inducible and reversible phenotypes in a novel mouse model of Friedreich’s Ataxia

PubMed Central

Gao, Kun; Swarup, Vivek; Versano, Revital; Dong, Hongmei; Jordan, Maria C

2017-01-01

Friedreich's ataxia (FRDA), the most common inherited ataxia, is caused by recessive mutations that reduce the levels of frataxin (FXN), a mitochondrial iron binding protein. We developed an inducible mouse model of Fxn deficiency that enabled us to control the onset and progression of disease phenotypes by the modulation of Fxn levels. Systemic knockdown of Fxn in adult mice led to multiple phenotypes paralleling those observed in human patients across multiple organ systems. By reversing knockdown after clinical features appear, we were able to determine to what extent observed phenotypes represent reversible cellular dysfunction. Remarkably, upon restoration of near wild-type FXN levels, we observed significant recovery of function, associated pathology and transcriptomic dysregulation even after substantial motor dysfunction and pathology were observed. This model will be of broad utility in therapeutic development and in refining our understanding of the relative contribution of reversible cellular dysfunction at different stages in disease. PMID:29257745

Monte Carlo simulation of a simple gene network yields new evolutionary insights.

PubMed

Andrecut, M; Cloud, D; Kauffman, S A

2008-02-07

Monte Carlo simulations of a genetic toggle switch show that its behavior can be more complex than analytic models would suggest. We show here that as a result of the interplay between frequent and infrequent reaction events, such a switch can have more stable states than an analytic model would predict, and that the number and character of these states depend to a large extent on the propensity of transcription factors to bind to and dissociate from promoters. The effects of gene duplications differ even more; in analytic models, these seem to result in the disappearance of bi-stability and thus a loss of the switching function, but a Monte Carlo simulation shows that they can result in the appearance of new stable states without the loss of old ones, and thus in an increase of the complexity of the switch's behavior which may facilitate the evolution of new cellular functions. These differences are of interest with respect to the evolution of gene networks, particularly in clonal lines of cancer cells, where the duplication of active genes is an extremely common event, and often seems to result in the appearance of viable new cellular phenotypes.

'Gardos Channelopathy': a variant of hereditary Stomatocytosis with complex molecular regulation.

PubMed

Fermo, Elisa; Bogdanova, Anna; Petkova-Kirova, Polina; Zaninoni, Anna; Marcello, Anna Paola; Makhro, Asya; Hänggi, Pascal; Hertz, Laura; Danielczok, Jens; Vercellati, Cristina; Mirra, Nadia; Zanella, Alberto; Cortelezzi, Agostino; Barcellini, Wilma; Kaestner, Lars; Bianchi, Paola

2017-05-11

The Gardos channel is a Ca 2+ sensitive, K + selective channel present in several tissues including RBCs, where it is involved in cell volume regulation. Recently, mutations at two different aminoacid residues in KCNN4 have been reported in patients with hereditary xerocytosis. We identified by whole exome sequencing a new family with two members affected by chronic hemolytic anemia carrying mutation R352H in the KCNN4 gene. No additional mutations in genes encoding for RBCs cytoskeletal, membrane or channel proteins were detected. We performed functional studies on patients' RBCs to evaluate the effects of R352H mutation on the cellular properties and eventually on the clinical phenotype. Gardos channel hyperactivation was demonstrated in circulating erythrocytes and erythroblasts differentiated ex-vivo from peripheral CD34+ cells. Pathological alterations in the function of multiple ion transport systems were observed, suggesting the presence of compensatory effects ultimately preventing cellular dehydration in patient's RBCs; moreover, flow cytometry and confocal fluorescence live-cell imaging showed Ca 2+ overload in the RBCs of both patients and hypersensitivity of Ca 2+ uptake by RBCs to swelling. Altogether these findings suggest that the 'Gardos channelopathy' is a complex pathology, to some extent different from the common hereditary xerocytosis.

A multi-scale convolutional neural network for phenotyping high-content cellular images.

PubMed

Godinez, William J; Hossain, Imtiaz; Lazic, Stanley E; Davies, John W; Zhang, Xian

2017-07-01

Identifying phenotypes based on high-content cellular images is challenging. Conventional image analysis pipelines for phenotype identification comprise multiple independent steps, with each step requiring method customization and adjustment of multiple parameters. Here, we present an approach based on a multi-scale convolutional neural network (M-CNN) that classifies, in a single cohesive step, cellular images into phenotypes by using directly and solely the images' pixel intensity values. The only parameters in the approach are the weights of the neural network, which are automatically optimized based on training images. The approach requires no a priori knowledge or manual customization, and is applicable to single- or multi-channel images displaying single or multiple cells. We evaluated the classification performance of the approach on eight diverse benchmark datasets. The approach yielded overall a higher classification accuracy compared with state-of-the-art results, including those of other deep CNN architectures. In addition to using the network to simply obtain a yes-or-no prediction for a given phenotype, we use the probability outputs calculated by the network to quantitatively describe the phenotypes. This study shows that these probability values correlate with chemical treatment concentrations. This finding validates further our approach and enables chemical treatment potency estimation via CNNs. The network specifications and solver definitions are provided in Supplementary Software 1. william_jose.godinez_navarro@novartis.com or xian-1.zhang@novartis.com. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Rubella viruses shift cellular bioenergetics to a more oxidative and glycolytic phenotype with a strain-specific requirement for glutamine.

PubMed

Bilz, Nicole C; Jahn, Kristin; Lorenz, Mechthild; Lüdtke, Anja; Hübschen, Judith M; Geyer, Henriette; Mankertz, Annette; Hübner, Denise; Liebert, Uwe G; Claus, Claudia

2018-06-27

The flexible regulation of cellular metabolic pathways enables cellular adaptation to changes in energy demand under conditions of stress such as posed by a virus infection. To analyze such an impact on cellular metabolism, rubella virus (RV) was used in this study. RV replication under selected substrate supplementation with glucose, pyruvate, and glutamine as essential nutrients for mammalian cells revealed its requirement for glutamine. The assessment of the mitochondrial respiratory (based on oxygen consumption rate, OCR) and glycolytic (based on extracellular acidification rate, ECAR) rate and capacity by respective stress tests through Seahorse technology enabled determination of the bioenergetic phenotype of RV-infected cells. Irrespective of the cellular metabolic background, RV infection induced a shift of the bioenergetic state of epithelial (Vero and A549) and endothelial (HUVEC) cells to a higher oxidative and glycolytic level. Interestingly there was a RV strain-specific, but genotype-independent demand for glutamine to induce a significant increase in metabolic activity. While glutaminolysis appeared to be rather negligible for RV replication, glutamine could serve as donor of its amide nitrogen in biosynthesis pathways for important metabolites. This study suggests that the capacity of rubella viruses to induce metabolic alterations could evolve differently during natural infection. Thus, changes in cellular bioenergetics represent an important component of virus-host interactions and could complement our understanding of the viral preference for a distinct host cell population. Importance RV pathologies, especially during embryonal development, could be connected with its impact on mitochondrial metabolism. With bioenergetic phenotyping we pursued a rather novel approach in virology. For the first time it was shown that a virus infection could shift the bioenergetics of its infected host cell to a higher energetic state. Notably, the capacity to induce such alterations varied among different RV isolates. Thus, our data adds viral adaptation of cellular metabolic activity to its specific needs as a novel aspect to virus-host evolution. Additionally, this study emphasizes the implementation of different viral strains in the study of virus-host interactions and the use of bioenergetic phenotyping of infected cells as a biomarker for virus-induced pathological alterations. Copyright © 2018 American Society for Microbiology.

Tumor Heterogenity Research Interactive Visualization Environment (THRIVE) | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

A platform for quantitative evaluation of intratumoral spatial heterogeneity in multiplexed immunofluorescence images, via characterization of the spatial interactions between different cellular phenotypes and non-cellular constituents in the tumor microenvironment.

A Herpesviral Immediate Early Protein Promotes Transcription Elongation of Viral Transcripts.

PubMed

Fox, Hannah L; Dembowski, Jill A; DeLuca, Neal A

2017-06-13

Herpes simplex virus 1 (HSV-1) genes are transcribed by cellular RNA polymerase II (RNA Pol II). While four viral immediate early proteins (ICP4, ICP0, ICP27, and ICP22) function in some capacity in viral transcription, the mechanism by which ICP22 functions remains unclear. We observed that the FACT complex (comprised of SSRP1 and Spt16) was relocalized in infected cells as a function of ICP22. ICP22 was also required for the association of FACT and the transcription elongation factors SPT5 and SPT6 with viral genomes. We further demonstrated that the FACT complex interacts with ICP22 throughout infection. We therefore hypothesized that ICP22 recruits cellular transcription elongation factors to viral genomes for efficient transcription elongation of viral genes. We reevaluated the phenotype of an ICP22 mutant virus by determining the abundance of all viral mRNAs throughout infection by transcriptome sequencing (RNA-seq). The accumulation of almost all viral mRNAs late in infection was reduced compared to the wild type, regardless of kinetic class. Using chromatin immunoprecipitation sequencing (ChIP-seq), we mapped the location of RNA Pol II on viral genes and found that RNA Pol II levels on the bodies of viral genes were reduced in the ICP22 mutant compared to wild-type virus. In contrast, the association of RNA Pol II with transcription start sites in the mutant was not reduced. Taken together, our results indicate that ICP22 plays a role in recruiting elongation factors like the FACT complex to the HSV-1 genome to allow for efficient viral transcription elongation late in viral infection and ultimately infectious virion production. IMPORTANCE HSV-1 interacts with many cellular proteins throughout productive infection. Here, we demonstrate the interaction of a viral protein, ICP22, with a subset of cellular proteins known to be involved in transcription elongation. We determined that ICP22 is required to recruit the FACT complex and other transcription elongation factors to viral genomes and that in the absence of ICP22 viral transcription is globally reduced late in productive infection, due to an elongation defect. This insight defines a fundamental role of ICP22 in HSV-1 infection and elucidates the involvement of cellular factors in HSV-1 transcription. Copyright © 2017 Fox et al.

Modulation of cardiac myocyte phenotype in vitro by the composition and orientation of the extracellular matrix.

PubMed

Simpson, D G; Terracio, L; Terracio, M; Price, R L; Turner, D C; Borg, T K

1994-10-01

Cellular phenotype is the result of a dynamic interaction between a cell's intrinsic genetic program and the morphogenetic signals that serve to modulate the extent to which that program is expressed. In the present study we have examined how morphogenetic information might be stored in the extracellular matrix (ECM) and communicated to the neonatal heart cell (NHC) by the cardiac alpha 1 beta 1 integrin molecule. A thin film of type I collagen (T1C) was prepared with a defined orientation. This was achieved by applying T1C to the peripheral edge of a 100 mm culture dish. The T1C was then drawn across the surface of the dish in a continuous stroke with a sterile cell scraper and allowed to polymerize. When NHCs were cultured on this substrate, they spread, as a population, along a common axis in parallel with the gel lattice and expressed an in vivo-like phenotype. Individual NHCs displayed an elongated, rod-like shape and disclosed parallel arrays of myofibrils. These phenotypic characteristics were maintained for at least 4 weeks in primary culture. The evolution of this tissue-like organizational pattern was dependent upon specific interactions between the NHCs and the collagen-based matrix that were mediated by the cardiac alpha 1 beta 1 integrin complex. This conclusion was supported by a variety of experimental results. Altering the tertiary structure of the matrix or blocking the extracellular domains of either the cardiac alpha 1 or beta 1 integrin chain inhibited the expression of the tissue-like pattern of organization. Neither cell-to-cell contact or contractile function were necessary to induce the formation of the rod-like cell shape. However, beating activity was necessary for the assembly of a well-differentiated myofibrillar apparatus. These data suggest that the cardiac alpha 1 beta 1 integrin complex serves to detect and transduce phenotypic information stored within the tertiary structure of the surrounding matrix.

Exome Sequencing and the Management of Neurometabolic Disorders.

PubMed

Tarailo-Graovac, Maja; Shyr, Casper; Ross, Colin J; Horvath, Gabriella A; Salvarinova, Ramona; Ye, Xin C; Zhang, Lin-Hua; Bhavsar, Amit P; Lee, Jessica J Y; Drögemöller, Britt I; Abdelsayed, Mena; Alfadhel, Majid; Armstrong, Linlea; Baumgartner, Matthias R; Burda, Patricie; Connolly, Mary B; Cameron, Jessie; Demos, Michelle; Dewan, Tammie; Dionne, Janis; Evans, A Mark; Friedman, Jan M; Garber, Ian; Lewis, Suzanne; Ling, Jiqiang; Mandal, Rupasri; Mattman, Andre; McKinnon, Margaret; Michoulas, Aspasia; Metzger, Daniel; Ogunbayo, Oluseye A; Rakic, Bojana; Rozmus, Jacob; Ruben, Peter; Sayson, Bryan; Santra, Saikat; Schultz, Kirk R; Selby, Kathryn; Shekel, Paul; Sirrs, Sandra; Skrypnyk, Cristina; Superti-Furga, Andrea; Turvey, Stuart E; Van Allen, Margot I; Wishart, David; Wu, Jiang; Wu, John; Zafeiriou, Dimitrios; Kluijtmans, Leo; Wevers, Ron A; Eydoux, Patrice; Lehman, Anna M; Vallance, Hilary; Stockler-Ipsiroglu, Sylvia; Sinclair, Graham; Wasserman, Wyeth W; van Karnebeek, Clara D

2016-06-09

Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level. To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patient's clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes. We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%). Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Children's Hospital Foundation and others.).

Exome Sequencing and the Management of Neurometabolic Disorders

PubMed Central

Tarailo-Graovac, M.; Shyr, C.; Ross, C.J.; Horvath, G.A.; Salvarinova, R.; Ye, X.C.; Zhang, L.-H.; Bhavsar, A.P.; Lee, J.J.Y.; Drögemöller, B.I.; Abdelsayed, M.; Alfadhel, M.; Armstrong, L.; Baumgartner, M.R.; Burda, P.; Connolly, M.B.; Cameron, J.; Demos, M.; Dewan, T.; Dionne, J.; Evans, A.M.; Friedman, J.M.; Garber, I.; Lewis, S.; Ling, J.; Mandal, R.; Mattman, A.; McKinnon, M.; Michoulas, A.; Metzger, D.; Ogunbayo, O.A.; Rakic, B.; Rozmus, J.; Ruben, P.; Sayson, B.; Santra, S.; Schultz, K.R.; Selby, K.; Shekel, P.; Sirrs, S.; Skrypnyk, C.; Superti-Furga, A.; Turvey, S.E.; Van Allen, M.I.; Wishart, D.; Wu, J.; Wu, J.; Zafeiriou, D.; Kluijtmans, L.; Wevers, R.A.; Eydoux, P.; Lehman, A.M.; Vallance, H.; Stockler-Ipsiroglu, S.; Sinclair, G.; Wasserman, W.W.; van Karnebeek, C.D.

2016-01-01

BACKGROUND Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level. METHODS To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patient’s clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes. RESULTS We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%). CONCLUSIONS Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Children’s Hospital Foundation and others.) PMID:27276562

Photo-induced toxic epidermal necrolysis caused by clobazam.

PubMed

Redondo, P; Vicente, J; España, A; Subira, M L; De Felipe, I; Quintanilla, E

1996-12-01

Toxic epidermal necrolysis (TEN) is a life-threatening disease, the pathogenesis of which remains largely unknown. We describe a 23-year-old woman under treatment with clobazam who developed lesions of TEN in light-exposed areas. Patch and photopatch tests with clobazam were negative. The cellular phenotype and cytokines were studied in blister fluid. The cellular infiltrate was composed mainly of T lymphocytes with a predominant cytotoxic phenotype. There was an increase in the level of tumour necrosis factor (TNF)-alpha in blister fluid compared with the control (a patient with bullous pemphigoid).

The evolution of cellular deficiency in GATA2 mutation

PubMed Central

Dickinson, Rachel E.; Milne, Paul; Jardine, Laura; Zandi, Sasan; Swierczek, Sabina I.; McGovern, Naomi; Cookson, Sharon; Ferozepurwalla, Zaveyna; Langridge, Alexander; Pagan, Sarah; Gennery, Andrew; Heiskanen-Kosma, Tarja; Hämäläinen, Sari; Seppänen, Mikko; Helbert, Matthew; Tholouli, Eleni; Gambineri, Eleonora; Reykdal, Sigrún; Gottfreðsson, Magnús; Thaventhiran, James E.; Morris, Emma; Hirschfield, Gideon; Richter, Alex G.; Jolles, Stephen; Bacon, Chris M.; Hambleton, Sophie; Haniffa, Muzlifah; Bryceson, Yenan; Allen, Carl; Prchal, Josef T.; Dick, John E.; Bigley, Venetia

2014-01-01

Constitutive heterozygous GATA2 mutation is associated with deafness, lymphedema, mononuclear cytopenias, infection, myelodysplasia (MDS), and acute myeloid leukemia. In this study, we describe a cross-sectional analysis of 24 patients and 6 relatives with 14 different frameshift or substitution mutations of GATA2. A pattern of dendritic cell, monocyte, B, and natural killer (NK) lymphoid deficiency (DCML deficiency) with elevated Fms-like tyrosine kinase 3 ligand (Flt3L) was observed in all 20 patients phenotyped, including patients with Emberger syndrome, monocytopenia with Mycobacterium avium complex (MonoMAC), and MDS. Four unaffected relatives had a normal phenotype indicating that cellular deficiency may evolve over time or is incompletely penetrant, while 2 developed subclinical cytopenias or elevated Flt3L. Patients with GATA2 mutation maintained higher hemoglobin, neutrophils, and platelets and were younger than controls with acquired MDS and wild-type GATA2. Frameshift mutations were associated with earlier age of clinical presentation than substitution mutations. Elevated Flt3L, loss of bone marrow progenitors, and clonal myelopoiesis were early signs of disease evolution. Clinical progression was associated with increasingly elevated Flt3L, depletion of transitional B cells, CD56bright NK cells, naïve T cells, and accumulation of terminally differentiated NK and CD8+ memory T cells. These studies provide a framework for clinical and laboratory monitoring of patients with GATA2 mutation and may inform therapeutic decision-making. PMID:24345756

Clinical and functional characterization of a patient carrying a compound heterozygous pericentrin mutation and a heterozygous IGF1 receptor mutation.

PubMed

Müller, Eva; Dunstheimer, Desiree; Klammt, Jürgen; Friebe, Daniela; Kiess, Wieland; Kratzsch, Jürgen; Kruis, Tassilo; Laue, Sandy; Pfäffle, Roland; Wallborn, Tillmann; Heidemann, Peter H

2012-01-01

Intrauterine and postnatal longitudinal growth is controlled by a strong genetic component that regulates a complex network of endocrine factors integrating them with cellular proliferation, differentiation and apoptotic processes in target tissues, particularly the growth centers of the long bones. Here we report on a patient born small for gestational age (SGA) with severe, proportionate postnatal growth retardation, discreet signs of skeletal dysplasia, microcephaly and moyamoya disease. Initial genetic evaluation revealed a novel heterozygous IGF1R p.Leu1361Arg mutation affecting a highly conserved residue with the insulin-like growth factor type 1 receptor suggestive for a disturbance within the somatotropic axis. However, because the mutation did not co-segregate with the phenotype and functional characterization did not reveal an obvious impairment of the ligand depending major IGF1R signaling capabilities a second-site mutation was assumed. Mutational screening of components of the somatotropic axis, constituents of the IGF signaling system and factors involved in cellular proliferation, which are described or suggested to provoke syndromic dwarfism phenotypes, was performed. Two compound heterozygous PCNT mutations (p.[Arg585X];[Glu1774X]) were identified leading to the specification of the diagnosis to MOPD II. These investigations underline the need for careful assessment of all available information to derive a firm diagnosis from a sequence aberration.

Clinical and Functional Characterization of a Patient Carrying a Compound Heterozygous Pericentrin Mutation and a Heterozygous IGF1 Receptor Mutation

PubMed Central

Klammt, Jürgen; Friebe, Daniela; Kiess, Wieland; Kratzsch, Jürgen; Kruis, Tassilo; Laue, Sandy; Pfäffle, Roland; Wallborn, Tillmann; Heidemann, Peter H.

2012-01-01

Intrauterine and postnatal longitudinal growth is controlled by a strong genetic component that regulates a complex network of endocrine factors integrating them with cellular proliferation, differentiation and apoptotic processes in target tissues, particularly the growth centers of the long bones. Here we report on a patient born small for gestational age (SGA) with severe, proportionate postnatal growth retardation, discreet signs of skeletal dysplasia, microcephaly and moyamoya disease. Initial genetic evaluation revealed a novel heterozygous IGF1R p.Leu1361Arg mutation affecting a highly conserved residue with the insulin-like growth factor type 1 receptor suggestive for a disturbance within the somatotropic axis. However, because the mutation did not co-segregate with the phenotype and functional characterization did not reveal an obvious impairment of the ligand depending major IGF1R signaling capabilities a second-site mutation was assumed. Mutational screening of components of the somatotropic axis, constituents of the IGF signaling system and factors involved in cellular proliferation, which are described or suggested to provoke syndromic dwarfism phenotypes, was performed. Two compound heterozygous PCNT mutations (p.[Arg585X];[Glu1774X]) were identified leading to the specification of the diagnosis to MOPD II. These investigations underline the need for careful assessment of all available information to derive a firm diagnosis from a sequence aberration. PMID:22693602

Hybrid proline-rich proteins: novel players in plant cell elongation?

PubMed Central

Dvořáková, Lenka; Srba, Miroslav; Opatrny, Zdenek; Fischer, Lukas

2012-01-01

Background and Aims Hybrid proline-rich proteins (HyPRPs) represent a large family of putative cell-wall proteins characterized by the presence of a variable N-terminal domain and a conserved C-terminal domain that is related to non-specific lipid transfer proteins. The function of HyPRPs remains unclear, but their widespread occurrence and abundant expression patterns indicate that they may be involved in a basic cellular process. Methods To elucidate the cellular function of HyPRPs, we modulated the expression of three HyPRP genes in tobacco (Nicotiana tabacum) BY-2 cell lines and in potato (Solanum tuberosum) plants. Key Results In BY-2 lines, over-expression of the three HyPRP genes with different types of N-terminal domains resulted in similar phenotypic changes, namely increased cell elongation, both in suspension culture and on solid media where the over-expression resulted in enhanced calli size. The over-expressing cells showed increased plasmolysis in a hypertonic mannitol solution and accelerated rate of protoplast release, suggesting loosening of the cell walls. In contrast to BY-2 lines, no phenotypic changes were observed in potato plants over-expressing the same or analogous HyPRP genes, presumably due to more complex compensatory mechanisms in planta. Conclusions Based on the results from BY-2 lines, we propose that HyPRPs, more specifically their C-terminal domains, represent a novel group of proteins involved in cell expansion. PMID:22028464

Integrated network analysis and effective tools in plant systems biology

PubMed Central

Fukushima, Atsushi; Kanaya, Shigehiko; Nishida, Kozo

2014-01-01

One of the ultimate goals in plant systems biology is to elucidate the genotype-phenotype relationship in plant cellular systems. Integrated network analysis that combines omics data with mathematical models has received particular attention. Here we focus on the latest cutting-edge computational advances that facilitate their combination. We highlight (1) network visualization tools, (2) pathway analyses, (3) genome-scale metabolic reconstruction, and (4) the integration of high-throughput experimental data and mathematical models. Multi-omics data that contain the genome, transcriptome, proteome, and metabolome and mathematical models are expected to integrate and expand our knowledge of complex plant metabolisms. PMID:25408696

Progress in Using Mouse Inbred Strains, Consomics, and Mutants to Identify Genes Related to Stress, Anxiety, and Alcohol Phenotypes

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

Goldowitz, Daniel; Matthews, Douglas B.; Hamre, Kristin M.

ALCOHOL ABUSE AND alcoholism result from the complex interplay of genetic and environmental factors. Stress is a factor that is widely thought to contribute to excessive drinking and alcoholism. One consequence of stressful experiences is anxiety, and there is a rich literature on the interactions between alcohol and anxiety. Less is known about brain mechanisms at the molecular, cellular, and system levels that mediate stress effects that contribute to excessive drinking and alcoholism. In addition, it is not clear whether and/or how genetic factors that contribute to excessive drinking interact with neural stress mechanisms.

Familial Hypercholesterolaemia

PubMed Central

Marais, A David

2004-01-01

Familial hypercholesterolaemia (FH), defined as the heritable occurrence of severe hypercholesterolaemia with cholesterol deposits in tendons and premature heart disease, is caused by at least four genes in sterol and lipoprotein pathways and displays varying gene-dose effects. The genes are the low-density lipoprotein (LDL) receptor, apolipoprotein (apo) B, proprotein convertase subtilisin/kexin 9, and the autosomal recessive hypercholesterolaemia (ARH) adaptor protein. All of these disorders have in common defective clearance of LDL within a complex system of lipid and lipoprotein metabolism and regulation. Normal cellular cholesterol and lipoprotein metabolism is reviewed before describing the disorders, their metabolic derangements and their clinical effects. FH is classified as two simplified phenotypes of disease according to the severity of the metabolic derangement. The dominantly inherited heterozygous phenotype comprises defects in the LDL receptor, apoB100, and neural apoptosis regulatory cleavage protein. The homozygous phenotype is co-dominant in defects of the LDL receptor, and occurs also as the ARH of adapter protein mutations. Defective binding of apoB100 does not result in a significant gene dose effect, but enhances the severity of heterozygotes for LDL receptor mutations. The genetic diagnosis of FH has provided greater accuracy in definition and detection of disease and exposes information about migration of populations. All of these disorders pose a high risk of atherosclerosis, especially in the homozygous phenotype. Studies of influences on the phenotype and responses to treatment are also discussed in the context of the metabolic derangements. PMID:18516203

A Genotypic-Oriented View of CFTR Genetics Highlights Specific Mutational Patterns Underlying Clinical Macrocategories of Cystic Fibrosis

PubMed Central

Lucarelli, Marco; Bruno, Sabina Maria; Pierandrei, Silvia; Ferraguti, Giampiero; Stamato, Antonella; Narzi, Fabiana; Amato, Annalisa; Cimino, Giuseppe; Bertasi, Serenella; Quattrucci, Serena; Strom, Roberto

2015-01-01

Cystic fibrosis (CF) is a monogenic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The genotype–phenotype relationship in this disease is still unclear, and diagnostic, prognostic and therapeutic challenges persist. We enrolled 610 patients with different forms of CF and studied them from a clinical, biochemical, microbiological and genetic point of view. Overall, there were 125 different mutated alleles (11 with novel mutations and 10 with complex mutations) and 225 genotypes. A strong correlation between mutational patterns at the genotypic level and phenotypic macrocategories emerged. This specificity appears to largely depend on rare and individual mutations, as well as on the varying prevalence of common alleles in different clinical macrocategories. However, 19 genotypes appeared to underlie different clinical forms of the disease. The dissection of the pathway from the CFTR mutated genotype to the clinical phenotype allowed to identify at least two components of the variability usually found in the genotype–phenotype relationship. One component seems to depend on the genetic variation of CFTR, the other component on the cumulative effect of variations in other genes and cellular pathways independent from CFTR. The experimental dissection of the overall biological CFTR pathway appears to be a powerful approach for a better comprehension of the genotype–phenotype relationship. However, a change from an allele-oriented to a genotypic-oriented view of CFTR genetics is mandatory, as well as a better assessment of sources of variability within the CFTR pathway. PMID:25910067

How well do you know your mutation? Complex effects of genetic background on expressivity, complementation, and ordering of allelic effects

PubMed Central

Choi, Lin; DeNieu, Michael; Sonnenschein, Anne; Hummel, Kristen; Marier, Christian; Victory, Andrew; Porter, Cody; Mammel, Anna; Holms, Julie; Sivaratnam, Gayatri

2017-01-01

For a given gene, different mutations influence organismal phenotypes to varying degrees. However, the expressivity of these variants not only depends on the DNA lesion associated with the mutation, but also on factors including the genetic background and rearing environment. The degree to which these factors influence related alleles, genes, or pathways similarly, and whether similar developmental mechanisms underlie variation in the expressivity of a single allele across conditions and among alleles is poorly understood. Besides their fundamental biological significance, these questions have important implications for the interpretation of functional genetic analyses, for example, if these factors alter the ordering of allelic series or patterns of complementation. We examined the impact of genetic background and rearing environment for a series of mutations spanning the range of phenotypic effects for both the scalloped and vestigial genes, which influence wing development in Drosophila melanogaster. Genetic background and rearing environment influenced the phenotypic outcome of mutations, including intra-genic interactions, particularly for mutations of moderate expressivity. We examined whether cellular correlates (such as cell proliferation during development) of these phenotypic effects matched the observed phenotypic outcome. While cell proliferation decreased with mutations of increasingly severe effects, surprisingly it did not co-vary strongly with the degree of background dependence. We discuss these findings and propose a phenomenological model to aid in understanding the biology of genes, and how this influences our interpretation of allelic effects in genetic analysis. PMID:29166655

Characterization and reproducibility of HepG2 hanging drop spheroids toxicology in vitro.

PubMed

Hurrell, Tracey; Ellero, Andrea Antonio; Masso, Zelie Flavienne; Cromarty, Allan Duncan

2018-02-21

Hepatotoxicity remains a major challenge in drug development despite preclinical toxicity screening using hepatocytes of human origin. To overcome some limitations of reproducing the hepatic phenotype, more structurally and functionally authentic cultures in vitro can be introduced by growing cells in 3D spheroid cultures. Characterisation and reproducibility of HepG2 spheroid cultures using a high-throughput hanging drop technique was performed and features contributing to potential phenotypic variation highlighted. Cultured HepG2 cells were seeded into Perfecta 3D® 96-well hanging drop plates and assessed over time for morphology, viability, cell cycle distribution, protein content and protein-mass profiles. Divergent aspects which were assessed included cell stocks, seeding density, volume of culture medium and use of extracellular matrix additives. Hanging drops are advantageous due to no complex culture matrix being present, enabling background free extractions for downstream experimentation. Varying characteristics were observed across cell stocks and batches, seeding density, culture medium volume and extracellular matrix when using immortalized HepG2 cells. These factors contribute to wide-ranging cellular responses and highlights concerns with respect to generating a reproducible phenotype in HepG2 hanging drop spheroids. Copyright © 2018 Elsevier Ltd. All rights reserved.

Solving Immunology?

PubMed

Vodovotz, Yoram; Xia, Ashley; Read, Elizabeth L; Bassaganya-Riera, Josep; Hafler, David A; Sontag, Eduardo; Wang, Jin; Tsang, John S; Day, Judy D; Kleinstein, Steven H; Butte, Atul J; Altman, Matthew C; Hammond, Ross; Sealfon, Stuart C

2017-02-01

Emergent responses of the immune system result from the integration of molecular and cellular networks over time and across multiple organs. High-content and high-throughput analysis technologies, concomitantly with data-driven and mechanistic modeling, hold promise for the systematic interrogation of these complex pathways. However, connecting genetic variation and molecular mechanisms to individual phenotypes and health outcomes has proven elusive. Gaps remain in data, and disagreements persist about the value of mechanistic modeling for immunology. Here, we present the perspectives that emerged from the National Institute of Allergy and Infectious Disease (NIAID) workshop 'Complex Systems Science, Modeling and Immunity' and subsequent discussions regarding the potential synergy of high-throughput data acquisition, data-driven modeling, and mechanistic modeling to define new mechanisms of immunological disease and to accelerate the translation of these insights into therapies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Distinct gene regulatory programs define the inhibitory effects of liver X receptors and PPARG on cancer cell proliferation.

PubMed

Savic, Daniel; Ramaker, Ryne C; Roberts, Brian S; Dean, Emma C; Burwell, Todd C; Meadows, Sarah K; Cooper, Sara J; Garabedian, Michael J; Gertz, Jason; Myers, Richard M

2016-07-11

The liver X receptors (LXRs, NR1H2 and NR1H3) and peroxisome proliferator-activated receptor gamma (PPARG, NR1C3) nuclear receptor transcription factors (TFs) are master regulators of energy homeostasis. Intriguingly, recent studies suggest that these metabolic regulators also impact tumor cell proliferation. However, a comprehensive temporal molecular characterization of the LXR and PPARG gene regulatory responses in tumor cells is still lacking. To better define the underlying molecular processes governing the genetic control of cellular growth in response to extracellular metabolic signals, we performed a comprehensive, genome-wide characterization of the temporal regulatory cascades mediated by LXR and PPARG signaling in HT29 colorectal cancer cells. For this analysis, we applied a multi-tiered approach that incorporated cellular phenotypic assays, gene expression profiles, chromatin state dynamics, and nuclear receptor binding patterns. Our results illustrate that the activation of both nuclear receptors inhibited cell proliferation and further decreased glutathione levels, consistent with increased cellular oxidative stress. Despite a common metabolic reprogramming, the gene regulatory network programs initiated by these nuclear receptors were widely distinct. PPARG generated a rapid and short-term response while maintaining a gene activator role. By contrast, LXR signaling was prolonged, with initial, predominantly activating functions that transitioned to repressive gene regulatory activities at late time points. Through the use of a multi-tiered strategy that integrated various genomic datasets, our data illustrate that distinct gene regulatory programs elicit common phenotypic effects, highlighting the complexity of the genome. These results further provide a detailed molecular map of metabolic reprogramming in cancer cells through LXR and PPARG activation. As ligand-inducible TFs, these nuclear receptors can potentially serve as attractive therapeutic targets for the treatment of various cancers.

Modeling the Transitions between Collective and Solitary Migration Phenotypes in Cancer Metastasis

PubMed Central

Huang, Bin; Jolly, Mohit Kumar; Lu, Mingyang; Tsarfaty, Ilan; Ben-Jacob, Eshel; Onuchic, Jose’ N

2015-01-01

Cellular plasticity during cancer metastasis is a major clinical challenge. Two key cellular plasticity mechanisms —Epithelial-to-Mesenchymal Transition (EMT) and Mesenchymal-to-Amoeboid Transition (MAT) – have been carefully investigated individually, yet a comprehensive understanding of their interconnections remains elusive. Previously, we have modeled the dynamics of the core regulatory circuits for both EMT (miR-200/ZEB/miR-34/SNAIL) and MAT (Rac1/RhoA). We now extend our previous work to study the coupling between these two core circuits by considering the two microRNAs (miR-200 and miR-34) as external signals to the core MAT circuit. We show that this coupled circuit enables four different stable steady states (phenotypes) that correspond to hybrid epithelial/mesenchymal (E/M), mesenchymal (M), amoeboid (A) and hybrid amoeboid/mesenchymal (A/M) phenotypes. Our model recapitulates the metastasis-suppressing role of the microRNAs even in the presence of EMT-inducing signals like Hepatocyte Growth Factor (HGF). It also enables mapping the microRNA levels to the transitions among various cell migration phenotypes. Finally, it offers a mechanistic understanding for the observed phenotypic transitions among different cell migration phenotypes, specifically the Collective-to-Amoeboid Transition (CAT). PMID:26627083

Cellular Retinoic Acid Binding Proteins: Genomic and Non-genomic Functions and their Regulation.

PubMed

Wei, Li-Na

Cellular retinoic acid binding proteins (CRABPs) are high-affinity retinoic acid (RA) binding proteins that mainly reside in the cytoplasm. In mammals, this family has two members, CRABPI and II, both highly conserved during evolution. The two proteins share a very similar structure that is characteristic of a "β-clam" motif built up from10-strands. The proteins are encoded by two different genes that share a very similar genomic structure. CRABPI is widely distributed and CRABPII has restricted expression in only certain tissues. The CrabpI gene is driven by a housekeeping promoter, but can be regulated by numerous factors, including thyroid hormones and RA, which engage a specific chromatin-remodeling complex containing either TRAP220 or RIP140 as coactivator and corepressor, respectively. The chromatin-remodeling complex binds the DR4 element in the CrabpI gene promoter to activate or repress this gene in different cellular backgrounds. The CrabpII gene promoter contains a TATA-box and is rapidly activated by RA through an RA response element. Biochemical and cell culture studies carried out in vitro show the two proteins have distinct biological functions. CRABPII mainly functions to deliver RA to the nuclear RA receptors for gene regulation, although recent studies suggest that CRABPII may also be involved in other cellular events, such as RNA stability. In contrast, biochemical and cell culture studies suggest that CRABPI functions mainly in the cytoplasm to modulate intracellular RA availability/concentration and to engage other signaling components such as ERK activity. However, these functional studies remain inconclusive because knocking out one or both genes in mice does not produce definitive phenotypes. Further studies are needed to unambiguously decipher the exact physiological activities of these two proteins.

Transmembrane 4 L Six Family Member 5 (TM4SF5)-Mediated Epithelial-Mesenchymal Transition in Liver Diseases.

PubMed

Lee, Jung Weon

2015-01-01

The membrane protein TM4SF5, a member of the transmembrane 4L six family, forms a tetraspanin-enriched microdomain (TEM) on the cell surface, where many different membrane proteins and receptors form a massive protein-protein complex to regulate cellular functions including transdifferentiation, migration, and invasion. We recently reported that TM4SF5 causes epithelial-mesenchymal transition (EMT), eventually contributing to aberrant multilayer cellular growth, drug resistance, enhanced migration, invasion, its circulation in the blood, tumor initiation for successful metastasis, and muscle development in zebrafish. In this review, I summarize the information on the role of TM4SF5 in EMT-related functions at TM4SF5-enriched microdomain (T5EM) on cell surface, where proteins such as TM4SF5, CD151, CD44, integrins, and epidermal growth factor receptor (EGFR) can form numerous protein complexes. TM4SF5-mediated EMT contributes to diverse cellular functions, leading to fibrotic phenotypes and initiating and maintaining tumors in primary and/or metastatic regions, in addition to its role in muscle development in zebrafish. Anti-TM4SF5 strategies for addressing the protein networks can lead to regulation of the fibrotic, tumorigenic, and tumor-maintaining functions of TM4SF5-positive hepatic cells. This review is for us to (re)consider the antifibrotic or antitumorigenic (i.e., anti-EMT-related diseases) strategies of dealing with protein networks that would be involved in cross-talks to regulate various cellular functions during TM4SF5-dependent progression from fibrotic to cancerous hepatic cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Automatic Classification of Cellular Expression by Nonlinear Stochastic Embedding (ACCENSE).

PubMed

Shekhar, Karthik; Brodin, Petter; Davis, Mark M; Chakraborty, Arup K

2014-01-07

Mass cytometry enables an unprecedented number of parameters to be measured in individual cells at a high throughput, but the large dimensionality of the resulting data severely limits approaches relying on manual "gating." Clustering cells based on phenotypic similarity comes at a loss of single-cell resolution and often the number of subpopulations is unknown a priori. Here we describe ACCENSE, a tool that combines nonlinear dimensionality reduction with density-based partitioning, and displays multivariate cellular phenotypes on a 2D plot. We apply ACCENSE to 35-parameter mass cytometry data from CD8(+) T cells derived from specific pathogen-free and germ-free mice, and stratify cells into phenotypic subpopulations. Our results show significant heterogeneity within the known CD8(+) T-cell subpopulations, and of particular note is that we find a large novel subpopulation in both specific pathogen-free and germ-free mice that has not been described previously. This subpopulation possesses a phenotypic signature that is distinct from conventional naive and memory subpopulations when analyzed by ACCENSE, but is not distinguishable on a biaxial plot of standard markers. We are able to automatically identify cellular subpopulations based on all proteins analyzed, thus aiding the full utilization of powerful new single-cell technologies such as mass cytometry.

Mechanobiological simulations of peri-acetabular bone ingrowth: a comparative analysis of cell-phenotype specific and phenomenological algorithms.

PubMed

Mukherjee, Kaushik; Gupta, Sanjay

2017-03-01

Several mechanobiology algorithms have been employed to simulate bone ingrowth around porous coated implants. However, there is a scarcity of quantitative comparison between the efficacies of commonly used mechanoregulatory algorithms. The objectives of this study are: (1) to predict peri-acetabular bone ingrowth using cell-phenotype specific algorithm and to compare these predictions with those obtained using phenomenological algorithm and (2) to investigate the influences of cellular parameters on bone ingrowth. The variation in host bone material property and interfacial micromotion of the implanted pelvis were mapped onto the microscale model of implant-bone interface. An overall variation of 17-88 % in peri-acetabular bone ingrowth was observed. Despite differences in predicted tissue differentiation patterns during the initial period, both the algorithms predicted similar spatial distribution of neo-tissue layer, after attainment of equilibrium. Results indicated that phenomenological algorithm, being computationally faster than the cell-phenotype specific algorithm, might be used to predict peri-prosthetic bone ingrowth. The cell-phenotype specific algorithm, however, was found to be useful in numerically investigating the influence of alterations in cellular activities on bone ingrowth, owing to biologically related factors. Amongst the host of cellular activities, matrix production rate of bone tissue was found to have predominant influence on peri-acetabular bone ingrowth.

ω-3 polyunsaturated fatty acids direct differentiation of the membrane phenotype in mesenchymal stem cells to potentiate osteogenesis

PubMed Central

Levental, Kandice R.; Surma, Michal A.; Skinkle, Allison D.; Lorent, Joseph H.; Zhou, Yong; Klose, Christian; Chang, Jeffrey T.; Hancock, John F.; Levental, Ilya

2017-01-01

Mammalian cells produce hundreds of dynamically regulated lipid species that are actively turned over and trafficked to produce functional membranes. These lipid repertoires are susceptible to perturbations from dietary sources, with potentially profound physiological consequences. However, neither the lipid repertoires of various cellular membranes, their modulation by dietary fats, nor their effects on cellular phenotypes have been widely explored. We report that differentiation of human mesenchymal stem cells (MSCs) into osteoblasts or adipocytes results in extensive remodeling of the plasma membrane (PM), producing cell-specific membrane compositions and biophysical properties. The distinct features of osteoblast PMs enabled rational engineering of membrane phenotypes to modulate differentiation in MSCs. Specifically, supplementation with docosahexaenoic acid (DHA), a lipid component characteristic of osteoblast membranes, induced broad lipidomic remodeling in MSCs that reproduced compositional and structural aspects of the osteoblastic PM phenotype. The PM changes induced by DHA supplementation potentiated osteogenic differentiation of MSCs concurrent with enhanced Akt activation at the PM. These observations prompt a model wherein the DHA-induced lipidome leads to more stable membrane microdomains, which serve to increase Akt activity and thereby enhance osteogenic differentiation. More broadly, our investigations suggest a general mechanism by which dietary fats affect cellular physiology through remodeling of membrane lipidomes, biophysical properties, and signaling. PMID:29134198

Monocyte Activation in Immunopathology: Cellular Test for Development of Diagnostics and Therapy.

PubMed

Ivanova, Ekaterina A; Orekhov, Alexander N

2016-01-01

Several highly prevalent human diseases are associated with immunopathology. Alterations in the immune system are found in such life-threatening disorders as cancer and atherosclerosis. Monocyte activation followed by macrophage polarization is an important step in normal immune response to pathogens and other relevant stimuli. Depending on the nature of the activation signal, macrophages can acquire pro- or anti-inflammatory phenotypes that are characterized by the expression of distinct patterns of secreted cytokines and surface antigens. This process is disturbed in immunopathologies resulting in abnormal monocyte activation and/or bias of macrophage polarization towards one or the other phenotype. Such alterations could be used as important diagnostic markers and also as possible targets for the development of immunomodulating therapy. Recently developed cellular tests are designed to analyze the phenotype and activity of living cells circulating in patient's bloodstream. Monocyte/macrophage activation test is a successful example of cellular test relevant for atherosclerosis and oncopathology. This test demonstrated changes in macrophage activation in subclinical atherosclerosis and breast cancer and could also be used for screening a panel of natural agents with immunomodulatory activity. Further development of cellular tests will allow broadening the scope of their clinical implication. Such tests may become useful tools for drug research and therapy optimization.

Genetic ablation of calcium-independent phospholipase A2gamma leads to alterations in mitochondrial lipid metabolism and function resulting in a deficient mitochondrial bioenergetic phenotype.

PubMed

Mancuso, David J; Sims, Harold F; Han, Xianlin; Jenkins, Christopher M; Guan, Shao Ping; Yang, Kui; Moon, Sung Ho; Pietka, Terri; Abumrad, Nada A; Schlesinger, Paul H; Gross, Richard W

2007-11-30

Previously, we identified a novel calcium-independent phospholipase, designated calcium-independent phospholipase A(2) gamma (iPLA(2)gamma), which possesses dual mitochondrial and peroxisomal subcellular localization signals. To identify the roles of iPLA(2)gamma in cellular bioenergetics, we generated mice null for the iPLA(2)gamma gene by eliminating the active site of the enzyme through homologous recombination. Mice null for iPLA(2)gamma display multiple bioenergetic dysfunctional phenotypes, including 1) growth retardation, 2) cold intolerance, 3) reduced exercise endurance, 4) greatly increased mortality from cardiac stress after transverse aortic constriction, 5) abnormal mitochondrial function with a 65% decrease in ascorbate-induced Complex IV-mediated oxygen consumption, and 6) a reduction in myocardial cardiolipin content accompanied by an altered cardiolipin molecular species composition. We conclude that iPLA(2)gamma is essential for maintaining efficient bioenergetic mitochondrial function through tailoring mitochondrial membrane lipid metabolism and composition.

The Flint Animal Cancer Center (FACC) Canine Tumour Cell Line Panel: a resource for veterinary drug discovery, comparative oncology and translational medicine.

PubMed

Fowles, J S; Dailey, D D; Gustafson, D L; Thamm, D H; Duval, D L

2017-06-01

Mammalian cell tissue culture has been a critical tool leading to our current understanding of cancer including many aspects of cellular transformation, growth and response to therapies. The current use of large panels of cell lines with associated phenotypic and genotypic information now allows for informatics approaches and in silico screens to rapidly test hypotheses based on simple as well as complex relationships. Current cell line panels with large amounts of associated drug sensitivity and genomics data are comprised of human cancer cell lines (i.e. NCI60 and GDSC). There is increased recognition of the contribution of canine cancer to comparative cancer research as a spontaneous large animal model with application in basic and translational studies. We have assembled a panel of canine cancer cell lines to facilitate studies in canine cancer and report here phenotypic and genotypic data associated with these cells. © 2016 John Wiley & Sons Ltd.

Discovery and validation of sub-threshold genome-wide association study loci using epigenomic signatures

PubMed Central

Wang, Xinchen; Tucker, Nathan R; Rizki, Gizem; Mills, Robert; Krijger, Peter HL; de Wit, Elzo; Subramanian, Vidya; Bartell, Eric; Nguyen, Xinh-Xinh; Ye, Jiangchuan; Leyton-Mange, Jordan; Dolmatova, Elena V; van der Harst, Pim; de Laat, Wouter; Ellinor, Patrick T; Newton-Cheh, Christopher; Milan, David J; Kellis, Manolis; Boyer, Laurie A

2016-01-01

Genetic variants identified by genome-wide association studies explain only a modest proportion of heritability, suggesting that meaningful associations lie 'hidden' below current thresholds. Here, we integrate information from association studies with epigenomic maps to demonstrate that enhancers significantly overlap known loci associated with the cardiac QT interval and QRS duration. We apply functional criteria to identify loci associated with QT interval that do not meet genome-wide significance and are missed by existing studies. We demonstrate that these 'sub-threshold' signals represent novel loci, and that epigenomic maps are effective at discriminating true biological signals from noise. We experimentally validate the molecular, gene-regulatory, cellular and organismal phenotypes of these sub-threshold loci, demonstrating that most sub-threshold loci have regulatory consequences and that genetic perturbation of nearby genes causes cardiac phenotypes in mouse. Our work provides a general approach for improving the detection of novel loci associated with complex human traits. DOI: http://dx.doi.org/10.7554/eLife.10557.001 PMID:27162171

De novo missense mutations in the NAA10 gene cause severe non-syndromic developmental delay in males and females

PubMed Central

Popp, Bernt; Støve, Svein I; Endele, Sabine; Myklebust, Line M; Hoyer, Juliane; Sticht, Heinrich; Azzarello-Burri, Silvia; Rauch, Anita; Arnesen, Thomas; Reis, André

2015-01-01

Recent studies revealed the power of whole-exome sequencing to identify mutations in sporadic cases with non-syndromic intellectual disability. We now identified de novo missense variants in NAA10 in two unrelated individuals, a boy and a girl, with severe global developmental delay but without any major dysmorphism by trio whole-exome sequencing. Both de novo variants were predicted to be deleterious, and we excluded other variants in this gene. This X-linked gene encodes N-alpha-acetyltransferase 10, the catalytic subunit of the NatA complex involved in multiple cellular processes. A single hypomorphic missense variant p.(Ser37Pro) was previously associated with Ogden syndrome in eight affected males from two different families. This rare disorder is characterized by a highly recognizable phenotype, global developmental delay and results in death during infancy. In an attempt to explain the discrepant phenotype, we used in vitro N-terminal acetylation assays which suggested that the severity of the phenotype correlates with the remaining catalytic activity. The variant in the Ogden syndrome patients exhibited a lower activity than the one seen in the boy with intellectual disability, while the variant in the girl was the most severe exhibiting only residual activity in the acetylation assays used. We propose that N-terminal acetyltransferase deficiency is clinically heterogeneous with the overall catalytic activity determining the phenotypic severity. PMID:25099252

Astrocytes acquire resistance to iron-dependent oxidative stress upon proinflammatory activation

PubMed Central

2013-01-01

Background Astrocytes respond to local insults within the brain and the spinal cord with important changes in their phenotype. This process, overall known as “activation”, is observed upon proinflammatory stimulation and leads astrocytes to acquire either a detrimental phenotype, thereby contributing to the neurodegenerative process, or a protective phenotype, thus supporting neuronal survival. Within the mechanisms responsible for inflammatory neurodegeneration, oxidative stress plays a major role and has recently been recognized to be heavily influenced by changes in cytosolic iron levels. In this work, we investigated how activation affects the competence of astrocytes to handle iron overload and the ensuing oxidative stress. Methods Cultures of pure cortical astrocytes were preincubated with proinflammatory cytokines (interleukin-1β and tumor necrosis factor α) or conditioned medium from lipopolysaccharide-activated microglia to promote activation and then exposed to a protocol of iron overload. Results We demonstrate that activated astrocytes display an efficient protection against iron-mediated oxidative stress and cell death. Based on this evidence, we performed a comprehensive biochemical and molecular analysis, including a transcriptomic approach, to identify the molecular basis of this resistance. Conclusions We propose the protective phenotype acquired after activation not to involve the most common astrocytic antioxidant pathway, based on the Nrf2 transcription factor, but to result from a complex change in the expression and activity of several genes involved in the control of cellular redox state. PMID:24160637

CHLORPYRIFOS DEVELOPMENTAL NEUROTOXICITY: INTERACTION WITH GLUCOCORTICOIDS IN PC12 CELLS

PubMed Central

Slotkin, Theodore A.; Card, Jennifer; Seidler, Frederic J.

2012-01-01

Prenatal coexposures to glucocorticoids and organophosphate pesticides are widespread. Glucocorticoids are elevated by maternal stress and are commonly given in preterm labor; organophosphate exposures are virtually ubiquitous. We used PC12 cells undergoing neurodifferentiation in order to assess whether dexamethasone enhances the developmental neurotoxicity of chlorpyrifos, focusing on concentrations relevant to human exposures. By themselves, each agent reduced the number of cells and the combined exposure elicited a correspondingly greater effect than with either agent alone. There was no general cytotoxicity, as cell growth was actually enhanced, and again, the combined treatment evoked greater cellular hypertrophy than with the individual compounds. The effects on neurodifferentiation were more complex. Chlorpyrifos alone had a promotional effect on neuri to genesis whereas dexamethasone impaired it; combined treatment showed an overall impairment greater than that seen with dexamethasone alone. The effect of chlorpyrifos on differentiation into specific neurotransmitter phenotypes was shifted by dexamethasone. Either agent alone promoted differentiation into the dopaminergic phenotype at the expense of the cholinergic phenotype. However, in dexamethasone-primed cells, chlorpyrifos actually enhanced cholinergic neurodifferentiation instead of suppressing this phenotype. Our results indicate that developmental exposure to glucocorticoids, either in the context of stress or the therapy of preterm labor, could enhance the developmental neurotoxicity of organophosphates and potentially of other neurotoxicants, as well as producing neurobehavioral outcomes distinct from those seen with either individual agent. PMID:22796634

Life is determined by its environment

NASA Astrophysics Data System (ADS)

Torday, John S.; Miller, William B.

2016-10-01

A well-developed theory of evolutionary biology requires understanding of the origins of life on Earth. However, the initial conditions (ontology) and causal (epistemology) bases on which physiology proceeded have more recently been called into question, given the teleologic nature of Darwinian evolutionary thinking. When evolutionary development is focused on cellular communication, a distinctly different perspective unfolds. The cellular communicative-molecular approach affords a logical progression for the evolutionary narrative based on the basic physiologic properties of the cell. Critical to this appraisal is recognition of the cell as a fundamental reiterative unit of reciprocating communication that receives information from and reacts to epiphenomena to solve problems. Following the course of vertebrate physiology from its unicellular origins instead of its overt phenotypic appearances and functional associations provides a robust, predictive picture for the means by which complex physiology evolved from unicellular organisms. With this foreknowledge of physiologic principles, we can determine the fundamentals of Physiology based on cellular first principles using a logical, predictable method. Thus, evolutionary creativity on our planet can be viewed as a paradoxical product of boundary conditions that permit homeostatic moments of varying length and amplitude that can productively absorb a variety of epigenetic impacts to meet environmental challenges.

Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (Ce3D)

PubMed Central

Li, Weizhe; Germain, Ronald N.

2017-01-01

Organ homeostasis, cellular differentiation, signal relay, and in situ function all depend on the spatial organization of cells in complex tissues. For this reason, comprehensive, high-resolution mapping of cell positioning, phenotypic identity, and functional state in the context of macroscale tissue structure is critical to a deeper understanding of diverse biological processes. Here we report an easy to use method, clearing-enhanced 3D (Ce3D), which generates excellent tissue transparency for most organs, preserves cellular morphology and protein fluorescence, and is robustly compatible with antibody-based immunolabeling. This enhanced signal quality and capacity for extensive probe multiplexing permits quantitative analysis of distinct, highly intermixed cell populations in intact Ce3D-treated tissues via 3D histo-cytometry. We use this technology to demonstrate large-volume, high-resolution microscopy of diverse cell types in lymphoid and nonlymphoid organs, as well as to perform quantitative analysis of the composition and tissue distribution of multiple cell populations in lymphoid tissues. Combined with histo-cytometry, Ce3D provides a comprehensive strategy for volumetric quantitative imaging and analysis that bridges the gap between conventional section imaging and disassociation-based techniques. PMID:28808033

Mitochondrial peptides modulate mitochondrial function during cellular senescence.

PubMed

Kim, Su-Jeong; Mehta, Hemal H; Wan, Junxiang; Kuehnemann, Chisaka; Chen, Jingcheng; Hu, Ji-Fan; Hoffman, Andrew R; Cohen, Pinchas

2018-06-10

Cellular senescence is a complex cell fate response that is thought to underlie several age-related pathologies. Despite a loss of proliferative potential, senescent cells are metabolically active and produce energy-consuming effectors, including senescence-associated secretory phenotypes (SASPs). Mitochondria play crucial roles in energy production and cellular signaling, but the key features of mitochondrial physiology and particularly of mitochondria-derived peptides (MDPs), remain underexplored in senescence responses. Here, we used primary human fibroblasts made senescent by replicative exhaustion, doxorubicin or hydrogen peroxide treatment, and examined the number of mitochondria and the levels of mitochondrial respiration, mitochondrial DNA methylation and the mitochondria-encoded peptides humanin, MOTS-c, SHLP2 and SHLP6. Senescent cells showed increased numbers of mitochondria and higher levels of mitochondrial respiration, variable changes in mitochondrial DNA methylation, and elevated levels of humanin and MOTS-c. Humanin and MOTS-c administration modestly increased mitochondrial respiration and selected components of the SASP in doxorubicin-induced senescent cells partially via JAK pathway. Targeting metabolism in senescence cells is an important strategy to reduce SASP production for eliminating the deleterious effects of senescence. These results provide insight into the role of MDPs in mitochondrial energetics and the production of SASP components by senescent cells.

Life is determined by its environment

PubMed Central

Torday, John S.; Miller, William B.

2016-01-01

A well-developed theory of evolutionary biology requires understanding of the origins of life on Earth. However, the initial conditions (ontology) and causal (epistemology) bases on which physiology proceeded have more recently been called into question, given the teleologic nature of Darwinian evolutionary thinking. When evolutionary development is focused on cellular communication, a distinctly different perspective unfolds. The cellular communicative-molecular approach affords a logical progression for the evolutionary narrative based on the basic physiologic properties of the cell. Critical to this appraisal is recognition of the cell as a fundamental reiterative unit of reciprocating communication that receives information from and reacts to epiphenomena to solve problems. Following the course of vertebrate physiology from its unicellular origins instead of its overt phenotypic appearances and functional associations provides a robust, predictive picture for the means by which complex physiology evolved from unicellular organisms. With this foreknowledge of physiologic principles, we can determine the fundamentals of Physiology based on cellular first principles using a logical, predictable method. Thus, evolutionary creativity on our planet can be viewed as a paradoxical product of boundary conditions that permit homeostatic moments of varying length and amplitude that can productively absorb a variety of epigenetic impacts to meet environmental challenges. PMID:27708547

New advances in probing cell–extracellular matrix interactions

PubMed Central

2017-01-01

The extracellular matrix (ECM) provides structural and biochemical support to cells within tissues. An emerging body of evidence has established that the ECM plays a key role in cell mechanotransduction – the study of coupling between mechanical inputs and cellular phenotype – through either mediating transmission of forces to the cells, or presenting mechanical cues that guide cellular behaviors. Recent progress in cell mechanotransduction research has been facilitated by advances of experimental tools, particularly microtechnologies, engineered biomaterials, and imaging and analytical methods. Microtechnologies have enabled the design and fabrication of controlled physical microenvironments for the study and measurement of cell–ECM interactions. Advances in engineered biomaterials have allowed researchers to develop synthetic ECMs that mimic tissue microenvironments and investigate the impact of altered physicochemical properties on various cellular processes. Finally, advanced imaging and spectroscopy techniques have facilitated the visualization of the complex interaction between cells and ECM in vitro and in living tissues. This review will highlight the application of recent innovations in these areas to probing cell–ECM interactions. We believe cross-disciplinary approaches, combining aspects of the different technologies reviewed here, will inspire innovative ideas to further elucidate the secrets of ECM-mediated cell control. PMID:28352896

Automatic Segmentation of High-Throughput RNAi Fluorescent Cellular Images

PubMed Central

Yan, Pingkum; Zhou, Xiaobo; Shah, Mubarak; Wong, Stephen T. C.

2010-01-01

High-throughput genome-wide RNA interference (RNAi) screening is emerging as an essential tool to assist biologists in understanding complex cellular processes. The large number of images produced in each study make manual analysis intractable; hence, automatic cellular image analysis becomes an urgent need, where segmentation is the first and one of the most important steps. In this paper, a fully automatic method for segmentation of cells from genome-wide RNAi screening images is proposed. Nuclei are first extracted from the DNA channel by using a modified watershed algorithm. Cells are then extracted by modeling the interaction between them as well as combining both gradient and region information in the Actin and Rac channels. A new energy functional is formulated based on a novel interaction model for segmenting tightly clustered cells with significant intensity variance and specific phenotypes. The energy functional is minimized by using a multiphase level set method, which leads to a highly effective cell segmentation method. Promising experimental results demonstrate that automatic segmentation of high-throughput genome-wide multichannel screening can be achieved by using the proposed method, which may also be extended to other multichannel image segmentation problems. PMID:18270043

Recursive expectation-maximization clustering: A method for identifying buffering mechanisms composed of phenomic modules

NASA Astrophysics Data System (ADS)

Guo, Jingyu; Tian, Dehua; McKinney, Brett A.; Hartman, John L.

2010-06-01

Interactions between genetic and/or environmental factors are ubiquitous, affecting the phenotypes of organisms in complex ways. Knowledge about such interactions is becoming rate-limiting for our understanding of human disease and other biological phenomena. Phenomics refers to the integrative analysis of how all genes contribute to phenotype variation, entailing genome and organism level information. A systems biology view of gene interactions is critical for phenomics. Unfortunately the problem is intractable in humans; however, it can be addressed in simpler genetic model systems. Our research group has focused on the concept of genetic buffering of phenotypic variation, in studies employing the single-cell eukaryotic organism, S. cerevisiae. We have developed a methodology, quantitative high throughput cellular phenotyping (Q-HTCP), for high-resolution measurements of gene-gene and gene-environment interactions on a genome-wide scale. Q-HTCP is being applied to the complete set of S. cerevisiae gene deletion strains, a unique resource for systematically mapping gene interactions. Genetic buffering is the idea that comprehensive and quantitative knowledge about how genes interact with respect to phenotypes will lead to an appreciation of how genes and pathways are functionally connected at a systems level to maintain homeostasis. However, extracting biologically useful information from Q-HTCP data is challenging, due to the multidimensional and nonlinear nature of gene interactions, together with a relative lack of prior biological information. Here we describe a new approach for mining quantitative genetic interaction data called recursive expectation-maximization clustering (REMc). We developed REMc to help discover phenomic modules, defined as sets of genes with similar patterns of interaction across a series of genetic or environmental perturbations. Such modules are reflective of buffering mechanisms, i.e., genes that play a related role in the maintenance of physiological homeostasis. To develop the method, 297 gene deletion strains were selected based on gene-drug interactions with hydroxyurea, an inhibitor of ribonucleotide reductase enzyme activity, which is critical for DNA synthesis. To partition the gene functions, these 297 deletion strains were challenged with growth inhibitory drugs known to target different genes and cellular pathways. Q-HTCP-derived growth curves were used to quantify all gene interactions, and the data were used to test the performance of REMc. Fundamental advantages of REMc include objective assessment of total number of clusters and assignment to each cluster a log-likelihood value, which can be considered an indicator of statistical quality of clusters. To assess the biological quality of clusters, we developed a method called gene ontology information divergence z-score (GOid_z). GOid_z summarizes total enrichment of GO attributes within individual clusters. Using these and other criteria, we compared the performance of REMc to hierarchical and K-means clustering. The main conclusion is that REMc provides distinct efficiencies for mining Q-HTCP data. It facilitates identification of phenomic modules, which contribute to buffering mechanisms that underlie cellular homeostasis and the regulation of phenotypic expression.

Signalling and the control of skeletal muscle size

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

Otto, Anthony; Patel, Ketan, E-mail: ketan.patel@reading.ac.uk

2010-11-01

Skeletal muscle is highly adaptive to environmental stimuli and can alter its mass accordingly. This tissue is almost unique in that it can increase its size through two distinct mechanisms. It can grow through a cellular process mediated by cell fusion, or it can increase its size simply by increasing its protein content. Understanding how these processes are regulated is crucial for the development of potential therapies against debilitating skeletal muscle wasting diseases. Two key signalling molecules, Insulin like Growth Factor (IGF) and GDF-8/myostatin, have emerged in recent years to be potent regulators of skeletal muscle size. In this reviewmore » we bring together recent data highlighting the important and novel aspects of both molecules and their signalling pathways, culminating in a discussion of the cellular and tissue phenotypic outcomes of their stimulation or antagonism. We emphasise the complex regulatory mechanisms and discuss the temporal and spatial differences that control their action, understanding of which is crucial to further their use as potential therapeutic targets.« less

Petri net-based method for the analysis of the dynamics of signal propagation in signaling pathways.

PubMed

Hardy, Simon; Robillard, Pierre N

2008-01-15

Cellular signaling networks are dynamic systems that propagate and process information, and, ultimately, cause phenotypical responses. Understanding the circuitry of the information flow in cells is one of the keys to understanding complex cellular processes. The development of computational quantitative models is a promising avenue for attaining this goal. Not only does the analysis of the simulation data based on the concentration variations of biological compounds yields information about systemic state changes, but it is also very helpful for obtaining information about the dynamics of signal propagation. This article introduces a new method for analyzing the dynamics of signal propagation in signaling pathways using Petri net theory. The method is demonstrated with the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) regulation network. The results constitute temporal information about signal propagation in the network, a simplified graphical representation of the network and of the signal propagation dynamics and a characterization of some signaling routes as regulation motifs.

Module Based Complexity Formation: Periodic Patterning in Feathers and Hairs

PubMed Central

Chuong, Cheng-Ming; Yeh, Chao-Yuan; Jiang, Ting-Xin; Widelitz, Randall

2012-01-01

Patterns describe order which emerges from homogeneity. Complex patterns on the integument are striking because of their visibility throughout an organism's lifespan. Periodic patterning is an effective design because the ensemble of hair or feather follicles (modules) allows the generation of complexity, including regional variations and cyclic regeneration, giving the skin appendages a new lease on life. Spatial patterns include the arrangements of feathers and hairs in specified number, size, and spacing. We explore how a field of equivalent progenitor cells can generate periodically arranged modules based on genetic information, physical-chemical rules and developmental timing. Reconstitution experiments suggest a competitive equilibrium regulated by activators / inhibitors involving Turing reaction-diffusion. Temporal patterns result from oscillating stem cell activities within each module (micro-environment regulation), reflected as growth (anagen) and resting (telogen) phases during the cycling of feather and hair follicles. Stimulating modules with activators initiates the spread of regenerative hair waves, while global inhibitors outside each module (macro-environment) prevent this. Different wave patterns can be simulated by Cellular Automata principles. Hormonal status and seasonal changes can modulate appendage phenotypes, leading to “organ metamorphosis”, with multiple ectodermal organ phenotypes generated from the same precursors. We discuss potential evolutionary novel steps using this module based complexity in several amniote integument organs, exemplified by the spectacular peacock feather pattern. We thus explore the application of the acquired knowledge of patterning in tissue engineering. New hair follicles can be generated after wounding. Hairs and feathers can be reconstituted through self-organization of dissociated progenitor cells. PMID:23539312

Module-based complexity formation: periodic patterning in feathers and hairs.

PubMed

Chuong, Cheng-Ming; Yeh, Chao-Yuan; Jiang, Ting-Xin; Widelitz, Randall

2013-01-01

Patterns describe order which emerges from homogeneity. Complex patterns on the integument are striking because of their visibility throughout an organism’s lifespan. Periodic patterning is an effective design because the ensemble of hair or feather follicles (modules) allows the generation of complexity, including regional variations and cyclic regeneration, giving the skin appendages a new lease on life. Spatial patterns include the arrangements of feathers and hairs in specific number, size, and spacing.We explorehowa field of equivalent progenitor cells can generate periodically arranged modules based on genetic information, physical–chemical rules and developmental timing. Reconstitution experiments suggest a competitive equilibrium regulated by activators/inhibitors involving Turing reaction-diffusion. Temporal patterns result from oscillating stem cell activities within each module (microenvironment regulation), reflected as growth (anagen) and resting (telogen) phases during the cycling of feather and hair follicles. Stimulating modules with activators initiates the spread of regenerative hair waves, while global inhibitors outside each module (macroenvironment) prevent this. Different wave patterns can be simulated by cellular automata principles. Hormonal status and seasonal changes can modulate appendage phenotypes, leading to ‘organ metamorphosis’, with multiple ectodermal organ phenotypes generated from the same precursors. We discuss potential novel evolutionary steps using this module-based complexity in several amniote integument organs, exemplified by the spectacular peacock feather pattern. We thus explore the application of the acquired knowledge of patterning in tissue engineering. New hair follicles can be generated after wounding. Hairs and feathers can be reconstituted through self-organization of dissociated progenitor cells. © 2012 Wiley Periodicals, Inc.

Predictive genomics: a cancer hallmark network framework for predicting tumor clinical phenotypes using genome sequencing data.

PubMed

Wang, Edwin; Zaman, Naif; Mcgee, Shauna; Milanese, Jean-Sébastien; Masoudi-Nejad, Ali; O'Connor-McCourt, Maureen

2015-02-01

Tumor genome sequencing leads to documenting thousands of DNA mutations and other genomic alterations. At present, these data cannot be analyzed adequately to aid in the understanding of tumorigenesis and its evolution. Moreover, we have little insight into how to use these data to predict clinical phenotypes and tumor progression to better design patient treatment. To meet these challenges, we discuss a cancer hallmark network framework for modeling genome sequencing data to predict cancer clonal evolution and associated clinical phenotypes. The framework includes: (1) cancer hallmarks that can be represented by a few molecular/signaling networks. 'Network operational signatures' which represent gene regulatory logics/strengths enable to quantify state transitions and measures of hallmark traits. Thus, sets of genomic alterations which are associated with network operational signatures could be linked to the state/measure of hallmark traits. The network operational signature transforms genotypic data (i.e., genomic alterations) to regulatory phenotypic profiles (i.e., regulatory logics/strengths), to cellular phenotypic profiles (i.e., hallmark traits) which lead to clinical phenotypic profiles (i.e., a collection of hallmark traits). Furthermore, the framework considers regulatory logics of the hallmark networks under tumor evolutionary dynamics and therefore also includes: (2) a self-promoting positive feedback loop that is dominated by a genomic instability network and a cell survival/proliferation network is the main driver of tumor clonal evolution. Surrounding tumor stroma and its host immune systems shape the evolutionary paths; (3) cell motility initiating metastasis is a byproduct of the above self-promoting loop activity during tumorigenesis; (4) an emerging hallmark network which triggers genome duplication dominates a feed-forward loop which in turn could act as a rate-limiting step for tumor formation; (5) mutations and other genomic alterations have specific patterns and tissue-specificity, which are driven by aging and other cancer-inducing agents. This framework represents the logics of complex cancer biology as a myriad of phenotypic complexities governed by a limited set of underlying organizing principles. It therefore adds to our understanding of tumor evolution and tumorigenesis, and moreover, potential usefulness of predicting tumors' evolutionary paths and clinical phenotypes. Strategies of using this framework in conjunction with genome sequencing data in an attempt to predict personalized drug targets, drug resistance, and metastasis for cancer patients, as well as cancer risks for healthy individuals are discussed. Accurate prediction of cancer clonal evolution and clinical phenotypes will have substantial impact on timely diagnosis, personalized treatment and personalized prevention of cancer. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

Integrating Genomic Analysis with the Genetic Basis of Gene Expression: Preliminary Evidence of the Identification of Causal Genes for Cardiovascular and Metabolic Traits Related to Nutrition in Mexicans123

PubMed Central

Bastarrachea, Raúl A.; Gallegos-Cabriales, Esther C.; Nava-González, Edna J.; Haack, Karin; Voruganti, V. Saroja; Charlesworth, Jac; Laviada-Molina, Hugo A.; Veloz-Garza, Rosa A.; Cardenas-Villarreal, Velia Margarita; Valdovinos-Chavez, Salvador B.; Gomez-Aguilar, Patricia; Meléndez, Guillermo; López-Alvarenga, Juan Carlos; Göring, Harald H. H.; Cole, Shelley A.; Blangero, John; Comuzzie, Anthony G.; Kent, Jack W.

2012-01-01

Whole-transcriptome expression profiling provides novel phenotypes for analysis of complex traits. Gene expression measurements reflect quantitative variation in transcript-specific messenger RNA levels and represent phenotypes lying close to the action of genes. Understanding the genetic basis of gene expression will provide insight into the processes that connect genotype to clinically significant traits representing a central tenet of system biology. Synchronous in vivo expression profiles of lymphocytes, muscle, and subcutaneous fat were obtained from healthy Mexican men. Most genes were expressed at detectable levels in multiple tissues, and RNA levels were correlated between tissue types. A subset of transcripts with high reliability of expression across tissues (estimated by intraclass correlation coefficients) was enriched for cis-regulated genes, suggesting that proximal sequence variants may influence expression similarly in different cellular environments. This integrative global gene expression profiling approach is proving extremely useful for identifying genes and pathways that contribute to complex clinical traits. Clearly, the coincidence of clinical trait quantitative trait loci and expression quantitative trait loci can help in the prioritization of positional candidate genes. Such data will be crucial for the formal integration of positional and transcriptomic information characterized as genetical genomics. PMID:22797999

Order, Disorder, Death: Lessons from a Superorganism

PubMed Central

Amdam, Gro V.; Seehuu, Siri-Christine

2008-01-01

Animal models contribute to the understanding of molecular mechanism of cancer, revealing complex roles of altered cellular-signaling networks and deficient surveillance systems. Analogous pathologies are documented in an unconventional model organism that receives attention in research on systems theory, evolution, and aging. The honeybee (Apis mellifera) colony is an advanced integrative unit, a “superorganism” in which order is controlled via complex signaling cascades and surveillance schemes. A facultatively sterile caste, the workers, regulates patterns of growth, differentiation, homeostasis, and death. Workers differentiate into temporal phenotypes in response to dynamic social cues; chemosensory signals that can translate into dramatic physiological responses, including programmed cell death. Temporal worker forms function together, and effectively identify and terminate abnormal colony members ranging from embryos to adults. As long as this regulatory system is operational at a colony level, the unit survives and propagates. However, if the worker phenotypes that collectively govern order become too few or change into malignant forms that bypass control mechanisms to replicate aberrantly; order is replaced by disorder that ultimately leads to the destruction of the society. In this chapter we describe fundamental properties of honeybee social organization, and explore conditions that lead to states of disorder. Our hope is that this chapter will be an inspirational source for ongoing and future work in the field of cancer research. PMID:16860655

Estimating genetic and phenotypic parameters of cellular immune-associated traits in dairy cows.

PubMed

Denholm, Scott J; McNeilly, Tom N; Banos, Georgios; Coffey, Mike P; Russell, George C; Bagnall, Ainsley; Mitchell, Mairi C; Wall, Eileen

2017-04-01

Data collected from an experimental Holstein-Friesian research herd were used to determine genetic and phenotypic parameters of innate and adaptive cellular immune-associated traits. Relationships between immune-associated traits and production, health, and fertility traits were also investigated. Repeated blood leukocyte records were analyzed in 546 cows for 9 cellular immune-associated traits, including percent T cell subsets, B cells, NK cells, and granulocytes. Variance components were estimated by univariate analysis. Heritability estimates were obtained for all 9 traits, the highest of which were observed in the T cell subsets percent CD4 + , percent CD8 + , CD4 + :CD8 + ratio, and percent NKp46 + cells (0.46, 0.41, 0.43 and 0.42, respectively), with between-individual variation accounting for 59 to 81% of total phenotypic variance. Associations between immune-associated traits and production, health, and fertility traits were investigated with bivariate analyses. Strong genetic correlations were observed between percent NKp46 + and stillbirth rate (0.61), and lameness episodes and percent CD8 + (-0.51). Regarding production traits, the strongest relationships were between CD4 + :CD8 + ratio and weight phenotypes (-0.52 for live weight; -0.51 for empty body weight). Associations between feed conversion traits and immune-associated traits were also observed. Our results provide evidence that cellular immune-associated traits are heritable and repeatable, and the noticeable variation between animals would permit selection for altered trait values, particularly in the case of the T cell subsets. The associations we observed between immune-associated, health, fertility, and production traits suggest that genetic selection for cellular immune-associated traits could provide a useful tool in improving animal health, fitness, and fertility. The Authors. Published by the Federation of Animal Science Societies and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY 2.0 license (http://creativecommons.org/licenses/by/2.0/).

A novel method for identifying disease associated protein complexes based on functional similarity protein complex networks.

PubMed

Le, Duc-Hau

2015-01-01

Protein complexes formed by non-covalent interaction among proteins play important roles in cellular functions. Computational and purification methods have been used to identify many protein complexes and their cellular functions. However, their roles in terms of causing disease have not been well discovered yet. There exist only a few studies for the identification of disease-associated protein complexes. However, they mostly utilize complicated heterogeneous networks which are constructed based on an out-of-date database of phenotype similarity network collected from literature. In addition, they only apply for diseases for which tissue-specific data exist. In this study, we propose a method to identify novel disease-protein complex associations. First, we introduce a framework to construct functional similarity protein complex networks where two protein complexes are functionally connected by either shared protein elements, shared annotating GO terms or based on protein interactions between elements in each protein complex. Second, we propose a simple but effective neighborhood-based algorithm, which yields a local similarity measure, to rank disease candidate protein complexes. Comparing the predictive performance of our proposed algorithm with that of two state-of-the-art network propagation algorithms including one we used in our previous study, we found that it performed statistically significantly better than that of these two algorithms for all the constructed functional similarity protein complex networks. In addition, it ran about 32 times faster than these two algorithms. Moreover, our proposed method always achieved high performance in terms of AUC values irrespective of the ways to construct the functional similarity protein complex networks and the used algorithms. The performance of our method was also higher than that reported in some existing methods which were based on complicated heterogeneous networks. Finally, we also tested our method with prostate cancer and selected the top 100 highly ranked candidate protein complexes. Interestingly, 69 of them were evidenced since at least one of their protein elements are known to be associated with prostate cancer. Our proposed method, including the framework to construct functional similarity protein complex networks and the neighborhood-based algorithm on these networks, could be used for identification of novel disease-protein complex associations.

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

Ha, Jung Min; Yun, Sung Ji; Kim, Young Whan

Mammalian target of rapamycin complex (mTORC) regulates various cellular processes including proliferation, growth, migration and differentiation. In this study, we showed that mTORC1 regulates platelet-derived growth factor (PDGF)-induced phenotypic conversion of vascular smooth muscle cells (VSMCs). Stimulation of contractile VSMCs with PDGF significantly reduced the expression of contractile marker proteins in a time- and dose-dependent manner. In addition, angiotensin II (AngII)-induced contraction of VSMCs was completely blocked by the stimulation of VSMCs with PDGF. PDGF-dependent suppression of VSMC marker gene expression was significantly blocked by inhibition of phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and mTOR whereas inhibition of p38more » MAPK had no effect. In particular, inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked the PDGF-dependent phenotypic change of VSMCs whereas silencing of Rictor had no effect. In addition, loss of AngII-dependent contraction by PDGF was significantly retained by silencing of Raptor. Inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked PDGF-induced proliferation of VSMCs. Taken together, we suggest that mTORC1 plays an essential role in PDGF-dependent phenotypic changes of VSMCs. - Graphical abstract: Regulation of VSMC phenotype by PDGF-dependent activation of mTORC1. - Highlights: • The expression of contractile marker proteins was reduced by PDGF stimulation. • PDGF-dependent phenotypic conversion of VSMCs was blocked by inhibition of mTOR. • PDGF-induced proliferation of VSMCs was attenuated by inhibition of mTORC1. • mTORC1 plays a critical role in PDGF-dependent phenotypic conversion of VSMCs.« less

Systems Biology Perspectives on Minimal and Simpler Cells

PubMed Central

Xavier, Joana C.; Patil, Kiran Raosaheb

2014-01-01

SUMMARY The concept of the minimal cell has fascinated scientists for a long time, from both fundamental and applied points of view. This broad concept encompasses extreme reductions of genomes, the last universal common ancestor (LUCA), the creation of semiartificial cells, and the design of protocells and chassis cells. Here we review these different areas of research and identify common and complementary aspects of each one. We focus on systems biology, a discipline that is greatly facilitating the classical top-down and bottom-up approaches toward minimal cells. In addition, we also review the so-called middle-out approach and its contributions to the field with mathematical and computational models. Owing to the advances in genomics technologies, much of the work in this area has been centered on minimal genomes, or rather minimal gene sets, required to sustain life. Nevertheless, a fundamental expansion has been taking place in the last few years wherein the minimal gene set is viewed as a backbone of a more complex system. Complementing genomics, progress is being made in understanding the system-wide properties at the levels of the transcriptome, proteome, and metabolome. Network modeling approaches are enabling the integration of these different omics data sets toward an understanding of the complex molecular pathways connecting genotype to phenotype. We review key concepts central to the mapping and modeling of this complexity, which is at the heart of research on minimal cells. Finally, we discuss the distinction between minimizing the number of cellular components and minimizing cellular complexity, toward an improved understanding and utilization of minimal and simpler cells. PMID:25184563

Single-Cell RNA Sequencing of Human T Cells.

PubMed

Villani, Alexandra-Chloé; Shekhar, Karthik

2017-01-01

Understanding how populations of human T cells leverage cellular heterogeneity, plasticity, and diversity to achieve a wide range of functional flexibility, particularly during dynamic processes such as development, differentiation, and antigenic response, is a core challenge that is well suited for single-cell analysis. Hypothesis-free evaluation of cellular states and subpopulations by transcriptional profiling of single T cells can identify relationships that may be obscured by targeted approaches such as FACS sorting on cell-surface antigens, or bulk expression analysis. While this approach is relevant to all cell types, it is of particular interest in the study of T cells for which classical phenotypic criteria are now viewed as insufficient for distinguishing different T cell subtypes and transitional states, and defining the changes associated with dysfunctional T cell states in autoimmunity and tumor-related exhaustion. This unit describes a protocol to generate single-cell transcriptomic libraries of human blood CD4 + and CD8 + T cells, and also introduces the basic bioinformatic steps to process the resulting sequence data for further computational analysis. We show how cellular subpopulations can be identified from transcriptional data, and derive characteristic gene expression signatures that distinguish these states. We believe single-cell RNA-seq is a powerful technique to study the cellular heterogeneity in complex tissues, a paradigm that will be of great value for the immune system.

Cyclin C influences the timing of mitosis in fission yeast

PubMed Central

Banyai, Gabor; Szilagyi, Zsolt; Baraznenok, Vera; Khorosjutina, Olga; Gustafsson, Claes M.

2017-01-01

The multiprotein Mediator complex is required for the regulated transcription of nearly all RNA polymerase II–dependent genes. Mediator contains the Cdk8 regulatory subcomplex, which directs periodic transcription and influences cell cycle progression in fission yeast. Here we investigate the role of CycC, the cognate cyclin partner of Cdk8, in cell cycle control. Previous reports suggested that CycC interacts with other cellular Cdks, but a fusion of CycC to Cdk8 reported here did not cause any obvious cell cycle phenotypes. We find that Cdk8 and CycC interactions are stabilized within the Mediator complex and the activity of Cdk8-CycC is regulated by other Mediator components. Analysis of a mutant yeast strain reveals that CycC, together with Cdk8, primarily affects M-phase progression but mutations that release Cdk8 from CycC control also affect timing of entry into S phase. PMID:28515143

Cryopreservation has no effect on function of natural killer cells differentiated in vitro from umbilical cord blood CD34(+) cells.

PubMed

Domogala, Anna; Madrigal, J Alejandro; Saudemont, Aurore

2016-06-01

Natural killer (NK) cells offer the potential for a powerful cellular immunotherapy because they can target malignant cells without being direct effectors of graft-versus-host disease. We have previously shown that high numbers of functional NK cells can be differentiated in vitro from umbilical cord blood (CB) CD34(+) cells. To develop a readily available, off-the-shelf cellular product, it is essential that NK cells differentiated in vitro can be frozen and thawed while maintaining the same phenotype and functions. We evaluated the phenotype and function of fresh and frozen NK cells differentiated in vitro. We also assessed whether the concentration of NK cells at the time of freezing had an impact on cell viability. We found that cell concentration of NK cells at the time of freezing did not have an impact on their viability and on cell recovery post-thaw. Moreover, freezing of differentiated NK cells in vitro did not affect their phenotype, cytotoxicity and degranulation capacity toward K562 cells, cytokine production and proliferation. We are therefore able to generate large numbers of functional NK cells from CB CD34(+) cells that maintain the same phenotype and function post-cryopreservation, which will allow for multiple infusions of a highly cytotoxic NK cell product. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Complex, multi-scale small intestinal topography replicated in cellular growth substrates fabricated via chemical vapor deposition of Parylene C.

PubMed

Koppes, Abigail N; Kamath, Megha; Pfluger, Courtney A; Burkey, Daniel D; Dokmeci, Mehmet; Wang, Lin; Carrier, Rebecca L

2016-08-22

Native small intestine possesses distinct multi-scale structures (e.g., crypts, villi) not included in traditional 2D intestinal culture models for drug delivery and regenerative medicine. The known impact of structure on cell function motivates exploration of the influence of intestinal topography on the phenotype of cultured epithelial cells, but the irregular, macro- to submicron-scale features of native intestine are challenging to precisely replicate in cellular growth substrates. Herein, we utilized chemical vapor deposition of Parylene C on decellularized porcine small intestine to create polymeric intestinal replicas containing biomimetic irregular, multi-scale structures. These replicas were used as molds for polydimethylsiloxane (PDMS) growth substrates with macro to submicron intestinal topographical features. Resultant PDMS replicas exhibit multiscale resolution including macro- to micro-scale folds, crypt and villus structures, and submicron-scale features of the underlying basement membrane. After 10 d of human epithelial colorectal cell culture on PDMS substrates, the inclusion of biomimetic topographical features enhanced alkaline phosphatase expression 2.3-fold compared to flat controls, suggesting biomimetic topography is important in induced epithelial differentiation. This work presents a facile, inexpensive method for precisely replicating complex hierarchal features of native tissue, towards a new model for regenerative medicine and drug delivery for intestinal disorders and diseases.

The Impact of the Emerging Genomics Data on the Management of Agerelated Phenotypes in the Context of Cellular Senescence.

PubMed

Montesanto, Alberto; Geracitano, Silvana; Garasto, Sabrina; Fusco, Sergio; Lattanzio, Fabrizia; Passarino, Giuseppe; Corsonello, Andrea

2016-01-01

Before the last decade, attempts to identify the genetic factors involved in the susceptibility to age-related complex diseases such as cardiovascular disease, diabetes and cancer had very limited success. Recently, two important advancements have provided new opportunities to improve our knowledge in this field. Firstly, it has emerged the concept of studying the molecular mechanisms underlying the age related decline of the organism (such as cellular senescence), rather than the genetics of single disorders. In addition, advances in DNA technology have uncovered an incredible number of common susceptibility variants for several complex traits. Despite these progresses, the translation of these discoveries into clinical practice has been very difficult. To date, several attempts in translating genomics to medicine are being carried out to look for the best way by which genomic discoveries may improve our understanding of fundamental issues in the prediction and prevention of some complex diseases. The successful strategy seems to be testing simultaneously multiple susceptibility variants in combination with traditional risk factors. In fact, such approach showed that genetic factors substantially improve the prediction of complex diseases especially for coronary heart disease and prostate cancer, making possible appropriate behavioural and medical interventions. In the future, the identification of new genetic variants and their inclusion into current risk profile models will probably improve the discrimination power of these models for other complex diseases such as type 2 diabetes mellitus and breast cancer. On the other hand, for traits with low heritability, this improvement will probably be negligible, and this will urge further researches on the role played by traditional and newly discovered non-genetic risk factors.

A Complex Interaction Between Reduced Reelin Expression and Prenatal Organophosphate Exposure Alters Neuronal Cell Morphology.

PubMed

Mullen, Brian R; Ross, Brennan; Chou, Joan Wang; Khankan, Rana; Khialeeva, Elvira; Bui, Kimberly; Carpenter, Ellen M

2016-06-01

Genetic and environmental factors are both likely to contribute to neurodevelopmental disorders including schizophrenia, autism spectrum disorders, and major depressive disorders. Prior studies from our laboratory and others have demonstrated that the combinatorial effect of two factors-reduced expression of reelin protein and prenatal exposure to the organophosphate pesticide chlorpyrifos oxon-gives rise to acute biochemical effects and to morphological and behavioral phenotypes in adolescent and young adult mice. In the current study, we examine the consequences of these factors on reelin protein expression and neuronal cell morphology in adult mice. While the cell populations that express reelin in the adult brain appear unchanged in location and distribution, the levels of full length and cleaved reelin protein show persistent reductions following prenatal exposure to chlorpyrifos oxon. Cell positioning and organization in the hippocampus and cerebellum are largely normal in animals with either reduced reelin expression or prenatal exposure to chlorpyrifos oxon, but cellular complexity and dendritic spine organization is altered, with a skewed distribution of immature dendritic spines in adult animals. Paradoxically, combinatorial exposure to both factors appears to generate a rescue of the dendritic spine phenotypes, similar to the mitigation of behavioral and morphological changes observed in our prior study. Together, our observations support an interaction between reelin expression and chlorpyrifos oxon exposure that is not simply additive, suggesting a complex interplay between genetic and environmental factors in regulating brain morphology. © The Author(s) 2016.

Mouse models of ageing and their relevance to disease.

PubMed

Kõks, Sulev; Dogan, Soner; Tuna, Bilge Guvenc; González-Navarro, Herminia; Potter, Paul; Vandenbroucke, Roosmarijn E

2016-12-01

Ageing is a process that gradually increases the organism's vulnerability to death. It affects different biological pathways, and the underlying cellular mechanisms are complex. In view of the growing disease burden of ageing populations, increasing efforts are being invested in understanding the pathways and mechanisms of ageing. We review some mouse models commonly used in studies on ageing, highlight the advantages and disadvantages of the different strategies, and discuss their relevance to disease susceptibility. In addition to addressing the genetics and phenotypic analysis of mice, we discuss examples of models of delayed or accelerated ageing and their modulation by caloric restriction. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

C. elegans network biology: a beginning.

PubMed Central

Piano, Fabio; Gunsalus, Kristin C; Hill, David E; Vidal, Marc

2006-01-01

The architecture and dynamics of molecular networks can provide an understanding of complex biological processes complementary to that obtained from the in-depth study of single genes and proteins. With a completely sequenced and well-annotated genome, a fully characterized cell lineage, and powerful tools available to dissect development, Caenorhabditis elegans, among metazoans, provides an optimal system to bridge cellular and organismal biology with the global properties of macromolecular networks. This chapter considers omic technologies available for C. elegans to describe molecular networks--encompassing transcriptional and phenotypic profiling as well as physical interaction mapping--and discusses how their individual and integrated applications are paving the way for a network-level understanding of C. elegans biology. PMID:18050437

Fine Tuning Gene Expression: The Epigenome

PubMed Central

Mohtat, Davoud; Susztak, Katalin

2011-01-01

An epigenetic trait is a stably inherited phenotype resulting from changes in a chromosome without alterations in the DNA sequence. Epigenetic modifications, such as; DNA methylation, together with covalent modification of histones, are thought to alter chromatin density and accessibility of the DNA to cellular machinery, thereby modulating the transcriptional potential of the underlying DNA sequence. As epigenetic marks under environmental influence, epigenetics provides an added layer of variation that might mediate the relationship between genotype and internal and external environmental factors. Integration of our knowledge in genetics, epigenomics and genomics with the use of systems biology tools may present investigators with new powerful tools to study many complex human diseases such as kidney disease. PMID:21044758

Profiling protein function with small molecule microarrays

PubMed Central

Winssinger, Nicolas; Ficarro, Scott; Schultz, Peter G.; Harris, Jennifer L.

2002-01-01

The regulation of protein function through posttranslational modification, local environment, and protein–protein interaction is critical to cellular function. The ability to analyze on a genome-wide scale protein functional activity rather than changes in protein abundance or structure would provide important new insights into complex biological processes. Herein, we report the application of a spatially addressable small molecule microarray to an activity-based profile of proteases in crude cell lysates. The potential of this small molecule-based profiling technology is demonstrated by the detection of caspase activation upon induction of apoptosis, characterization of the activated caspase, and inhibition of the caspase-executed apoptotic phenotype using the small molecule inhibitor identified in the microarray-based profile. PMID:12167675

Endothelial-regenerating cells: an expanding universe.

PubMed

Steinmetz, Martin; Nickenig, Georg; Werner, Nikos

2010-03-01

Atherosclerosis is the most common cause for cardiovascular diseases and is based on endothelial dysfunction. A growing body of evidence suggests the contribution of bone marrow-derived endothelial progenitor cells, monocytic cells, and mature endothelial cells to vessel formation and endothelial rejuvenation. To this day, various subsets of these endothelial-regenerating cells have been identified according to cellular origin, phenotype, and properties in vivo and in vitro. However, the definition and biology, especially of endothelial progenitor cells, is complex and under heavy debate. In this review, we focus on current definitions of endothelial progenitor cells, highlight the clinical relevance of endothelial-regenerating cells, and provide new insights into cell-cell interactions involved in endothelial cell rejuvenation.

High-Density Genetic Mapping Identifies New Susceptibility Variants in Sarcoidosis Phenotypes and Shows Genomic-driven Phenotypic Differences

PubMed Central

Ronninger, Marcus; Shchetynsky, Klementy; Franke, Andre; Nöthen, Markus M.; Müller-Quernheim, Joachim; Schreiber, Stefan; Adrianto, Indra; Karakaya, Bekir; van Moorsel, Coline H. M.; Navratilova, Zdenka; Kolek, Vitezslav; Rybicki, Benjamin A.; Iannuzzi, Michael C.; Petrek, Martin; Grutters, Jan C.; Montgomery, Courtney; Fischer, Annegret; Eklund, Anders; Padyukov, Leonid; Grunewald, Johan

2016-01-01

Rationale: Sarcoidosis is a multisystem disease of unknown cause. Löfgren’s syndrome (LS) is a characteristic subgroup of sarcoidosis that is associated with a good prognosis in sarcoidosis. However, little is known about its genetic architecture or its broader phenotype, non-LS sarcoidosis. Objectives: To address the genetic architecture of sarcoidosis phenotypes, LS and non-LS. Methods: An association study in a white Swedish cohort of 384 LS, 664 non-LS, and 2,086 control subjects, totaling 3,134 subjects using a fine-mapping genotyping platform was conducted. Replication was performed in four independent cohorts, three of white European descent (Germany, n = 4,975; the Netherlands, n = 613; and Czech Republic, n = 521), and one of black African descent (United States, n = 1,657), totaling 7,766 subjects. Measurements and Main Results: A total of 727 LS-associated variants expanding throughout the extended major histocompatibility complex (MHC) region and 68 non-LS–associated variants located in the MHC class II region were identified and confirmed. A shared overlap between LS and non-LS defined by 17 variants located in the MHC class II region was found. Outside the MHC region, two LS-associated loci, in ADCY3 and between CSMD1 and MCPH1, were observed and replicated. Conclusions: Comprehensive and integrative analyses of genetics, transcription, and pathway modeling on LS and non-LS indicates that these sarcoidosis phenotypes have different genetic susceptibility, genomic distributions, and cellular activities, suggesting distinct molecular mechanisms in pathways related to immune response with a common region. PMID:26651848

Proteomics to assess the role of phenotypic plasticity in aquatic organisms exposed to pollution and global warming.

PubMed

Silvestre, Frédéric; Gillardin, Virginie; Dorts, Jennifer

2012-11-01

Nowadays, the unprecedented rates of anthropogenic changes in ecosystems suggest that organisms have to migrate to new distributional ranges or to adapt commensurately quickly to new conditions to avoid becoming extinct. Pollution and global warming are two of the most important threats aquatic organisms will have to face in the near future. If genetic changes in a population in response to natural selection are extensively studied, the role of acclimation through phenotypic plasticity (the property of a given genotype to produce different phenotypes in response to particular environmental conditions) in a species to deal with new environmental conditions remains largely unknown. Proteomics is the extensive study of the protein complement of a genome. It is dynamic and depends on the specific tissue, developmental stage, and environmental conditions. As the final product of gene expression, it is subjected to several regulatory steps from gene transcription to the functional protein. Consequently, there is a discrepancy between the abundance of mRNA and the abundance of the corresponding protein. Moreover, proteomics is closer to physiology and gives a more functional knowledge of the regulation of gene expression than does transcriptomics. The study of protein-expression profiles, however, gives a better portrayal of the cellular phenotype and is considered as a key link between the genotype and the organismal phenotype. Under new environmental conditions, we can observe a shift of the protein-expression pattern defining a new cellular phenotype that can possibly improve the fitness of the organism. It is now necessary to define a proteomic norm of reaction for organisms acclimating to environmental stressors. Its link to fitness will give new insights into how organisms can evolve in a changing environment. The proteomic literature bearing on chronic exposure to pollutants and on acclimation to heat stress in aquatic organisms, as well as potential application of proteomics in evolutionary issues, are outlined. While the transcriptome responses are commonly investigated, proteomics approaches now need to be intensified, with the new perspective of integrating the cellular phenotype with the organismal phenotype and with the mechanisms of the regulation of gene expression, such as epigenetics.

Genetic Variation in the Nuclear and Organellar Genomes Modulates Stochastic Variation in the Metabolome, Growth, and Defense

PubMed Central

Joseph, Bindu; Corwin, Jason A.; Kliebenstein, Daniel J.

2015-01-01

Recent studies are starting to show that genetic control over stochastic variation is a key evolutionary solution of single celled organisms in the face of unpredictable environments. This has been expanded to show that genetic variation can alter stochastic variation in transcriptional processes within multi-cellular eukaryotes. However, little is known about how genetic diversity can control stochastic variation within more non-cell autonomous phenotypes. Using an Arabidopsis reciprocal RIL population, we showed that there is significant genetic diversity influencing stochastic variation in the plant metabolome, defense chemistry, and growth. This genetic diversity included loci specific for the stochastic variation of each phenotypic class that did not affect the other phenotypic classes or the average phenotype. This suggests that the organism's networks are established so that noise can exist in one phenotypic level like metabolism and not permeate up or down to different phenotypic levels. Further, the genomic variation within the plastid and mitochondria also had significant effects on the stochastic variation of all phenotypic classes. The genetic influence over stochastic variation within the metabolome was highly metabolite specific, with neighboring metabolites in the same metabolic pathway frequently showing different levels of noise. As expected from bet-hedging theory, there was more genetic diversity and a wider range of stochastic variation for defense chemistry than found for primary metabolism. Thus, it is possible to begin dissecting the stochastic variation of whole organismal phenotypes in multi-cellular organisms. Further, there are loci that modulate stochastic variation at different phenotypic levels. Finding the identity of these genes will be key to developing complete models linking genotype to phenotype. PMID:25569687

Genetic variation in the nuclear and organellar genomes modulates stochastic variation in the metabolome, growth, and defense.

PubMed

Joseph, Bindu; Corwin, Jason A; Kliebenstein, Daniel J

2015-01-01

Recent studies are starting to show that genetic control over stochastic variation is a key evolutionary solution of single celled organisms in the face of unpredictable environments. This has been expanded to show that genetic variation can alter stochastic variation in transcriptional processes within multi-cellular eukaryotes. However, little is known about how genetic diversity can control stochastic variation within more non-cell autonomous phenotypes. Using an Arabidopsis reciprocal RIL population, we showed that there is significant genetic diversity influencing stochastic variation in the plant metabolome, defense chemistry, and growth. This genetic diversity included loci specific for the stochastic variation of each phenotypic class that did not affect the other phenotypic classes or the average phenotype. This suggests that the organism's networks are established so that noise can exist in one phenotypic level like metabolism and not permeate up or down to different phenotypic levels. Further, the genomic variation within the plastid and mitochondria also had significant effects on the stochastic variation of all phenotypic classes. The genetic influence over stochastic variation within the metabolome was highly metabolite specific, with neighboring metabolites in the same metabolic pathway frequently showing different levels of noise. As expected from bet-hedging theory, there was more genetic diversity and a wider range of stochastic variation for defense chemistry than found for primary metabolism. Thus, it is possible to begin dissecting the stochastic variation of whole organismal phenotypes in multi-cellular organisms. Further, there are loci that modulate stochastic variation at different phenotypic levels. Finding the identity of these genes will be key to developing complete models linking genotype to phenotype.

Characterizing Protein Interactions Employing a Genome-Wide siRNA Cellular Phenotyping Screen

PubMed Central

Suratanee, Apichat; Schaefer, Martin H.; Betts, Matthew J.; Soons, Zita; Mannsperger, Heiko; Harder, Nathalie; Oswald, Marcus; Gipp, Markus; Ramminger, Ellen; Marcus, Guillermo; Männer, Reinhard; Rohr, Karl; Wanker, Erich; Russell, Robert B.; Andrade-Navarro, Miguel A.; Eils, Roland; König, Rainer

2014-01-01

Characterizing the activating and inhibiting effect of protein-protein interactions (PPI) is fundamental to gain insight into the complex signaling system of a human cell. A plethora of methods has been suggested to infer PPI from data on a large scale, but none of them is able to characterize the effect of this interaction. Here, we present a novel computational development that employs mitotic phenotypes of a genome-wide RNAi knockdown screen and enables identifying the activating and inhibiting effects of PPIs. Exemplarily, we applied our technique to a knockdown screen of HeLa cells cultivated at standard conditions. Using a machine learning approach, we obtained high accuracy (82% AUC of the receiver operating characteristics) by cross-validation using 6,870 known activating and inhibiting PPIs as gold standard. We predicted de novo unknown activating and inhibiting effects for 1,954 PPIs in HeLa cells covering the ten major signaling pathways of the Kyoto Encyclopedia of Genes and Genomes, and made these predictions publicly available in a database. We finally demonstrate that the predicted effects can be used to cluster knockdown genes of similar biological processes in coherent subgroups. The characterization of the activating or inhibiting effect of individual PPIs opens up new perspectives for the interpretation of large datasets of PPIs and thus considerably increases the value of PPIs as an integrated resource for studying the detailed function of signaling pathways of the cellular system of interest. PMID:25255318

TORC1 signaling inhibition by rapamycin and caffeine affect lifespan, global gene expression, and cell proliferation of fission yeast.

PubMed

Rallis, Charalampos; Codlin, Sandra; Bähler, Jürg

2013-08-01

Target of rapamycin complex 1 (TORC1) is implicated in growth control and aging from yeast to humans. Fission yeast is emerging as a popular model organism to study TOR signaling, although rapamycin has been thought to not affect cell growth in this organism. Here, we analyzed the effects of rapamycin and caffeine, singly and combined, on multiple cellular processes in fission yeast. The two drugs led to diverse and specific phenotypes that depended on TORC1 inhibition, including prolonged chronological lifespan, inhibition of global translation, inhibition of cell growth and division, and reprograming of global gene expression mimicking nitrogen starvation. Rapamycin and caffeine differentially affected these various TORC1-dependent processes. Combined drug treatment augmented most phenotypes and effectively blocked cell growth. Rapamycin showed a much more subtle effect on global translation than did caffeine, while both drugs were effective in prolonging chronological lifespan. Rapamycin and caffeine did not affect the lifespan via the pH of the growth media. Rapamycin prolonged the lifespan of nongrowing cells only when applied during the growth phase but not when applied after cells had stopped proliferation. The doses of rapamycin and caffeine strongly correlated with growth inhibition and with lifespan extension. This comprehensive analysis will inform future studies into TORC1 function and cellular aging in fission yeast and beyond. © 2013 The Authors. Aging Cell published by John Wiley & Sons Ltd and the Anatomical Society.

Epigenetics in Paediatric Gastroenterology, Hepatology, and Nutrition: Present Trends and Future Perspectives.

PubMed

Zilbauer, Matthias; Zellos, Aglaia; Heuschkel, Robert; Gasparetto, Marco; Kraiczy, Judith; Postberg, Jan; Greco, Luigi; Auricchio, Renata; Galatola, Martina; Embleton, Nicholas; Wirth, Stefan; Jenke, Andreas

2016-04-01

Epigenetics can be defined as stable, potentially heritable changes in the cellular phenotype caused by mechanisms other than alterations to the underlying DNA sequence. As such, any observed phenotypic changes including organ development, aging, and the occurrence of disease could be driven by epigenetic mechanisms in the presence of stable cellular DNA sequences. Indeed, with the exception of rare mutations, the human genome-sequence has remained remarkably stable over the past centuries. In contrast, substantial changes to our environment as part of our modern life style have not only led to a significant reduction of certain infectious diseases but also seen the exponential increase in complex traits including obesity and multifactorial diseases such as autoimmune disorders. It is becoming increasingly clear that epigenetic mechanisms operate at the interface between the genetic code and our environment, and a large body of existing evidence supports the importance of environmental factors such as diet and nutrition, infections, and exposure to toxins on human health. This seems to be particularly the case during vulnerable periods of human development such as pregnancy and early life. Importantly, as the first point of contact for many of such environmental factors including nutrition, the digestive system is being increasingly linked to a number of "modern" pathologies. In this review article, we aim to give a brief introduction to the basic molecular principals of epigenetics and provide a concise summary of the existing evidence for the role of epigenetic mechanisms in gastrointestinal health and disease, hepatology, and nutrition.

Modeling schizophrenia using hiPSC neurons

PubMed Central

Brennand, Kristen; Simone, Anthony; Jou, Jessica; Gelboin-Burkhart, Chelsea; Tran, Ngoc; Sangar, Sarah; Li, Yan; Mu, Yangling; Chen, Gong; Yu, Diana; McCarthy, Shane; Sebat, Jonathan; Gage, Fred H.

2012-01-01

SUMMARY Schizophrenia (SCZD) is a debilitating neurological disorder with a world-wide prevalence of 1%; there is a strong genetic component, with an estimated heritability of 80–85%1. Though postmortem studies have revealed reduced brain volume, cell size, spine density and abnormal neural distribution in the prefrontal cortex and hippocampus of SCZD brain tissue2 and neuropharmacological studies have implicated dopaminergic, glutamatergic and GABAergic activity in SCZD3, the cell types affected in SCZD and the molecular mechanisms underlying the disease state remain unclear. To elucidate the cellular and molecular defects of SCZD, we directly reprogrammed fibroblasts from SCZD patients into human induced pluripotent stem cells (hiPSCs) and subsequently differentiated these disorder-specific hiPSCs into neurons (SI Fig. 1). SCZD hiPSC neurons showed diminished neuronal connectivity in conjunction with decreased neurite number, PSD95-protein levels and glutamate receptor expression. Gene expression profiles of SCZD hiPSC neurons identified altered expression of many components of the cAMP and WNT signaling pathways. Key cellular and molecular elements of the SCZD phenotype were ameliorated following treatment of SCZD hiPSC neurons with the antipsychotic Loxapine. To date, hiPSC neuronal pathology has only been demonstrated in diseases characterized by both the loss of function of a single gene product and rapid disease progression in early childhood4–6. We now report hiPSC neuronal phenotypes and gene expression changes associated with SCZD, a complex genetic psychiatric disorder (SI Table 1). PMID:21490598

Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer

DTIC Science & Technology

2015-09-01

AWARD NUMBER: W81XWH-14-1-0177 TITLE: Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer PRINCIPAL INVESTIGATOR: Katerina Politi...CONTRACT NUMBER Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer 5b. GRANT NUMBER W81XWH-14-1-0177 5c. PROGRAM ELEMENT NUMBER 6...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Phenotypic changes have been observed in EGFR mutant lung cancers that become resistant to targeted

General and craniofacial development are complex adaptive processes influenced by diversity.

PubMed

Brook, A H; O'Donnell, M Brook; Hone, A; Hart, E; Hughes, T E; Smith, R N; Townsend, G C

2014-06-01

Complex systems are present in such diverse areas as social systems, economies, ecosystems and biology and, therefore, are highly relevant to dental research, education and practice. A Complex Adaptive System in biological development is a dynamic process in which, from interacting components at a lower level, higher level phenomena and structures emerge. Diversity makes substantial contributions to the performance of complex adaptive systems. It enhances the robustness of the process, allowing multiple responses to external stimuli as well as internal changes. From diversity comes variation in outcome and the possibility of major change; outliers in the distribution enhance the tipping points. The development of the dentition is a valuable, accessible model with extensive and reliable databases for investigating the role of complex adaptive systems in craniofacial and general development. The general characteristics of such systems are seen during tooth development: self-organization; bottom-up emergence; multitasking; self-adaptation; variation; tipping points; critical phases; and robustness. Dental findings are compatible with the Random Network Model, the Threshold Model and also with the Scale Free Network Model which has a Power Law distribution. In addition, dental development shows the characteristics of Modularity and Clustering to form Hierarchical Networks. The interactions between the genes (nodes) demonstrate Small World phenomena, Subgraph Motifs and Gene Regulatory Networks. Genetic mechanisms are involved in the creation and evolution of variation during development. The genetic factors interact with epigenetic and environmental factors at the molecular level and form complex networks within the cells. From these interactions emerge the higher level tissues, tooth germs and mineralized teeth. Approaching development in this way allows investigation of why there can be variations in phenotypes from identical genotypes; the phenotype is the outcome of perturbations in the cellular systems and networks, as well as of the genotype. Understanding and applying complexity theory will bring about substantial advances not only in dental research and education but also in the organization and delivery of oral health care. © 2014 Australian Dental Association.

Integrating mRNA and Protein Sequencing Enables the Detection and Quantitative Profiling of Natural Protein Sequence Variants of Populus trichocarpa.

PubMed

Abraham, Paul E; Wang, Xiaojing; Ranjan, Priya; Nookaew, Intawat; Zhang, Bing; Tuskan, Gerald A; Hettich, Robert L

2015-12-04

Next-generation sequencing has transformed the ability to link genotypes to phenotypes and facilitates the dissection of genetic contribution to complex traits. However, it is challenging to link genetic variants with the perturbed functional effects on proteins encoded by such genes. Here we show how RNA sequencing can be exploited to construct genotype-specific protein sequence databases to assess natural variation in proteins, providing information about the molecular toolbox driving cellular processes. For this study, we used two natural genotypes selected from a recent genome-wide association study of Populus trichocarpa, an obligate outcrosser with tremendous phenotypic variation across the natural population. This strategy allowed us to comprehensively catalogue proteins containing single amino acid polymorphisms (SAAPs), as well as insertions and deletions. We profiled the frequency of 128 types of naturally occurring amino acid substitutions, including both expected (neutral) and unexpected (non-neutral) SAAPs, with a subset occurring in regions of the genome having strong polymorphism patterns consistent with recent positive and/or divergent selection. By zeroing in on the molecular signatures of these important regions that might have previously been uncharacterized, we now provide a high-resolution molecular inventory that should improve accessibility and subsequent identification of natural protein variants in future genotype-to-phenotype studies.

Time series modeling of live-cell shape dynamics for image-based phenotypic profiling.

PubMed

Gordonov, Simon; Hwang, Mun Kyung; Wells, Alan; Gertler, Frank B; Lauffenburger, Douglas A; Bathe, Mark

2016-01-01

Live-cell imaging can be used to capture spatio-temporal aspects of cellular responses that are not accessible to fixed-cell imaging. As the use of live-cell imaging continues to increase, new computational procedures are needed to characterize and classify the temporal dynamics of individual cells. For this purpose, here we present the general experimental-computational framework SAPHIRE (Stochastic Annotation of Phenotypic Individual-cell Responses) to characterize phenotypic cellular responses from time series imaging datasets. Hidden Markov modeling is used to infer and annotate morphological state and state-switching properties from image-derived cell shape measurements. Time series modeling is performed on each cell individually, making the approach broadly useful for analyzing asynchronous cell populations. Two-color fluorescent cells simultaneously expressing actin and nuclear reporters enabled us to profile temporal changes in cell shape following pharmacological inhibition of cytoskeleton-regulatory signaling pathways. Results are compared with existing approaches conventionally applied to fixed-cell imaging datasets, and indicate that time series modeling captures heterogeneous dynamic cellular responses that can improve drug classification and offer additional important insight into mechanisms of drug action. The software is available at http://saphire-hcs.org.

Dynamics of phenotypic switching of bacterial cells with temporal fluctuations in pressure

NASA Astrophysics Data System (ADS)

Nepal, Sudip; Kumar, Pradeep

2018-05-01

Phenotypic switching is one of the mechanisms by which bacteria thrive in ever changing environmental conditions around them. Earlier studies have shown that the application of steady high hydrostatic pressure leads to stochastic switching of mesophilic bacteria from a cellular phenotype having a normal cell cycle to another phenotype lacking cell division. Here, we have studied the dynamics of this phenotypic switching with fluctuating periodic pressure using a set of experiments and a theoretical model. Our results suggest that the phenotypic switching rate from high-pressure phenotype to low-pressure phenotype in the reversible regime is larger as compared to the switching rate from low-pressure phenotype to high-pressure phenotype. Furthermore, we find that even though the cell division and elongation are presumably regulated by a large number of genes the underlying physics of the dynamics of stochastic switching at high pressure is captured reasonably well by a simple two-state model.

Humanity in a Dish: Population Genetics with iPSCs.

PubMed

Warren, Curtis R; Cowan, Chad A

2018-01-01

Induced pluripotent stem cells (iPSCs) are powerful tools for investigating the relationship between genotype and phenotype. Recent publications have described iPSC cohort studies of common genetic variants and their effects on gene expression and cellular phenotypes. These in vitro quantitative trait locus (QTL) studies are the first experiments in a new paradigm with great potential: iPSC-based functional population genetic studies. iPSC collections from large cohorts are currently under development to facilitate the next wave of these studies, which have the potential to discover the effects of common genetic variants on cellular phenotypes and to uncover the molecular basis of common genetic diseases. Here, we describe the recent advances in this developing field, and provide a road map for future in vitro functional population genetic studies and trial-in-a-dish experiments. Copyright © 2017 Elsevier Ltd. All rights reserved.

Modeling integrated cellular machinery using hybrid Petri-Boolean networks.

PubMed

Berestovsky, Natalie; Zhou, Wanding; Nagrath, Deepak; Nakhleh, Luay

2013-01-01

The behavior and phenotypic changes of cells are governed by a cellular circuitry that represents a set of biochemical reactions. Based on biological functions, this circuitry is divided into three types of networks, each encoding for a major biological process: signal transduction, transcription regulation, and metabolism. This division has generally enabled taming computational complexity dealing with the entire system, allowed for using modeling techniques that are specific to each of the components, and achieved separation of the different time scales at which reactions in each of the three networks occur. Nonetheless, with this division comes loss of information and power needed to elucidate certain cellular phenomena. Within the cell, these three types of networks work in tandem, and each produces signals and/or substances that are used by the others to process information and operate normally. Therefore, computational techniques for modeling integrated cellular machinery are needed. In this work, we propose an integrated hybrid model (IHM) that combines Petri nets and Boolean networks to model integrated cellular networks. Coupled with a stochastic simulation mechanism, the model simulates the dynamics of the integrated network, and can be perturbed to generate testable hypotheses. Our model is qualitative and is mostly built upon knowledge from the literature and requires fine-tuning of very few parameters. We validated our model on two systems: the transcriptional regulation of glucose metabolism in human cells, and cellular osmoregulation in S. cerevisiae. The model produced results that are in very good agreement with experimental data, and produces valid hypotheses. The abstract nature of our model and the ease of its construction makes it a very good candidate for modeling integrated networks from qualitative data. The results it produces can guide the practitioner to zoom into components and interconnections and investigate them using such more detailed mathematical models.

Modeling Integrated Cellular Machinery Using Hybrid Petri-Boolean Networks

PubMed Central

Berestovsky, Natalie; Zhou, Wanding; Nagrath, Deepak; Nakhleh, Luay

2013-01-01

The behavior and phenotypic changes of cells are governed by a cellular circuitry that represents a set of biochemical reactions. Based on biological functions, this circuitry is divided into three types of networks, each encoding for a major biological process: signal transduction, transcription regulation, and metabolism. This division has generally enabled taming computational complexity dealing with the entire system, allowed for using modeling techniques that are specific to each of the components, and achieved separation of the different time scales at which reactions in each of the three networks occur. Nonetheless, with this division comes loss of information and power needed to elucidate certain cellular phenomena. Within the cell, these three types of networks work in tandem, and each produces signals and/or substances that are used by the others to process information and operate normally. Therefore, computational techniques for modeling integrated cellular machinery are needed. In this work, we propose an integrated hybrid model (IHM) that combines Petri nets and Boolean networks to model integrated cellular networks. Coupled with a stochastic simulation mechanism, the model simulates the dynamics of the integrated network, and can be perturbed to generate testable hypotheses. Our model is qualitative and is mostly built upon knowledge from the literature and requires fine-tuning of very few parameters. We validated our model on two systems: the transcriptional regulation of glucose metabolism in human cells, and cellular osmoregulation in S. cerevisiae. The model produced results that are in very good agreement with experimental data, and produces valid hypotheses. The abstract nature of our model and the ease of its construction makes it a very good candidate for modeling integrated networks from qualitative data. The results it produces can guide the practitioner to zoom into components and interconnections and investigate them using such more detailed mathematical models. PMID:24244124

Interaction of Epstein-Barr virus (EBV) with human B-lymphocytes

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

Klein, George, E-mail: Georg.Klein@ki.se; Klein, Eva; Kashuba, Elena

Epstein-Barr virus, EBV, and humans have a common history that reaches back to our primate ancestors. The virus co-evolved with man and has established a largely harmless and highly complex co-existence. It is carried as silent infection by almost all human adults. A serendipitous discovery established that it is the causative agent of infectious mononucleosis. Still, EBV became known first in 1964, in a rare, geographically prevalent malignant lymphoma of B-cell origin, Burkitt lymphoma BL. Its association with a malignancy prompted intensive studies and its capacity to immortalize B-lymphocytes in vitro was soon demonstrated. Consequently EBV was classified therefore asmore » a potentially tumorigenic virus. Despite of this property however, the virus carrier state itself does not lead to malignancies because the transformed cells are recognized by the immune response. Consequently the EBV induced proliferation of EBV carrying B-lymphocytes is manifested only under immunosuppressive conditions. The expression of EBV encoded genes is regulated by the cell phenotype. The virus genome can be found in malignancies originating from cell types other than the B-lymphocyte. Even in the EBV infected B-cell, the direct transforming capacity is restricted to a defined window of differentiation. A complex interaction between virally encoded proteins and B-cell specific cellular proteins constitute the proliferation inducing program. In this short review we touch upon aspects which are the subject of our present work. We describe the mechanisms of some of the functional interactions between EBV encoded and cellular proteins that determine the phenotype of latently infected B-cells. The growth promoting EBV encoded genes are not expressed in the virus carrying BL cells. Still, EBV seems to contribute to the etiology of this tumor by modifying events that influence cell survival and proliferation. We describe a possible growth promoting mechanism in the genesis of Burkitt lymphoma that depends on the presence of EBV.« less

The biological role of actinin-4 (ACTN4) in malignant phenotypes of cancer.

PubMed

Honda, Kazufumi

2015-01-01

Invasion and metastasis are malignant phenotypes in cancer that lead to patient death. Cell motility is involved in these processes. In 1998, we identified overexpression of the actin-bundling protein actinin-4 in several types of cancer. Protein expression of actinin-4 is closely associated with the invasive phenotypes of cancers. Actinin-4 is predominantly expressed in the cellular protrusions that stimulate the invasive phenotype in cancer cells and is essential for formation of cellular protrusions such as filopodia and lamellipodia. ACTN4 (gene name encoding actinin-4 protein) is located on human chromosome 19q. ACTN4 amplification is frequently observed in patients with carcinomas of the pancreas, ovary, lung, and salivary gland, and patients with ACTN4 amplifications have worse outcomes than patients without amplification. In addition, nuclear distribution of actinin-4 is frequently observed in small cell lung, breast, and ovarian cancer. Actinin-4, when expressed in cancer cell nuclei, functions as a transcriptional co-activator. In this review, we summarize recent developments regarding the biological roles of actinin-4 in cancer invasion.

Oxidative Stress, Bone Marrow Failure, and Genome Instability in Hematopoietic Stem Cells

PubMed Central

Richardson, Christine; Yan, Shan; Vestal, C. Greer

2015-01-01

Reactive oxygen species (ROS) can be generated by defective endogenous reduction of oxygen by cellular enzymes or in the mitochondrial respiratory pathway, as well as by exogenous exposure to UV or environmental damaging agents. Regulation of intracellular ROS levels is critical since increases above normal concentrations lead to oxidative stress and DNA damage. A growing body of evidence indicates that the inability to regulate high levels of ROS leading to alteration of cellular homeostasis or defective repair of ROS-induced damage lies at the root of diseases characterized by both neurodegeneration and bone marrow failure as well as cancer. That these diseases may be reflective of the dynamic ability of cells to respond to ROS through developmental stages and aging lies in the similarities between phenotypes at the cellular level. This review summarizes work linking the ability to regulate intracellular ROS to the hematopoietic stem cell phenotype, aging, and disease. PMID:25622253

Enhanced conversion of induced neuronal cells (iN cells) from human fibroblasts: utility in uncovering cellular deficits in mental illness-associated chromosomal abnormalities

PubMed Central

Passeri, Eleonora; Wilson, Ashley M.; Primerano, Amedeo; Kondo, Mari A.; Sengupta, Srona; Srivastava, Rupali; Koga, Minori; Obie, Cassandra; Zandi, Peter P.; Goes, Fernando S.; Valle, David; Rapoport, Judith L.; Sawa, Akira; Kano, Shin-ichi; Ishizuka, Koko

2016-01-01

The novel technology of induced neuronal cells (iN cells) is promising for translational neuroscience, as it allows the conversion of human fibroblasts into cells with postmitotic neuronal traits. However, a major technical barrier is the low conversion rate. To overcome this problem, we optimized the conversion media. Using our improved formulation, we studied how major mental illness-associated chromosomal abnormalities may impact the characteristics of iN cells. We demonstrated that our new iN cell culture protocol enabled us to obtain more precise measurement of neuronal cellular phenotypes than previous iN cell methods. Thus, this iN cell culture provides a platform to efficiently obtain possible cellular phenotypes caused by genetic differences, which can be more thoroughly studied in research using other human cell models such as induced pluripotent stem cells. PMID:26260244

Nonlinear ghost waves accelerate the progression of high-grade brain tumors

NASA Astrophysics Data System (ADS)

Pardo, Rosa; Martínez-González, Alicia; Pérez-García, Víctor M.

2016-10-01

We study a reduced continuous model describing the evolution of high grade gliomas in response to hypoxic events through the interplay of different cellular phenotypes. We show that hypoxic events, even when sporadic and/or limited in space, may have a crucial role on the acceleration of high grade gliomas growth. Our modeling approach is based on two cellular phenotypes. One of them is more migratory and a second one is more proliferative. Transitions between both phenotypes are driven by the local oxygen values, assumed in this simple model to be uniform. Surprisingly, even very localized in time hypoxia events leading to transient migratory populations have the potential to accelerate the tumor's invasion speed up to speeds close to those of the migratory phenotype. The high invasion speed persists for times much longer than the lifetime of the hypoxic event. Moreover, the phenomenon is observed both when the migratory cells form a persistent wave of cells located on the invasion front and when they form a evanescent "ghost" wave disappearing after a short time by decay to the more proliferative phenotype. Our findings are obtained through numerical simulations of the model equations both in 1D and higher dimensional scenarios. We also provide a deeper mathematical analysis of some aspects of the problem such as the conditions for the existence of persistent waves of cells with a more migratory phenotype.

Semi-synthesis of a HGF/SF kringle one (K1) domain scaffold generates a potent in vivo MET receptor agonist.

PubMed

Simonneau, Claire; Bérénice Leclercq; Mougel, Alexandra; Adriaenssens, Eric; Paquet, Charlotte; Raibaut, Laurent; Ollivier, Nathalie; Drobecq, Hervé; Marcoux, Julien; Cianférani, Sarah; Tulasne, David; de Jonge, Hugo; Melnyk, Oleg; Vicogne, Jérôme

2015-03-01

The development of MET receptor agonists is an important goal in regenerative medicine, but is limited by the complexity and incomplete understanding of its interaction with HGF/SF (Hepatocyte Growth Factor/Scatter Factor). NK1 is a natural occurring agonist comprising the N-terminal (N) and the first kringle (K1) domains of HGF/SF. In the presence of heparin, NK1 can self-associate into a "head to tail" dimer which is considered as the minimal structural module able to trigger MET dimerization and activation whereas isolated K1 and N domains showed a weak or a complete lack of agonistic activity respectively. Starting from these structural and biological observations, we investigated whether it was possible to recapitulate the biological properties of NK1 using a new molecular architecture of isolated N or K1 domains. Therefore, we engineered multivalent N or K1 scaffolds by combining synthetic and homogeneous site-specifically biotinylated N and K1 domains (NB and K1B) and streptavidin (S). NB alone or in complex failed to activate MET signaling and to trigger cellular phenotypes. Importantly and to the contrary of K1B alone, the semi-synthetic K1B/S complex mimicked NK1 MET agonist activity in cell scattering, morphogenesis and survival phenotypic assays. Impressively, K1B/S complex stimulated in vivo angiogenesis and, when injected in mice, protected the liver against fulminant hepatitis in a MET dependent manner whereas NK1 and HGF were substantially less potent. These data reveal that without N domain, proper multimerization of K1 domain is a promising strategy for the rational design of powerful MET agonists.

Induction of vascular endothelial phenotype and cellular proliferation from human cord blood stem cells cultured in simulated microgravity

NASA Astrophysics Data System (ADS)

Chiu, Brian; Z-M Wan, Jim; Abley, Doris; Akabutu, John

2005-05-01

Recent studies have demonstrated that stem cells derived from adult hematopoietic tissues are capable of trans-differentiation into non-hematopoietic cells, and that the culture in microgravity ( μg) may modulate the proliferation and differentiation. We investigated the application of μg to human umbilical cord blood stem cells (CBSC) in the induction of vascular endothelial phenotype expression and cellular proliferation. CD34+ mononuclear cells were isolated from waste human umbilical cord blood samples and cultured in simulated μg for 14 days. The cells were seeded in rotary wall vessels (RWV) with or without microcarrier beads (MCB) and vascular endothelial growth factor was added during culture. Controls consisted of culture in 1 G. The cell cultures in RWV were examined by inverted microscopy. Cell counts, endothelial cell and leukocyte markers performed by flow-cytometry and FACS scan were assayed at days 1, 4, 7 and at the termination of the experiments. Culture in RWV revealed significantly increased cellular proliferation with three-dimensional (3D) tissue-like aggregates. At day 4, CD34+ cells cultured in RWV bioreactor without MCB developed vascular tubular assemblies and exhibited endothelial phenotypic markers. These data suggest that CD34+ human umbilical cord blood progenitors are capable of trans-differentiation into vascular endothelial cell phenotype and assemble into 3D tissue structures. Culture of CBSC in simulated μg may be potentially beneficial in the fields of stem cell biology and somatic cell therapy.

Requirement for the Murine Zinc Finger Protein ZFR in Perigastrulation Growth and Survival

PubMed Central

Meagher, Madeleine J.; Braun, Robert E.

2001-01-01

The transition from preimplantation to postimplantation development leads to the initiation of complex cellular differentiation and morphogenetic movements, a dramatic decrease in cell cycle length, and a commensurate increase in the size of the embryo. Accompanying these changes is the need for the transfer of nutrients from the mother to the embryo and the elaboration of sophisticated genetic networks that monitor genomic integrity and the homeostatic control of cellular growth, differentiation, and programmed cell death. To determine the function of the murine zinc finger protein ZFR in these events, we generated mice carrying a null mutation in the gene encoding it. Homozygous mutant embryos form normal-appearing blastocysts that implant and initiate the process of gastrulation. Mutant embryos form mesoderm but they are delayed in their development and fail to form normal anterior embryonic structures. Loss of ZFR function leads to both an increase in programmed cell death and a decrease in mitotic index, especially in the region of the distal tip of the embryonic ectoderm. Mutant embryos also have an apparent reduction in apical vacuoles in the columnar visceral endoderm cells in the extraembryonic region. Together, these cellular phenotypes lead to a dramatic development delay and embryonic death by 8 to 9 days of gestation, which are independent of p53 function. PMID:11283266

Juxtaposed Polycomb complexes co-regulate vertebral identity.

PubMed

Kim, Se Young; Paylor, Suzanne W; Magnuson, Terry; Schumacher, Armin

2006-12-01

Best known as epigenetic repressors of developmental Hox gene transcription, Polycomb complexes alter chromatin structure by means of post-translational modification of histone tails. Depending on the cellular context, Polycomb complexes of diverse composition and function exhibit cooperative interaction or hierarchical interdependency at target loci. The present study interrogated the genetic, biochemical and molecular interaction of BMI1 and EED, pivotal constituents of heterologous Polycomb complexes, in the regulation of vertebral identity during mouse development. Despite a significant overlap in dosage-sensitive homeotic phenotypes and co-repression of a similar set of Hox genes, genetic analysis implicated eed and Bmi1 in parallel pathways, which converge at the level of Hox gene regulation. Whereas EED and BMI1 formed separate biochemical entities with EzH2 and Ring1B, respectively, in mid-gestation embryos, YY1 engaged in both Polycomb complexes. Strikingly, methylated lysine 27 of histone H3 (H3-K27), a mediator of Polycomb complex recruitment to target genes, stably associated with the EED complex during the maintenance phase of Hox gene repression. Juxtaposed EED and BMI1 complexes, along with YY1 and methylated H3-K27, were detected in upstream regulatory regions of Hoxc8 and Hoxa5. The combined data suggest a model wherein epigenetic and genetic elements cooperatively recruit and retain juxtaposed Polycomb complexes in mammalian Hox gene clusters toward co-regulation of vertebral identity.

Potential Role of Lateral Gene Transfer in the Evolution of Biofilm Communities at the Lost City Hydrothermal Field and in the Earliest Stages of Cellular Evolution

NASA Astrophysics Data System (ADS)

Brazelton, W. J.; Mehta, M. P.; Baross, J. A.

2010-04-01

DNA sequencing and metabolic activity measurements show that lateral gene transfer promotes phenotypic diversity in single-species archaeal biofilms attached to hydrothermal chimneys. This system may be a useful model for early cellular evolution.

In-vitro analysis of Quantum Molecular Resonance effects on human mesenchymal stromal cells

PubMed Central

Sella, Sabrina; Adami, Valentina; Amati, Eliana; Bernardi, Martina; Chieregato, Katia; Gatto, Pamela; Menarin, Martina; Pozzato, Alessandro; Pozzato, Gianantonio; Astori, Giuseppe

2018-01-01

Electromagnetic fields play an essential role in cellular functions interfering with cellular pathways and tissue physiology. In this context, Quantum Molecular Resonance (QMR) produces waves with a specific form at high-frequencies (4–64 MHz) and low intensity through electric fields. We evaluated the effects of QMR stimulation on bone marrow derived mesenchymal stromal cells (MSC). MSC were treated with QMR for 10 minutes for 4 consecutive days for 2 weeks at different nominal powers. Cell morphology, phenotype, multilineage differentiation, viability and proliferation were investigated. QMR effects were further investigated by cDNA microarray validated by real-time PCR. After 1 and 2 weeks of QMR treatment morphology, phenotype and multilineage differentiation were maintained and no alteration of cellular viability and proliferation were observed between treated MSC samples and controls. cDNA microarray analysis evidenced more transcriptional changes on cells treated at 40 nominal power than 80 ones. The main enrichment lists belonged to development processes, regulation of phosphorylation, regulation of cellular pathways including metabolism, kinase activity and cellular organization. Real-time PCR confirmed significant increased expression of MMP1, PLAT and ARHGAP22 genes while A2M gene showed decreased expression in treated cells compared to controls. Interestingly, differentially regulated MMP1, PLAT and A2M genes are involved in the extracellular matrix (ECM) remodelling through the fibrinolytic system that is also implicated in embryogenesis, wound healing and angiogenesis. In our model QMR-treated MSC maintained unaltered cell phenotype, viability, proliferation and the ability to differentiate into bone, cartilage and adipose tissue. Microarray analysis may suggest an involvement of QMR treatment in angiogenesis and in tissue regeneration probably through ECM remodelling. PMID:29293552

Hypothesis: solid tumours behave as systemic metabolic dictators.

PubMed

Lee, Yang-Ming; Chang, Wei-Chun; Ma, Wen-Lung

2016-06-01

Current knowledge regarding mechanisms of carcinogenesis in human beings centres around the accumulation of genetic instability, amplified cellular signalling, disturbed cellular energy metabolism and microenvironmental regulation governed by complicated cell-cell interactions. In this article, we provide an alternative view of cancer biology. We propose that cancer behaves as a systemic dictator that interacts with tissues throughout the body to control their metabolism and eventually homeostasis. The mechanism of development of this endocrine organ-like tumour (EOLT) tissue might be the driving force for cancer progression. Here, we review the literature that led to the development of this hypothesis. The EOLT phenotype can be defined as a tumour that alters systemic homeostasis. The literature indicates that the EOLT phenotype is present throughout cancer progression. The feedback mechanism that governs the interaction between tumours and various organs is unknown. We believe that investigating the mechanism of EOLT development may advance the current knowledge of regulation within the tumour macroenvironment and consequently lead to new diagnostic methods and therapy. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Compound annotation with real time cellular activity profiles to improve drug discovery.

PubMed

Fang, Ye

2016-01-01

In the past decade, a range of innovative strategies have been developed to improve the productivity of pharmaceutical research and development. In particular, compound annotation, combined with informatics, has provided unprecedented opportunities for drug discovery. In this review, a literature search from 2000 to 2015 was conducted to provide an overview of the compound annotation approaches currently used in drug discovery. Based on this, a framework related to a compound annotation approach using real-time cellular activity profiles for probe, drug, and biology discovery is proposed. Compound annotation with chemical structure, drug-like properties, bioactivities, genome-wide effects, clinical phenotypes, and textural abstracts has received significant attention in early drug discovery. However, these annotations are mostly associated with endpoint results. Advances in assay techniques have made it possible to obtain real-time cellular activity profiles of drug molecules under different phenotypes, so it is possible to generate compound annotation with real-time cellular activity profiles. Combining compound annotation with informatics, such as similarity analysis, presents a good opportunity to improve the rate of discovery of novel drugs and probes, and enhance our understanding of the underlying biology.

The genotype-phenotype map of an evolving digital organism.

PubMed

Fortuna, Miguel A; Zaman, Luis; Ofria, Charles; Wagner, Andreas

2017-02-01

To understand how evolving systems bring forth novel and useful phenotypes, it is essential to understand the relationship between genotypic and phenotypic change. Artificial evolving systems can help us understand whether the genotype-phenotype maps of natural evolving systems are highly unusual, and it may help create evolvable artificial systems. Here we characterize the genotype-phenotype map of digital organisms in Avida, a platform for digital evolution. We consider digital organisms from a vast space of 10141 genotypes (instruction sequences), which can form 512 different phenotypes. These phenotypes are distinguished by different Boolean logic functions they can compute, as well as by the complexity of these functions. We observe several properties with parallels in natural systems, such as connected genotype networks and asymmetric phenotypic transitions. The likely common cause is robustness to genotypic change. We describe an intriguing tension between phenotypic complexity and evolvability that may have implications for biological evolution. On the one hand, genotypic change is more likely to yield novel phenotypes in more complex organisms. On the other hand, the total number of novel phenotypes reachable through genotypic change is highest for organisms with simple phenotypes. Artificial evolving systems can help us study aspects of biological evolvability that are not accessible in vastly more complex natural systems. They can also help identify properties, such as robustness, that are required for both human-designed artificial systems and synthetic biological systems to be evolvable.

The genotype-phenotype map of an evolving digital organism

PubMed Central

Zaman, Luis; Wagner, Andreas

2017-01-01

To understand how evolving systems bring forth novel and useful phenotypes, it is essential to understand the relationship between genotypic and phenotypic change. Artificial evolving systems can help us understand whether the genotype-phenotype maps of natural evolving systems are highly unusual, and it may help create evolvable artificial systems. Here we characterize the genotype-phenotype map of digital organisms in Avida, a platform for digital evolution. We consider digital organisms from a vast space of 10141 genotypes (instruction sequences), which can form 512 different phenotypes. These phenotypes are distinguished by different Boolean logic functions they can compute, as well as by the complexity of these functions. We observe several properties with parallels in natural systems, such as connected genotype networks and asymmetric phenotypic transitions. The likely common cause is robustness to genotypic change. We describe an intriguing tension between phenotypic complexity and evolvability that may have implications for biological evolution. On the one hand, genotypic change is more likely to yield novel phenotypes in more complex organisms. On the other hand, the total number of novel phenotypes reachable through genotypic change is highest for organisms with simple phenotypes. Artificial evolving systems can help us study aspects of biological evolvability that are not accessible in vastly more complex natural systems. They can also help identify properties, such as robustness, that are required for both human-designed artificial systems and synthetic biological systems to be evolvable. PMID:28241039

A de novo FOXP1 variant in a patient with autism, intellectual disability and severe speech and language impairment.

PubMed

Lozano, Reymundo; Vino, Arianna; Lozano, Cristina; Fisher, Simon E; Deriziotis, Pelagia

2015-12-01

FOXP1 (forkhead box protein P1) is a transcription factor involved in the development of several tissues, including the brain. An emerging phenotype of patients with protein-disrupting FOXP1 variants includes global developmental delay, intellectual disability and mild to severe speech/language deficits. We report on a female child with a history of severe hypotonia, autism spectrum disorder and mild intellectual disability with severe speech/language impairment. Clinical exome sequencing identified a heterozygous de novo FOXP1 variant c.1267_1268delGT (p.V423Hfs*37). Functional analyses using cellular models show that the variant disrupts multiple aspects of FOXP1 activity, including subcellular localization and transcriptional repression properties. Our findings highlight the importance of performing functional characterization to help uncover the biological significance of variants identified by genomics approaches, thereby providing insight into pathways underlying complex neurodevelopmental disorders. Moreover, our data support the hypothesis that de novo variants represent significant causal factors in severe sporadic disorders and extend the phenotype seen in individuals with FOXP1 haploinsufficiency.

The retinal phenotype of Usher syndrome: pathophysiological insights from animal models.

PubMed

El-Amraoui, Aziz; Petit, Christine

2014-03-01

The Usher syndrome (USH) is the most prevalent cause of inherited deaf-blindness. Three clinical subtypes, USH1-3, have been defined, and ten USH genes identified. The hearing impairment due to USH gene defects has been shown to result from improper organisation of the hair bundle, the sound receptive structure of sensory hair cells. In contrast, the cellular basis of the visual defect is less well understood as this phenotype is absent in almost all the USH mouse models that faithfully mimic the human hearing impairment. Structural and molecular interspecies discrepancies regarding photoreceptor calyceal processes and the association with the distribution of USH1 proteins have recently been unravelled, and have led to the conclusion that a defect in the USH1 protein complex-mediated connection between the photoreceptor outer segment and the surrounding calyceal processes (in both rods and cones), and the inner segment (in rods only), probably causes the USH1 retinal dystrophy in humans. Copyright © 2013 Académie des sciences. Published by Elsevier SAS. All rights reserved.

Generation of Isogenic Human iPS Cell Line Precisely Corrected by Genome Editing Using the CRISPR/Cas9 System.

PubMed

Grobarczyk, Benjamin; Franco, Bénédicte; Hanon, Kevin; Malgrange, Brigitte

2015-10-01

Genome engineering and human iPS cells are two powerful technologies, which can be combined to highlight phenotypic differences and identify pathological mechanisms of complex diseases by providing isogenic cellular material. However, very few data are available regarding precise gene correction in human iPS cells. Here, we describe an optimized stepwise protocol to deliver CRISPR/Cas9 plasmids in human iPS cells. We highlight technical issues especially those associated to human stem cell culture and to the correction of a point mutation to obtain isogenic iPS cell line, without inserting any resistance cassette. Based on a two-steps clonal isolation protocol (mechanical picking followed by enzymatic dissociation), we succeed to select and expand corrected human iPS cell line with a great efficiency (more than 2% of the sequenced colonies). This protocol can also be used to obtain knock-out cell line from healthy iPS cell line by the NHEJ pathway (with about 15% efficiency) and reproduce disease phenotype. In addition, we also provide protocols for functional validation tests after every critical step.

Divergent and nonuniform gene expression patterns in mouse brain

PubMed Central

Morris, John A.; Royall, Joshua J.; Bertagnolli, Darren; Boe, Andrew F.; Burnell, Josh J.; Byrnes, Emi J.; Copeland, Cathy; Desta, Tsega; Fischer, Shanna R.; Goldy, Jeff; Glattfelder, Katie J.; Kidney, Jolene M.; Lemon, Tracy; Orta, Geralyn J.; Parry, Sheana E.; Pathak, Sayan D.; Pearson, Owen C.; Reding, Melissa; Shapouri, Sheila; Smith, Kimberly A.; Soden, Chad; Solan, Beth M.; Weller, John; Takahashi, Joseph S.; Overly, Caroline C.; Lein, Ed S.; Hawrylycz, Michael J.; Hohmann, John G.; Jones, Allan R.

2010-01-01

Considerable progress has been made in understanding variations in gene sequence and expression level associated with phenotype, yet how genetic diversity translates into complex phenotypic differences remains poorly understood. Here, we examine the relationship between genetic background and spatial patterns of gene expression across seven strains of mice, providing the most extensive cellular-resolution comparative analysis of gene expression in the mammalian brain to date. Using comprehensive brainwide anatomic coverage (more than 200 brain regions), we applied in situ hybridization to analyze the spatial expression patterns of 49 genes encoding well-known pharmaceutical drug targets. Remarkably, over 50% of the genes examined showed interstrain expression variation. In addition, the variability was nonuniformly distributed across strain and neuroanatomic region, suggesting certain organizing principles. First, the degree of expression variance among strains mirrors genealogic relationships. Second, expression pattern differences were concentrated in higher-order brain regions such as the cortex and hippocampus. Divergence in gene expression patterns across the brain could contribute significantly to variations in behavior and responses to neuroactive drugs in laboratory mouse strains and may help to explain individual differences in human responsiveness to neuroactive drugs. PMID:20956311

The Immortal Senescence.

PubMed

Bianchi-Smiraglia, Anna; Lipchick, Brittany C; Nikiforov, Mikhail A

2017-01-01

Activation of oncogenic signaling paradoxically results in the permanent withdrawal from cell cycle and induction of senescence (oncogene-induced senescence (OIS)). OIS is a fail-safe mechanism used by the cells to prevent uncontrolled tumor growth, and, as such, it is considered as the first barrier against cancer. In order to progress, tumor cells thus need to first overcome the senescent phenotype. Despite the increasing attention gained by OIS in the past 20 years, this field is still rather young due to continuous emergence of novel pathways and processes involved in OIS. Among the many factors contributing to incomplete understanding of OIS are the lack of unequivocal markers for senescence and the complexity of the phenotypes revealed by senescent cells in vivo and in vitro. OIS has been shown to play major roles at both the cellular and organismal levels in biological processes ranging from embryonic development to barrier to cancer progression. Here we will briefly outline major advances in methodologies that are being utilized for induction, identification, and characterization of molecular processes in cells undergoing oncogene-induced senescence. The full description of such methodologies is provided in the corresponding chapters of the book.

Evaluation of Cellular Phenotypes Implicated in Immunopathogenesis and Monitoring Immune Reconstitution Inflammatory Syndrome in HIV/Leprosy Cases

PubMed Central

Giacoia-Gripp, Carmem Beatriz Wagner; Sales, Anna Maria; Nery, José Augusto da Costa; Santos-Oliveira, Joanna Reis; de Oliveira, Ariane Leite; Sarno, Euzenir Nunes; Morgado, Mariza Gonçalves

2011-01-01

Background It is now evident that HAART-associated immunological improvement often leads to a variety of new clinical manifestations, collectively termed immune reconstitution inflammatory syndrome, or IRIS. This phenomenon has already been described in cases of HIV coinfection with Mycobacterium leprae, most of them belonging to the tuberculoid spectrum of leprosy disease, as observed in leprosy reversal reaction (RR). However, the events related to the pathogenesis of this association need to be clarified. This study investigated the immunological profile of HIV/leprosy patients, with special attention to the cellular activation status, to better understand the mechanisms related to IRIS/RR immunopathogenesis, identifying any potential biomarkers for IRIS/RR intercurrence. Methods/Principal Findings Eighty-five individuals were assessed in this study: HIV/leprosy and HIV-monoinfected patients, grouped according to HIV-viral load levels, leprosy patients without HIV coinfection, and healthy controls. Phenotypes were evaluated by flow cytometry for T cell subsets and immune differentiation/activation markers. As expected, absolute counts of the CD4+ and CD8+ T cells from the HIV-infected individuals changed in relation to those of the leprosy patients and controls. However, there were no significant differences among the groups, whether in the expression of cellular differentiation phenotypes or cellular activation, as reflected by the expression of CD38 and HLA-DR. Six HIV/leprosy patients identified as IRIS/RR were analyzed during IRIS/RR episodes and after prednisone treatment. These patients presented high cellular activation levels regarding the expression of CD38 in CD8+ cells T during IRIS/RR (median: 77,15%), dropping significantly (p<0,05) during post-IRIS/RR moments (median: 29,7%). Furthermore, an increase of cellular activation seems to occur prior to IRIS/RR. Conclusion/Significance These data suggest CD38 expression in CD8+ T cells interesting tool identifying HIV/leprosy individuals at risk for IRIS/RR. So, a comparative investigation to leprosy patients at RR should be conducted. PMID:22205964

Evaluation of cellular phenotypes implicated in immunopathogenesis and monitoring immune reconstitution inflammatory syndrome in HIV/leprosy cases.

PubMed

Giacoia-Gripp, Carmem Beatriz Wagner; Sales, Anna Maria; Nery, José Augusto da Costa; Santos-Oliveira, Joanna Reis; de Oliveira, Ariane Leite; Sarno, Euzenir Nunes; Morgado, Mariza Gonçalves

2011-01-01

It is now evident that HAART-associated immunological improvement often leads to a variety of new clinical manifestations, collectively termed immune reconstitution inflammatory syndrome, or IRIS. This phenomenon has already been described in cases of HIV coinfection with Mycobacterium leprae, most of them belonging to the tuberculoid spectrum of leprosy disease, as observed in leprosy reversal reaction (RR). However, the events related to the pathogenesis of this association need to be clarified. This study investigated the immunological profile of HIV/leprosy patients, with special attention to the cellular activation status, to better understand the mechanisms related to IRIS/RR immunopathogenesis, identifying any potential biomarkers for IRIS/RR intercurrence. Eighty-five individuals were assessed in this study: HIV/leprosy and HIV-monoinfected patients, grouped according to HIV-viral load levels, leprosy patients without HIV coinfection, and healthy controls. Phenotypes were evaluated by flow cytometry for T cell subsets and immune differentiation/activation markers. As expected, absolute counts of the CD4+ and CD8+ T cells from the HIV-infected individuals changed in relation to those of the leprosy patients and controls. However, there were no significant differences among the groups, whether in the expression of cellular differentiation phenotypes or cellular activation, as reflected by the expression of CD38 and HLA-DR. Six HIV/leprosy patients identified as IRIS/RR were analyzed during IRIS/RR episodes and after prednisone treatment. These patients presented high cellular activation levels regarding the expression of CD38 in CD8+ cells T during IRIS/RR (median: 77,15%), dropping significantly (p<0,05) during post-IRIS/RR moments (median: 29,7%). Furthermore, an increase of cellular activation seems to occur prior to IRIS/RR. These data suggest CD38 expression in CD8+ T cells interesting tool identifying HIV/leprosy individuals at risk for IRIS/RR. So, a comparative investigation to leprosy patients at RR should be conducted.

Systems biology perspectives on minimal and simpler cells.

PubMed

Xavier, Joana C; Patil, Kiran Raosaheb; Rocha, Isabel

2014-09-01

The concept of the minimal cell has fascinated scientists for a long time, from both fundamental and applied points of view. This broad concept encompasses extreme reductions of genomes, the last universal common ancestor (LUCA), the creation of semiartificial cells, and the design of protocells and chassis cells. Here we review these different areas of research and identify common and complementary aspects of each one. We focus on systems biology, a discipline that is greatly facilitating the classical top-down and bottom-up approaches toward minimal cells. In addition, we also review the so-called middle-out approach and its contributions to the field with mathematical and computational models. Owing to the advances in genomics technologies, much of the work in this area has been centered on minimal genomes, or rather minimal gene sets, required to sustain life. Nevertheless, a fundamental expansion has been taking place in the last few years wherein the minimal gene set is viewed as a backbone of a more complex system. Complementing genomics, progress is being made in understanding the system-wide properties at the levels of the transcriptome, proteome, and metabolome. Network modeling approaches are enabling the integration of these different omics data sets toward an understanding of the complex molecular pathways connecting genotype to phenotype. We review key concepts central to the mapping and modeling of this complexity, which is at the heart of research on minimal cells. Finally, we discuss the distinction between minimizing the number of cellular components and minimizing cellular complexity, toward an improved understanding and utilization of minimal and simpler cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Fluorescence-based detection and quantification of features of cellular senescence.

PubMed

Cho, Sohee; Hwang, Eun Seong

2011-01-01

Cellular senescence is a spontaneous organismal defense mechanism against tumor progression which is raised upon the activation of oncoproteins or other cellular environmental stresses that must be circumvented for tumorigenesis to occur. It involves growth-arrest state of normal cells after a number of active divisions. There are multiple experimental routes that can drive cells into a state of senescence. Normal somatic cells and cancer cells enter a state of senescence upon overexpression of oncogenic Ras or Raf protein or by imposing certain kinds of stress such as cellular tumor suppressor function. Both flow cytometry and confocal imaging analysis techniques are very useful in quantitative analysis of cellular senescence phenomenon. They allow quantitative estimates of multiple different phenotypes expressed in multiple cell populations simultaneously. Here we review the various types of fluorescence methodologies including confocal imaging and flow cytometry that are frequently utilized to study a variety of senescence. First, we discuss key cell biological changes occurring during senescence and review the current understanding on the mechanisms of these changes with the goal of improving existing protocols and further developing new ones. Next, we list specific senescence phenotypes associated with each cellular trait along with the principles of their assay methods and the significance of the assay outcomes. We conclude by selecting appropriate references that demonstrate a typical example of each method. Copyright © 2011 Elsevier Inc. All rights reserved.

Effects of hypo-O-GlcNAcylation on Drosophila development.

PubMed

Mariappa, Daniel; Ferenbach, Andrew T; van Aalten, Daan M F

2018-05-11

Post-translational modification of serine/threonine residues in nucleocytoplasmic proteins with GlcNAc ( O -GlcNAcylation) is an essential regulatory mechanism in many cellular processes. In Drosophila , null mutants of the Polycomb gene O -GlcNAc transferase ( OGT ; also known as super sex combs ( sxc )) display homeotic phenotypes. To dissect the requirement for O -GlcNAc signaling in Drosophila development, we used CRISPR/Cas9 gene editing to generate rationally designed sxc catalytically hypomorphic or null point mutants. Of the fertile males derived from embryos injected with the CRISPR/Cas9 reagents, 25% produced progeny carrying precise point mutations with no detectable off-target effects. One of these mutants, the catalytically inactive sxc K872M , was recessive lethal, whereas a second mutant, the hypomorphic sxc H537A , was homozygous viable. We observed that reduced total protein O -GlcNAcylation in the sxc H537A mutant is associated with a wing vein phenotype and temperature-dependent lethality. Genetic interaction between sxc H537A and a null allele of Drosophila host cell factor ( dHcf ), encoding an extensively O -GlcNAcylated transcriptional coactivator, resulted in abnormal scutellar bristle numbers. A similar phenotype was also observed in sxc H537A flies lacking a copy of skuld ( skd ), a Mediator complex gene known to affect scutellar bristle formation. Interestingly, this phenotype was independent of OGT Polycomb function or dHcf downstream targets. In conclusion, the generation of the endogenous OGT hypomorphic mutant sxc H537A enabled us to identify pleiotropic effects of globally reduced protein O -GlcNAc during Drosophila development. The mutants generated and phenotypes observed in this study provide a platform for discovery of OGT substrates that are critical for Drosophila development. © 2018 Mariappa et al.

Resolving TYK2 locus genotype-to-phenotype differences in autoimmunity

PubMed Central

Dendrou, Calliope A.; Cortes, Adrian; Shipman, Lydia; Evans, Hayley G.; Attfield, Kathrine E.; Jostins, Luke; Barber, Thomas; Kaur, Gurman; Kuttikkatte, Subita Balaram; Leach, Oliver A.; Desel, Christiane; Faergeman, Soren L.; Cheeseman, Jane; Neville, Matt J.; Sawcer, Stephen; Compston, Alastair; Johnson, Adam R.; Everett, Christine; Bell, John I.; Karpe, Fredrik; Ultsch, Mark; Eigenbrot, Charles; McVean, Gil; Fugger, Lars

2017-01-01

Thousands of genetic variants have been identified that contribute to the development of complex diseases, but determining how to fully elucidate their biological consequences for translation into clinical benefit is challenging. Conflicting evidence regarding the functional impact of genetic variants in the tyrosine kinase 2 (TYK2) gene, which is differentially associated with common autoimmune diseases, currently obscures the potential of TYK2 as a therapeutic target. We aimed to resolve this conflict by performing genetic meta-analysis across disorders, subsequent molecular, cellular, in vivo and structural functional follow-up and epidemiological studies. Our data revealed a protective homozygous effect that defined a signaling optimum between autoimmunity and immunodeficiency and identified TYK2 as a potential drug target for multiple autoimmune disorders. PMID:27807284

The critical protein interactions and structures that elicit growth deregulation in cancer and viral replication

PubMed Central

Ou, Horng D.; May, Andrew P.

2010-01-01

One of the greatest challenges in biomedicine is to define the critical targets and network interactions that are subverted to elicit growth deregulation in human cells. Understanding and developing rational treatments for cancer requires a definition of the key molecular targets and how they interact to elicit the complex growth deregulation phenotype. Viral proteins provide discerning and powerful probes to understand both how cells work and how they can be manipulated using a minimal number of components. The small DNA viruses have evolved to target inherent weaknesses in cellular protein interaction networks to hijack the cellular DNA and protein replication machinery. In the battle to escape the inevitability of senescence and programmed cell death, cancers have converged on similar mechanisms, through the acquisition and selection of somatic mutations that drive unchecked cellular replication in tumors. Understanding the dynamic mechanisms through which a minimal number of viral proteins promote host cells to undergo unscheduled and pathological replication is a powerful strategy to identify critical targets that are also disrupted in cancer. Viruses can therefore be used as tools to probe the system-wide protein-protein interactions and structures that drive growth deregulation in human cells. Ultimately this can provide a path for developing system context-dependent therapeutics. This review will describe ongoing experimental approaches using viruses to study pathways deregulated in cancer, with a particular focus on viral cellular protein-protein interactions and structures. PMID:21061422

Metadata Standard and Data Exchange Specifications to Describe, Model, and Integrate Complex and Diverse High-Throughput Screening Data from the Library of Integrated Network-based Cellular Signatures (LINCS).

PubMed

Vempati, Uma D; Chung, Caty; Mader, Chris; Koleti, Amar; Datar, Nakul; Vidović, Dušica; Wrobel, David; Erickson, Sean; Muhlich, Jeremy L; Berriz, Gabriel; Benes, Cyril H; Subramanian, Aravind; Pillai, Ajay; Shamu, Caroline E; Schürer, Stephan C

2014-06-01

The National Institutes of Health Library of Integrated Network-based Cellular Signatures (LINCS) program is generating extensive multidimensional data sets, including biochemical, genome-wide transcriptional, and phenotypic cellular response signatures to a variety of small-molecule and genetic perturbations with the goal of creating a sustainable, widely applicable, and readily accessible systems biology knowledge resource. Integration and analysis of diverse LINCS data sets depend on the availability of sufficient metadata to describe the assays and screening results and on their syntactic, structural, and semantic consistency. Here we report metadata specifications for the most important molecular and cellular components and recommend them for adoption beyond the LINCS project. We focus on the minimum required information to model LINCS assays and results based on a number of use cases, and we recommend controlled terminologies and ontologies to annotate assays with syntactic consistency and semantic integrity. We also report specifications for a simple annotation format (SAF) to describe assays and screening results based on our metadata specifications with explicit controlled vocabularies. SAF specifically serves to programmatically access and exchange LINCS data as a prerequisite for a distributed information management infrastructure. We applied the metadata specifications to annotate large numbers of LINCS cell lines, proteins, and small molecules. The resources generated and presented here are freely available. © 2014 Society for Laboratory Automation and Screening.

A Modified MuDPIT Separation Identified 4,488 Proteins in a System Wide Analysis of Quiescence in Yeast

PubMed Central

Webb, Kristofor J.; Xu, Tao; Park, Sung Kyu; Yates, John R.

2013-01-01

A modified multidimensional protein identification technology (MudPIT) separation was coupled to an LTQ Orbitrap Velos mass spectrometer and used to rapidly identify the near complete yeast proteome from a whole cell tryptic digest. This modified on-line two dimensional liquid chromatography separation consists of 39 strong cation exchange steps followed by a short 18.5 min reversed-phase (RP) gradient. A total of 4,269 protein identifications were made from 4,189 distinguishable protein families from yeast during log phase growth. The “Micro” MudPIT separation performed as well as a standard MudPIT separation in 40% less gradient time. The majority of the yeast proteome can now be routinely covered in less than a days’ time with high reproducibility and sensitivity. The newly devised separation method was used to detect changes in protein expression during cellular quiescence in yeast. An enrichment in the GO annotations ‘oxidation reduction’, ‘catabolic processing’ and ‘cellular response to oxidative stress’ was seen in the quiescent cellular fraction, consistent with their long lived stress resistant phenotypes. Heterogeneity was observed in the stationary phase fraction with a less dense cell population showing reductions in KEGG pathway categories of ‘Ribosome’ and ‘Proteasome’, further defining the complex nature of yeast populations present during stationary phase growth. In total 4,488 distinguishable protein families were identified in all cellular conditions tested. PMID:23540446

Increased CYFIP1 dosage alters cellular and dendritic morphology and dysregulates mTOR.

PubMed

Oguro-Ando, A; Rosensweig, C; Herman, E; Nishimura, Y; Werling, D; Bill, B R; Berg, J M; Gao, F; Coppola, G; Abrahams, B S; Geschwind, D H

2015-09-01

Rare maternally inherited duplications at 15q11-13 are observed in ~1% of individuals with an autism spectrum disorder (ASD), making it among the most common causes of ASD. 15q11-13 comprises a complex region, and as this copy number variation encompasses many genes, it is important to explore individual genotype-phenotype relationships. Cytoplasmic FMR1-interacting protein 1 (CYFIP1) is of particular interest because of its interaction with Fragile X mental retardation protein (FMRP), its upregulation in transformed lymphoblastoid cell lines from patients with duplications at 15q11-13 and ASD and the presence of smaller overlapping deletions of CYFIP1 in patients with schizophrenia and intellectual disability. Here, we confirm that CYFIP1 is upregulated in transformed lymphoblastoid cell lines and demonstrate its upregulation in the post-mortem brain from 15q11-13 duplication patients for the first time. To investigate how increased CYFIP1 dosage might predispose to neurodevelopmental disease, we studied the consequence of its overexpression in multiple systems. We show that overexpression of CYFIP1 results in morphological abnormalities including cellular hypertrophy in SY5Y cells and differentiated mouse neuronal progenitors. We validate these results in vivo by generating a BAC transgenic mouse, which overexpresses Cyfip1 under the endogenous promotor, observing an increase in the proportion of mature dendritic spines and dendritic spine density. Gene expression profiling on embryonic day 15 suggested the dysregulation of mammalian target of rapamycin (mTOR) signaling, which was confirmed at the protein level. Importantly, similar evidence of mTOR-related dysregulation was seen in brains from 15q11-13 duplication patients with ASD. Finally, treatment of differentiated mouse neuronal progenitors with an mTOR inhibitor (rapamycin) rescued the morphological abnormalities resulting from CYFIP1 overexpression. Together, these data show that CYFIP1 overexpression results in specific cellular phenotypes and implicate modulation by mTOR signaling, further emphasizing its role as a potential convergent pathway in some forms of ASD.

Emergence of HGF/SF-Induced Coordinated Cellular Motility

PubMed Central

Zaritsky, Assaf; Natan, Sari; Ben-Jacob, Eshel; Tsarfaty, Ilan

2012-01-01

Collective cell migration plays a major role in embryonic morphogenesis, tissue remodeling, wound repair and cancer invasion. Despite many decades of extensive investigations, only few analytical tools have been developed to enhance the biological understanding of this important phenomenon. Here we present a novel quantitative approach to analyze long term kinetics of bright field time-lapse wound healing. Fully-automated spatiotemporal measures and visualization of cells' motility and implicit morphology were proven to be sound, repetitive and highly informative compared to single-cell tracking analysis. We study cellular collective migration induced by tyrosine kinase-growth factor signaling (Met-Hepatocyte Growth Factor/Scatter Factor (HGF/SF)). Our quantitative approach is applied to demonstrate that collective migration of the adenocarcinoma cell lines is characterized by simple morpho-kinetics. HGF/SF induces complex morpho-kinetic coordinated collective migration: cells at the front move faster and are more spread than those further away from the wound edge. As the wound heals, distant cells gradually accelerate and enhance spread and elongation –resembling the epithelial to mesenchymal transition (EMT), and then the cells become more spread and maintain higher velocity than cells located closer to the wound. Finally, upon wound closure, front cells halt, shrink and round up (resembling mesenchymal to epithelial transition (MET) phenotype) while distant cells undergo the same process gradually. Met inhibition experiments further validate that Met signaling dramatically alters the morpho-kinetic dynamics of the healing wound. Machine-learning classification was applied to demonstrate the generalization of our findings, revealing even subtle changes in motility patterns induced by Met-inhibition. It is concluded that activation of Met-signaling induces an elaborated model in which cells lead a coordinated increased motility along with gradual differentiation-based collective cell motility dynamics. Our quantitative phenotypes may guide future investigation on the molecular and cellular mechanisms of tyrosine kinase-induced coordinate cell motility and morphogenesis in metastasis. PMID:22970283

A rare complex DNA rearrangement in the murine Steel gene results in exon duplication and a lethal phenotype.

PubMed

Chandra, Saurabh; Kapur, Reuben; Chuzhanova, Nadia; Summey, Victoria; Prentice, David; Barker, Jane; Cooper, David N; Williams, David A

2003-11-15

Kit ligand (Kitl), encoded by the Steel (Sl) locus, plays an essential role in hematopoiesis, gametogenesis, and melanogenesis during both embryonic and adult life. We have characterized a new spontaneous mutant of the Sl locus in mice designated KitlSl-20J that arose in the breeding colony at Jackson Laboratories. Heterozygous KitlSl-20J mice display a white belly spot and intercrossing results in an embryonic lethal phenotype in the homozygous state. Analysis of homozygous embryos demonstrated a significant reduction in fetal liver cellularity, colony forming unit-erythroid (CFU-E) progenitors, and a total absence of germ cells. Although expressed in vivo, recombinant mutant protein demonstrated loss of bioactivity that was correlated with lack of receptor binding. Analysis of the Sl gene transcripts in heterozygous KitlSl-20J mice revealed an in-frame tandem duplication of exon 3. A long-range polymerase chain reaction (PCR) strategy using overlapping primers in exon 3 amplified an approximately 7-kilobase (kb) product from DNA isolated from heterozygous KitlSl-20J mice but not from wild-type DNA that contained sequences from both introns 2 and 3 and an inverted intron 2 sequence, suggesting a complex rearrangement as the mechanism of the mutation. "Complexity analysis" of the sequence of the amplified product strongly suggests that local DNA motifs may have contributed to the generation of this spontaneous KitlSl-20J allele, likely mediated by a 2-step process. The KitlSl-20J mutation is a unique KitlSl allele and represents an unusual mechanism of mutation.

How to Train a Cell - Cutting-Edge Molecular Tools

NASA Astrophysics Data System (ADS)

Czapiński, Jakub; Kiełbus, Michał; Kałafut, Joanna; Kos, Michał; Stepulak, Andrzej; Rivero-Müller, Adolfo

2017-03-01

In biological systems, the formation of molecular complexes is the currency for all cellular processes. Traditionally, functional experimentation was targeted to single molecular players in order to understand its effects in a cell or animal phenotype. In the last few years, we have been experiencing rapid progress in the development of ground-breaking molecular biology tools that affect the metabolic, structural, morphological, and (epi)genetic instructions of cells by chemical, optical (optogenetic) and mechanical inputs. Such precise dissection of cellular processes is not only essential for a better understanding of biological systems, but will also allow us to better diagnose and fix common dysfunctions. Here, we present several of these emerging and innovative techniques by providing the reader with elegant examples on how these tools have been implemented in cells, and, in some cases, organisms, to unravel molecular processes in minute detail. We also discuss their advantages and disadvantages with particular focus on their translation to multicellular organisms for in vivo spatiotemporal regulation. We envision that further developments of these tools will not only help solve the processes of life, but will give rise to novel clinical and industrial applications.

A cellular model for sporadic ALS using patient-derived induced pluripotent stem cells

PubMed Central

Burkhardt, Matthew F; Martinez, Fernando J; Wright, Sarah; Ramos, Carla; Volfson, Dmitri; Mason, Michael; Garnes, Jeff; Dang, Vu; Lievers, Jeffery; Shoukat-Mumtaz, Uzma; Martinez, Rita; Gai, Hui; Blake, Robert; Vaisberg, Eugeni; Grskovic, Marica; Johnson, Charles; Irion, Stefan; Bright, Jessica; Cooper, Bonnie; Nguyen, Leane; Griswold-Prenner, Irene; Javaherian, Ashkan

2016-01-01

Development of therapeutics for genetically complex neurodegenerative diseases such as sporadic amyotrophic lateral sclerosis (ALS) has largely been hampered by lack of relevant disease models. Reprogramming of sporadic ALS patients’ fibroblasts into induced pluripotent stem cells (iPSC) and differentiation into affected neurons that show a disease phenotype could provide a cellular model for disease mechanism studies and drug discovery. Here we report the reprogramming to pluripotency of fibroblasts from a large cohort of healthy controls and ALS patients and their differentiation into motor neurons. We demonstrate that motor neurons derived from three sALS patients show de novo TDP-43 aggregation and that the aggregates recapitulate pathology in postmortem tissue from one of the same patients from which the iPSC were derived. We configured a high-content chemical screen using the TDP-43 aggregate endpoint both in lower motor neurons and upper motor neuron like cells and identified FDA-approved small molecule modulators including Digoxin demonstrating the feasibility of patient-derived iPSC-based disease modelling for drug screening. PMID:23891805

Common Chemical Inductors of Replication Stress:  Focus on Cell-Based Studies.

PubMed

Vesela, Eva; Chroma, Katarina; Turi, Zsofia; Mistrik, Martin

2017-02-21

DNA replication is a highly demanding process regarding the energy and material supply and must be precisely regulated, involving multiple cellular feedbacks. The slowing down or stalling of DNA synthesis and/or replication forks is referred to as replication stress (RS). Owing to the complexity and requirements of replication, a plethora of factors may interfere and challenge the genome stability, cell survival or affect the whole organism. This review outlines chemical compounds that are known inducers of RS and commonly used in laboratory research. These compounds act on replication by direct interaction with DNA causing DNA crosslinks and bulky lesions (cisplatin), chemical interference with the metabolism of deoxyribonucleotide triphosphates (hydroxyurea), direct inhibition of the activity of replicative DNA polymerases (aphidicolin) and interference with enzymes dealing with topological DNA stress (camptothecin, etoposide). As a variety of mechanisms can induce RS, the responses of mammalian cells also vary. Here, we review the activity and mechanism of action of these compounds based on recent knowledge, accompanied by examples of induced phenotypes, cellular readouts and commonly used doses.

Common Chemical Inductors of Replication Stress: Focus on Cell-Based Studies

PubMed Central

Vesela, Eva; Chroma, Katarina; Turi, Zsofia; Mistrik, Martin

2017-01-01

DNA replication is a highly demanding process regarding the energy and material supply and must be precisely regulated, involving multiple cellular feedbacks. The slowing down or stalling of DNA synthesis and/or replication forks is referred to as replication stress (RS). Owing to the complexity and requirements of replication, a plethora of factors may interfere and challenge the genome stability, cell survival or affect the whole organism. This review outlines chemical compounds that are known inducers of RS and commonly used in laboratory research. These compounds act on replication by direct interaction with DNA causing DNA crosslinks and bulky lesions (cisplatin), chemical interference with the metabolism of deoxyribonucleotide triphosphates (hydroxyurea), direct inhibition of the activity of replicative DNA polymerases (aphidicolin) and interference with enzymes dealing with topological DNA stress (camptothecin, etoposide). As a variety of mechanisms can induce RS, the responses of mammalian cells also vary. Here, we review the activity and mechanism of action of these compounds based on recent knowledge, accompanied by examples of induced phenotypes, cellular readouts and commonly used doses. PMID:28230817

Open PHACTS computational protocols for in silico target validation of cellular phenotypic screens: knowing the knowns† †The authors declare no competing interests. ‡ ‡Electronic supplementary information (ESI) available: Pipeline Pilot protocols, xls file with the output of the Pipeline Pilot protocols, KNIME workflows, and supplementary figures showing the Pipeline Pilot protocols. See DOI: 10.1039/c6md00065g Click here for additional data file.

PubMed Central

Zdrazil, B.; Neefs, J.-M.; Van Vlijmen, H.; Herhaus, C.; Caracoti, A.; Brea, J.; Roibás, B.; Loza, M. I.; Queralt-Rosinach, N.; Furlong, L. I.; Gaulton, A.; Bartek, L.; Senger, S.; Chichester, C.; Engkvist, O.; Evelo, C. T.; Franklin, N. I.; Marren, D.; Ecker, G. F.

2016-01-01

Phenotypic screening is in a renaissance phase and is expected by many academic and industry leaders to accelerate the discovery of new drugs for new biology. Given that phenotypic screening is per definition target agnostic, the emphasis of in silico and in vitro follow-up work is on the exploration of possible molecular mechanisms and efficacy targets underlying the biological processes interrogated by the phenotypic screening experiments. Herein, we present six exemplar computational protocols for the interpretation of cellular phenotypic screens based on the integration of compound, target, pathway, and disease data established by the IMI Open PHACTS project. The protocols annotate phenotypic hit lists and allow follow-up experiments and mechanistic conclusions. The annotations included are from ChEMBL, ChEBI, GO, WikiPathways and DisGeNET. Also provided are protocols which select from the IUPHAR/BPS Guide to PHARMACOLOGY interaction file selective compounds to probe potential targets and a correlation robot which systematically aims to identify an overlap of active compounds in both the phenotypic as well as any kinase assay. The protocols are applied to a phenotypic pre-lamin A/C splicing assay selected from the ChEMBL database to illustrate the process. The computational protocols make use of the Open PHACTS API and data and are built within the Pipeline Pilot and KNIME workflow tools. PMID:27774140

Mapping the Complex Morphology of Cell Interactions with Nanowire Substrates Using FIB-SEM

PubMed Central

Jensen, Mikkel R. B.; Łopacińska, Joanna; Schmidt, Michael S.; Skolimowski, Maciej; Abeille, Fabien; Qvortrup, Klaus; Mølhave, Kristian

2013-01-01

Using high resolution focused ion beam scanning electron microscopy (FIB-SEM) we study the details of cell-nanostructure interactions using serial block face imaging. 3T3 Fibroblast cellular monolayers are cultured on flat glass as a control surface and on two types of nanostructured scaffold substrates made from silicon black (Nanograss) with low- and high nanowire density. After culturing for 72 hours the cells were fixed, heavy metal stained, embedded in resin, and processed with FIB-SEM block face imaging without removing the substrate. The sample preparation procedure, image acquisition and image post-processing were specifically optimised for cellular monolayers cultured on nanostructured substrates. Cells display a wide range of interactions with the nanostructures depending on the surface morphology, but also greatly varying from one cell to another on the same substrate, illustrating a wide phenotypic variability. Depending on the substrate and cell, we observe that cells could for instance: break the nanowires and engulf them, flatten the nanowires or simply reside on top of them. Given the complexity of interactions, we have categorised our observations and created an overview map. The results demonstrate that detailed nanoscale resolution images are required to begin understanding the wide variety of individual cells’ interactions with a structured substrate. The map will provide a framework for light microscopy studies of such interactions indicating what modes of interactions must be considered. PMID:23326412

Targeting of the hydrophobic metabolome by pathogens.

PubMed

Helms, J Bernd; Kaloyanova, Dora V; Strating, Jeroen R P; van Hellemond, Jaap J; van der Schaar, Hilde M; Tielens, Aloysius G M; van Kuppeveld, Frank J M; Brouwers, Jos F

2015-05-01

The hydrophobic molecules of the metabolome - also named the lipidome - constitute a major part of the entire metabolome. Novel technologies show the existence of a staggering number of individual lipid species, the biological functions of which are, with the exception of only a few lipid species, unknown. Much can be learned from pathogens that have evolved to take advantage of the complexity of the lipidome to escape the immune system of the host organism and to allow their survival and replication. Different types of pathogens target different lipids as shown in interaction maps, allowing visualization of differences between different types of pathogens. Bacterial and viral pathogens target predominantly structural and signaling lipids to alter the cellular phenotype of the host cell. Fungal and parasitic pathogens have complex lipidomes themselves and target predominantly the release of polyunsaturated fatty acids from the host cell lipidome, resulting in the generation of eicosanoids by either the host cell or the pathogen. Thus, whereas viruses and bacteria induce predominantly alterations in lipid metabolites at the host cell level, eukaryotic pathogens focus on interference with lipid metabolites affecting systemic inflammatory reactions that are part of the immune system. A better understanding of the interplay between host-pathogen interactions will not only help elucidate the fundamental role of lipid species in cellular physiology, but will also aid in the generation of novel therapeutic drugs. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Nutrient-dependent increased dendritic arborization of somatosensory neurons.

PubMed

Watanabe, Kaori; Furumizo, Yuki; Usui, Tadao; Hattori, Yukako; Uemura, Tadashi

2017-01-01

Suboptimal nutrition imposes developmental constraints on infant animals, which marshal adaptive responses to eventually become mature adults. Such responses are mounted at multiple levels from systemic to cellular. At the cellular level, the underlying mechanisms of cell proliferation control have been intensively studied. However, less is known about how growth of postmitotic and morphologically complex cells, such as neurons, is controlled by nutritional status. We address this question using Class I and Class IV dendritic arborization neurons in Drosophila larvae. Class IV neurons have been shown to sense nociceptive thermal, mechanical and light stimuli, whereas Class I neurons are proprioceptors. We reared larvae on diets with different protein and carbohydrate content throughout larval stages and examined how morphologies of Class I or Class IV neurons were affected. Dendritic arbors of Class IV neurons became more complex when larvae were reared on a low-yeast diet, which contains lower amounts of amino acids and other ingredients, compared to a high-yeast diet. In contrast, such low-yeast-dependent hyperarborization was not seen in Class I neurons. The physiological and metabolic implications of the hyperarborization phenotype are discussed in relation to a recent hypothesis that Class IV neurons sense protein-deficient stress and to our characterization of how the dietary yeast contents impacted larval metabolism. © 2016 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Sod1 Loss Induces Intrinsic Superoxide Accumulation Leading to p53-Mediated Growth Arrest and Apoptosis

PubMed Central

Watanabe, Kenji; Shibuya, Shuichi; Koyama, Hirofumi; Ozawa, Yusuke; Toda, Toshihiko; Yokote, Koutaro; Shimizu, Takahiko

2013-01-01

Oxidative damages induced by a redox imbalance cause age-related changes in cells and tissues. Superoxide dismutase (SOD) enzymes play a major role in the antioxidant system and they also catalyze superoxide radicals (O2•−). Since the loss of cytoplasmic SOD (SOD1) resulted in aging-like phenotypes in several types of mouse tissue, SOD1 is essential for the maintenance of tissue homeostasis. To clarify the cellular function of SOD1, we investigated the cellular phenotypes of Sod1-deficient fibroblasts. We demonstrated that Sod1 deficiency impaired proliferation and induced apoptosis associated with O2•− accumulation in the cytoplasm and mitochondria in fibroblasts. Sod1 loss also decreased the mitochondrial membrane potential and led to DNA damage-mediated p53 activation. Antioxidant treatments effectively improved the cellular phenotypes through suppression of both intracellular O2•− accumulation and p53 activation in Sod1-deficient fibroblasts. In vivo experiments revealed that transdermal treatment with a vitamin C derivative significantly reversed the skin thinning commonly associated with the upregulated p53 action in the skin. Our findings revealed that intrinsic O2•− accumulation promoted p53-mediated growth arrest and apoptosis as well as mitochondrial disfunction in the fibroblasts. PMID:23708100

Loss of the Mechanotransducer Zyxin Promotes a Synthetic Phenotype of Vascular Smooth Muscle Cells

PubMed Central

Ghosh, Subhajit; Kollar, Branislav; Nahar, Taslima; Suresh Babu, Sahana; Wojtowicz, Agnieszka; Sticht, Carsten; Gretz, Norbert; Wagner, Andreas H; Korff, Thomas; Hecker, Markus

2015-01-01

Background Exposure of vascular smooth muscle cells (VSMCs) to excessive cyclic stretch such as in hypertension causes a shift in their phenotype. The focal adhesion protein zyxin can transduce such biomechanical stimuli to the nucleus of both endothelial cells and VSMCs, albeit with different thresholds and kinetics. However, there is no distinct vascular phenotype in young zyxin-deficient mice, possibly due to functional redundancy among other gene products belonging to the zyxin family. Analyzing zyxin function in VSMCs at the cellular level might thus offer a better mechanistic insight. We aimed to characterize zyxin-dependent changes in gene expression in VSMCs exposed to biomechanical stretch and define the functional role of zyxin in controlling the resultant VSMC phenotype. Methods and Results DNA microarray analysis was used to identify genes and pathways that were zyxin regulated in static and stretched human umbilical artery–derived and mouse aortic VSMCs. Zyxin-null VSMCs showed a remarkable shift to a growth-promoting, less apoptotic, promigratory and poorly contractile phenotype with ≈90% of the stretch-responsive genes being zyxin dependent. Interestingly, zyxin-null cells already seemed primed for such a synthetic phenotype, with mechanical stretch further accentuating it. This could be accounted for by higher RhoA activity and myocardin-related transcription factor-A mainly localized to the nucleus of zyxin-null VSMCs, and a condensed and localized accumulation of F-actin upon stretch. Conclusions At the cellular level, zyxin is a key regulator of stretch-induced gene expression. Loss of zyxin drives VSMCs toward a synthetic phenotype, a process further consolidated by exaggerated stretch. PMID:26071033

Thioredoxin 1 and glutaredoxin 2 contribute to maintain the phenotype and integrity of neurons following perinatal asphyxia.

PubMed

Romero, Juan Ignacio; Hanschmann, Eva-Maria; Gellert, Manuela; Eitner, Susanne; Holubiec, Mariana Inés; Blanco-Calvo, Eduardo; Lillig, Christopher Horst; Capani, Francisco

2015-06-01

Thioredoxin (Trx) family proteins are crucial mediators of cell functions via regulation of the thiol redox state of various key proteins and the levels of the intracellular second messenger hydrogen peroxide. Their expression, localization and functions are altered in various pathologies. Here, we have analyzed the impact of Trx family proteins in neuronal development and recovery, following hypoxia/ischemia and reperfusion. We have analyzed the regulation and potential functions of Trx family proteins during hypoxia/ischemia and reoxygenation of the developing brain in both an animal and a cellular model of perinatal asphyxia. We have analyzed the distribution of 14 Trx family and related proteins in the cerebellum, striatum, and hippocampus, three areas of the rat brain that are especially susceptible to hypoxia. Using SH-SY5Y cells subjected to hypoxia and reoxygenation, we have analyzed the functions of some redoxins suggested by the animal experiment. We have described/discovered a complex, cell-type and tissue-specific expression pattern following the hypoxia/ischemia and reoxygenation. Particularly, Grx2 and Trx1 showed distinct changes during tissue recovery following hypoxia/ischemia and reoxygenation. Silencing of these proteins in SH-SY5Y cells subjected to hypoxia-reoxygenation confirmed that these proteins are required to maintain the normal neuronal phenotype. These findings demonstrate the significance of redox signaling in cellular pathways. Grx2 and Trx1 contribute significantly to neuronal integrity and could be clinically relevant in neuronal damage following perinatal asphyxia and other neuronal disorders. Copyright © 2015 Elsevier B.V. All rights reserved.

Protocol for Identifying Natural Agents That Selectively Affect Adhesion, Thickness, Architecture, Cellular Phenotypes, Extracellular Matrix, and Human White Blood Cell Impenetrability of Candida albicans Biofilms

PubMed Central

Park, Yang-Nim; Srikantha, Thyagarajan; Daniels, Karla J.; Jacob, Melissa R.; Agarwal, Ameeta K.; Li, Xing-Cong

2017-01-01

ABSTRACT In the screening of natural plant extracts for antifungal activity, assessment of their effects on the growth of cells in suspension or in the wells of microtiter plates is expedient. However, microorganisms, including Candida albicans, grow in nature as biofilms, which are organized cellular communities with a complex architecture capable of conditioning their microenvironment, communicating, and excluding low- and high-molecular-weight molecules and white blood cells. Here, a confocal laser scanning microscopy (CLSM) protocol for testing the effects of large numbers of agents on biofilm development is described. The protocol assessed nine parameters from a single z-stack series of CLSM scans for each individual biofilm analyzed. The parameters included adhesion, thickness, formation of a basal yeast cell polylayer, hypha formation, the vertical orientation of hyphae, the hyphal bend point, pseudohypha formation, calcofluor white staining of the extracellular matrix (ECM), and human white blood cell impenetrability. The protocol was applied first to five plant extracts and derivative compounds and then to a collection of 88 previously untested plant extracts. They were found to cause a variety of phenotypic profiles, as was the case for 64 of the 88 extracts (73%). Half of the 46 extracts that did not affect biofilm thickness affected other biofilm parameters. Correlations between specific effects were revealed. The protocol will be useful not only in the screening of chemical libraries but also in the analysis of compounds with known effects and mutations. PMID:28893778

Understanding the cancer cell phenotype beyond the limitations of current omics analyses.

PubMed

Moreno-Sánchez, Rafael; Saavedra, Emma; Gallardo-Pérez, Juan Carlos; Rumjanek, Franklin D; Rodríguez-Enríquez, Sara

2016-01-01

Efforts to understand the mechanistic principles driving cancer metabolism and proliferation have been lately governed by genomic, transcriptomic and proteomic studies. This paper analyzes the caveats of these approaches. As molecular biology's central dogma proposes a unidirectional flux of information from genes to mRNA to proteins, it has frequently been assumed that monitoring the changes in the gene sequences and in mRNA and protein contents is sufficient to explain complex cellular processes. Such a stance commonly disregards that post-translational modifications can alter the protein function/activity and also that regulatory mechanisms enter into action, to coordinate the protein activities of pathways/cellular processes, in order to keep the cellular homeostasis. Hence, the actual protein activities (as enzymes/transporters/receptors) and their regulatory mechanisms ultimately dictate the final outcomes of a pathway/cellular process. In this regard, it is here documented that the mRNA levels of many metabolic enzymes and transcriptional factors have no correlation with the respective protein contents and activities. The validity of current clinical mRNA-based tests and proposed metabolite biomarkers for cancer detection/prognosis is also discussed. Therefore, it is proposed that, to achieve a thorough understanding of the modifications undergone by proliferating cancer cells, it is mandatory to experimentally analyze the cellular processes at the functional level. This could be achieved (a) locally, by examining the actual protein activities in the cell and their kinetic properties (or at least kinetically characterize the most controlling steps of the pathway/cellular process); (b) systemically, by analyzing the main fluxes of the pathway/cellular process, and how they are modulated by metabolites, all which should contribute to comprehending the regulatory mechanisms that have been altered in cancer cells. By adopting a more holistic approach it may become possible to improve the design of therapeutic strategies that would target cancer cells more specifically. © 2015 FEBS.

Genotypic Complexity of Fisher’s Geometric Model

PubMed Central

Hwang, Sungmin; Park, Su-Chan; Krug, Joachim

2017-01-01

Fisher’s geometric model was originally introduced to argue that complex adaptations must occur in small steps because of pleiotropic constraints. When supplemented with the assumption of additivity of mutational effects on phenotypic traits, it provides a simple mechanism for the emergence of genotypic epistasis from the nonlinear mapping of phenotypes to fitness. Of particular interest is the occurrence of reciprocal sign epistasis, which is a necessary condition for multipeaked genotypic fitness landscapes. Here we compute the probability that a pair of randomly chosen mutations interacts sign epistatically, which is found to decrease with increasing phenotypic dimension n, and varies nonmonotonically with the distance from the phenotypic optimum. We then derive expressions for the mean number of fitness maxima in genotypic landscapes comprised of all combinations of L random mutations. This number increases exponentially with L, and the corresponding growth rate is used as a measure of the complexity of the landscape. The dependence of the complexity on the model parameters is found to be surprisingly rich, and three distinct phases characterized by different landscape structures are identified. Our analysis shows that the phenotypic dimension, which is often referred to as phenotypic complexity, does not generally correlate with the complexity of fitness landscapes and that even organisms with a single phenotypic trait can have complex landscapes. Our results further inform the interpretation of experiments where the parameters of Fisher’s model have been inferred from data, and help to elucidate which features of empirical fitness landscapes can be described by this model. PMID:28450460

Phenotypic Characterization of Retinoic Acid Differentiated SH-SY5Y Cells by Transcriptional Profiling

PubMed Central

Korecka, Joanna A.; van Kesteren, Ronald E.; Blaas, Eva; Spitzer, Sonia O.; Kamstra, Jorke H.; Smit, August B.; Swaab, Dick F.; Verhaagen, Joost; Bossers, Koen

2013-01-01

Multiple genetic and environmental factors play a role in the development and progression of Parkinson’s disease (PD). The main neuropathological hallmark of PD is the degeneration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta. To study genetic and molecular contributors to the disease process, there is a great need for readily accessible cells with prominent DAergic features that can be used for reproducible in vitro cellular screening. Here, we investigated the molecular phenotype of retinoic acid (RA) differentiated SH-SY5Y cells using genome wide transcriptional profiling combined with gene ontology, transcription factor and molecular pathway analysis. We demonstrated that RA induces a general neuronal differentiation program in SH-SY5Y cells and that these cells develop a predominantly mature DAergic-like neurotransmitter phenotype. This phenotype is characterized by increased dopamine levels together with a substantial suppression of other neurotransmitter phenotypes, such as those for noradrenaline, acetylcholine, glutamate, serotonin and histamine. In addition, we show that RA differentiated SH-SY5Y cells express the dopamine and noradrenalin neurotransmitter transporters that are responsible for uptake of MPP(+), a well known DAergic cell toxicant. MPP(+) treatment alters mitochondrial activity according to its proposed cytotoxic effect in DAergic neurons. Taken together, RA differentiated SH-SY5Y cells have a DAergic-like phenotype, and provide a good cellular screening tool to find novel genes or compounds that affect cytotoxic processes that are associated with PD. PMID:23724009

Cellular microparticle and thrombogram phenotypes in the Prospective Observational Multicenter Major Trauma Transfusion (PROMMTT) Study: correlation with coagulopathy

PubMed Central

Matijevic, Nena; Wang, Yao-Wei W.; Wade, Charles E.; Holcomb, John B.; Cotton, Bryan A.; Schreiber, Martin A.; Muskat, Peter; Fox, Erin E.; del Junco, Deborah J.; Cardenas, Jessica C.; Rahbar, Mohammad H.; Cohen, Mitchell Jay

2014-01-01

Background Trauma-induced coagulopathy following severe injury is associated with increased bleeding and mortality. Injury may result in alteration of cellular phenotypes and release of cell-derived microparticles (MP). Circulating MPs are procoagulant and support thrombin generation (TG) and clotting. We evaluated MP and TG phenotypes in severely injured patients at admission, in relation to coagulopathy and bleeding. Methods As part of the Prospective Observational Multicenter Major Trauma Transfusion (PROMMTT) study, research blood samples were obtained from 180 trauma patients requiring transfusions at 5 participating centers. Twenty five healthy controls and 40 minimally injured patients were analyzed for comparisons. Laboratory criteria for coagulopathy was activated partial thromboplastin time (APTT) ≥35 sec. Samples were analyzed by Calibrated Automated Thrombogram to assess TG, and by flow cytometry for MP phenotypes [platelet (PMP), erythrocyte (RMP), leukocyte (LMP), endothelial (EMP), tissue factor (TFMP), and Annexin V positive (AVMP)]. Results 21.7% of patients were coagulopathic with the median (IQR) APTT of 44 sec (37, 53), and an Injury Severity Score of 26 (17, 35). Compared to controls, patients had elevated EMP, RMP, LMP, and TFMP (all p<0.001), and enhanced TG (p<0.0001). However, coagulopathic PROMMTT patients had significantly lower PMP, TFMP, and TG, higher substantial bleeding, and higher mortality compared to non-coagulopathic patients (all p<0.001). Conclusions Cellular activation and enhanced TG are predominant after trauma and independent of injury severity. Coagulopathy was associated with lower thrombin peak and rate compared to non-coagulopathic patients, while lower levels of TF-bearing PMPs were associated with substantial bleeding. PMID:25086657

Semiautomated confocal imaging of fungal pathogenesis on plants: Microscopic analysis of macroscopic specimens.

PubMed

Minker, Katharine R; Biedrzycki, Meredith L; Kolagunda, Abhishek; Rhein, Stephen; Perina, Fabiano J; Jacobs, Samuel S; Moore, Michael; Jamann, Tiffany M; Yang, Qin; Nelson, Rebecca; Balint-Kurti, Peter; Kambhamettu, Chandra; Wisser, Randall J; Caplan, Jeffrey L

2018-02-01

The study of phenotypic variation in plant pathogenesis provides fundamental information about the nature of disease resistance. Cellular mechanisms that alter pathogenesis can be elucidated with confocal microscopy; however, systematic phenotyping platforms-from sample processing to image analysis-to investigate this do not exist. We have developed a platform for 3D phenotyping of cellular features underlying variation in disease development by fluorescence-specific resolution of host and pathogen interactions across time (4D). A confocal microscopy phenotyping platform compatible with different maize-fungal pathosystems (fungi: Setosphaeria turcica, Cochliobolus heterostrophus, and Cercospora zeae-maydis) was developed. Protocols and techniques were standardized for sample fixation, optical clearing, species-specific combinatorial fluorescence staining, multisample imaging, and image processing for investigation at the macroscale. The sample preparation methods presented here overcome challenges to fluorescence imaging such as specimen thickness and topography as well as physiological characteristics of the samples such as tissue autofluorescence and presence of cuticle. The resulting imaging techniques provide interesting qualitative and quantitative information not possible with conventional light or electron 2D imaging. Microsc. Res. Tech., 81:141-152, 2018. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The antiproliferative aspects of mortalin (review).

PubMed

Wadhwa, R; Mitsui, Y; Ide, T; Kaul, S

1995-07-01

Cellular mortal and immortal phenotypes as defined by the limited and the infinite capacity of cells to divide are the characteristics of normal and cancerous cells in culture. Numerous strategies that have been employed to understand the mechanism(s) of normal as well as tumor cell growth have revealed that these are genetically controlled, however, the genes and the synchronized regulations remain largely undefined so far. The present report reviews the identification of mortalin, a novel member of murine hsp70 family of proteins, as a gene involved in pathways that determine divisional phenotype of cells in vitro. In the present study, the anti-proliferative activity of mortalin is demonstrated also in human skin fibroblasts (TIG-73PD) by microinjection of anti-mortalin antibody. Furthermore, studies on the mortalin immunofluorescence patterns in SV40-immortalized pre-crisis and post-crisis human cells have revealed that the change in the intracellular distribution of mortalin is linked to the change in the divisional phenotype of cells. Thus, the studies to resolve the molecular basis of association of the cytosolically distributed form of mortalin with cellular mortal phenotype would be important in understanding of the mechanism(s) that determine replicative potential of cells in culture.

A platform for high-throughput bioenergy production phenotype characterization in single cells

PubMed Central

Kelbauskas, Laimonas; Glenn, Honor; Anderson, Clifford; Messner, Jacob; Lee, Kristen B.; Song, Ganquan; Houkal, Jeff; Su, Fengyu; Zhang, Liqiang; Tian, Yanqing; Wang, Hong; Bussey, Kimberly; Johnson, Roger H.; Meldrum, Deirdre R.

2017-01-01

Driven by an increasing number of studies demonstrating its relevance to a broad variety of disease states, the bioenergy production phenotype has been widely characterized at the bulk sample level. Its cell-to-cell variability, a key player associated with cancer cell survival and recurrence, however, remains poorly understood due to ensemble averaging of the current approaches. We present a technology platform for performing oxygen consumption and extracellular acidification measurements of several hundreds to 1,000 individual cells per assay, while offering simultaneous analysis of cellular communication effects on the energy production phenotype. The platform comprises two major components: a tandem optical sensor for combined oxygen and pH detection, and a microwell device for isolation and analysis of single and few cells in hermetically sealed sub-nanoliter chambers. Our approach revealed subpopulations of cells with aberrant energy production profiles and enables determination of cellular response variability to electron transfer chain inhibitors and ion uncouplers. PMID:28349963

Microenvironmental independence associated with tumor progression.

PubMed

Anderson, Alexander R A; Hassanein, Mohamed; Branch, Kevin M; Lu, Jenny; Lobdell, Nichole A; Maier, Julie; Basanta, David; Weidow, Brandy; Narasanna, Archana; Arteaga, Carlos L; Reynolds, Albert B; Quaranta, Vito; Estrada, Lourdes; Weaver, Alissa M

2009-11-15

Tumor-microenvironment interactions are increasingly recognized to influence tumor progression. To understand the competitive dynamics of tumor cells in diverse microenvironments, we experimentally parameterized a hybrid discrete-continuum mathematical model with phenotypic trait data from a set of related mammary cell lines with normal, transformed, or tumorigenic properties. Surprisingly, in a resource-rich microenvironment, with few limitations on proliferation or migration, transformed (but not tumorigenic) cells were most successful and outcompeted other cell types in heterogeneous tumor simulations. Conversely, constrained microenvironments with limitations on space and/or growth factors gave a selective advantage to phenotypes derived from tumorigenic cell lines. Analysis of the relative performance of each phenotype in constrained versus unconstrained microenvironments revealed that, although all cell types grew more slowly in resource-constrained microenvironments, the most aggressive cells were least affected by microenvironmental constraints. A game theory model testing the relationship between microenvironment resource availability and competitive cellular dynamics supports the concept that microenvironmental independence is an advantageous cellular trait in resource-limited microenvironments.

Mechanism of hard-nanomaterial clearance by the liver.

PubMed

Tsoi, Kim M; MacParland, Sonya A; Ma, Xue-Zhong; Spetzler, Vinzent N; Echeverri, Juan; Ouyang, Ben; Fadel, Saleh M; Sykes, Edward A; Goldaracena, Nicolas; Kaths, Johann M; Conneely, John B; Alman, Benjamin A; Selzner, Markus; Ostrowski, Mario A; Adeyi, Oyedele A; Zilman, Anton; McGilvray, Ian D; Chan, Warren C W

2016-11-01

The liver and spleen are major biological barriers to translating nanomedicines because they sequester the majority of administered nanomaterials and prevent delivery to diseased tissue. Here we examined the blood clearance mechanism of administered hard nanomaterials in relation to blood flow dynamics, organ microarchitecture and cellular phenotype. We found that nanomaterial velocity reduces 1,000-fold as they enter and traverse the liver, leading to 7.5 times more nanomaterial interaction with hepatic cells relative to peripheral cells. In the liver, Kupffer cells (84.8 ± 6.4%), hepatic B cells (81.5 ± 9.3%) and liver sinusoidal endothelial cells (64.6 ± 13.7%) interacted with administered PEGylated quantum dots, but splenic macrophages took up less material (25.4 ± 10.1%) due to differences in phenotype. The uptake patterns were similar for two other nanomaterial types and five different surface chemistries. Potential new strategies to overcome off-target nanomaterial accumulation may involve manipulating intra-organ flow dynamics and modulating the cellular phenotype to alter hepatic cell interactions.

Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping.

PubMed

Treweek, Jennifer B; Chan, Ken Y; Flytzanis, Nicholas C; Yang, Bin; Deverman, Benjamin E; Greenbaum, Alon; Lignell, Antti; Xiao, Cheng; Cai, Long; Ladinsky, Mark S; Bjorkman, Pamela J; Fowlkes, Charless C; Gradinaru, Viviana

2015-11-01

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1-2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks.

Interactome Analyses of Mature γ-Secretase Complexes Reveal Distinct Molecular Environments of Presenilin (PS) Paralogs and Preferential Binding of Signal Peptide Peptidase to PS2*

PubMed Central

Jeon, Amy Hye Won; Böhm, Christopher; Chen, Fusheng; Huo, Hairu; Ruan, Xueying; Ren, Carl He; Ho, Keith; Qamar, Seema; Mathews, Paul M.; Fraser, Paul E.; Mount, Howard T. J.; St George-Hyslop, Peter; Schmitt-Ulms, Gerold

2013-01-01

γ-Secretase plays a pivotal role in the production of neurotoxic amyloid β-peptides (Aβ) in Alzheimer disease (AD) and consists of a heterotetrameric core complex that includes the aspartyl intramembrane protease presenilin (PS). The human genome codes for two presenilin paralogs. To understand the causes for distinct phenotypes of PS paralog-deficient mice and elucidate whether PS mutations associated with early-onset AD affect the molecular environment of mature γ-secretase complexes, quantitative interactome comparisons were undertaken. Brains of mice engineered to express wild-type or mutant PS1, or HEK293 cells stably expressing PS paralogs with N-terminal tandem-affinity purification tags served as biological source materials. The analyses revealed novel interactions of the γ-secretase core complex with a molecular machinery that targets and fuses synaptic vesicles to cellular membranes and with the H+-transporting lysosomal ATPase macrocomplex but uncovered no differences in the interactomes of wild-type and mutant PS1. The catenin/cadherin network was almost exclusively found associated with PS1. Another intramembrane protease, signal peptide peptidase, predominantly co-purified with PS2-containing γ-secretase complexes and was observed to influence Aβ production. PMID:23589300

Usher syndrome: molecular links of pathogenesis, proteins and pathways.

PubMed

Kremer, Hannie; van Wijk, Erwin; Märker, Tina; Wolfrum, Uwe; Roepman, Ronald

2006-10-15

Usher syndrome is the most common form of deaf-blindness. The syndrome is both clinically and genetically heterogeneous, and to date, eight causative genes have been identified. The proteins encoded by these genes are part of a dynamic protein complex that is present in hair cells of the inner ear and in photoreceptor cells of the retina. The localization of the Usher proteins and the phenotype in animal models indicate that the Usher protein complex is essential in the morphogenesis of the stereocilia bundle in hair cells and in the calycal processes of photoreceptor cells. In addition, the Usher proteins are important in the synaptic processes of both cell types. The association of other proteins with the complex indicates functional links to a number of basic cell-biological processes. Prominently present is the connection to the dynamics of the actin cytoskeleton, involved in cellular morphology, cell polarity and cell-cell interactions. The Usher protein complex can also be linked to the cadherins/catenins in the adherens junction-associated protein complexes, suggesting a role in cell polarity and tissue organization. A third link can be established to the integrin transmembrane signaling network. The Usher interactome, as outlined in this review, participates in pathways common in inner ear and retina that are disrupted in the Usher syndrome.

Alteration of ROS Homeostasis and Decreased Lifespan in S. cerevisiae Elicited by Deletion of the Mitochondrial Translocator FLX1

PubMed Central

Giancaspero, Teresa Anna; Dipalo, Emilia; Miccolis, Angelica; Boles, Eckhard; Caselle, Michele; Barile, Maria

2014-01-01

This paper deals with the control exerted by the mitochondrial translocator FLX1, which catalyzes the movement of the redox cofactor FAD across the mitochondrial membrane, on the efficiency of ATP production, ROS homeostasis, and lifespan of S. cerevisiae. The deletion of the FLX1 gene resulted in respiration-deficient and small-colony phenotype accompanied by a significant ATP shortage and ROS unbalance in glycerol-grown cells. Moreover, the flx1Δ strain showed H2O2 hypersensitivity and decreased lifespan. The impaired biochemical phenotype found in the flx1Δ strain might be justified by an altered expression of the flavoprotein subunit of succinate dehydrogenase, a key enzyme in bioenergetics and cell regulation. A search for possible cis-acting consensus motifs in the regulatory region upstream SDH1-ORF revealed a dozen of upstream motifs that might respond to induced metabolic changes by altering the expression of Flx1p. Among these motifs, two are present in the regulatory region of genes encoding proteins involved in flavin homeostasis. This is the first evidence that the mitochondrial flavin cofactor status is involved in controlling the lifespan of yeasts, maybe by changing the cellular succinate level. This is not the only case in which the homeostasis of redox cofactors underlies complex phenotypical behaviours, as lifespan in yeasts. PMID:24895546

BAC-FISH assays delineate complex chromosomal rearrangements in a case of post-Chernobyl childhood thyroid cancer

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

Kwan, Johnson; Baumgartner, Adolf; Lu, Chun-Mei

2009-03-09

Structural chromosome aberrations are known hallmarks of many solid tumors. In the papillary form of thyroid cancer (PTC), for example, activation of the receptor tyrosine kinase (RTK) genes, RET and neurotrophic tyrosine kinase receptor type I (NTRK1) by intra- and interchromosomal rearrangements has been suggested as a cause of the disease. However, many phenotypically similar tumors do not carry an activated RET or NTRK-1 gene or express abnormal ret or NTRK-1 transcripts. Thus, we hypothesize that other cellular RTK-type genes are aberrantly expressed in these tumors. Using fluorescence in situ hybridization-based methods, we are studying karyotype changes in a relativelymore » rare subgroup of PTCs, i.e., tumors that arose in children following the 1986 nuclear accident in Chernobyl, Ukraine. Here, we report our technical developments and progress in deciphering complex chromosome aberrations in case S48TK, an aggressively growing PTC cell line, which shows an unusual high number of unbalanced translocations.« less

A multiplexed single-cell CRISPR screening platform enables systematic dissection of the unfolded protein response

PubMed Central

Adamson, Britt; Norman, Thomas M.; Jost, Marco; Cho, Min Y.; Nuñez, James K.; Chen, Yuwen; Villalta, Jacqueline E.; Gilbert, Luke A.; Horlbeck, Max A.; Hein, Marco Y.; Pak, Ryan A.; Gray, Andrew N.; Gross, Carol A.; Dixit, Atray; Parnas, Oren; Regev, Aviv; Weissman, Jonathan S.

2016-01-01

SUMMARY Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ~100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses. PMID:27984733

Genomic agonism and phenotypic antagonism between estrogen and progesterone receptors in breast cancer

PubMed Central

Singhal, Hari; Greene, Marianne E.; Tarulli, Gerard; Zarnke, Allison L.; Bourgo, Ryan J.; Laine, Muriel; Chang, Ya-Fang; Ma, Shihong; Dembo, Anna G.; Raj, Ganesh V.; Hickey, Theresa E.; Tilley, Wayne D.; Greene, Geoffrey L.

2016-01-01

The functional role of progesterone receptor (PR) and its impact on estrogen signaling in breast cancer remain controversial. In primary ER+ (estrogen receptor–positive)/PR+ human tumors, we report that PR reprograms estrogen signaling as a genomic agonist and a phenotypic antagonist. In isolation, estrogen and progestin act as genomic agonists by regulating the expression of common target genes in similar directions, but at different levels. Similarly, in isolation, progestin is also a weak phenotypic agonist of estrogen action. However, in the presence of both hormones, progestin behaves as a phenotypic estrogen antagonist. PR remodels nucleosomes to noncompetitively redirect ER genomic binding to distal enhancers enriched for BRCA1 binding motifs and sites that link PR and ER/PR complexes. When both hormones are present, progestin modulates estrogen action, such that responsive transcriptomes, cellular processes, and ER/PR recruitment to genomic sites correlate with those observed with PR alone, but not ER alone. Despite this overall correlation, the transcriptome patterns modulated by dual treatment are sufficiently different from individual treatments, such that antagonism of oncogenic processes is both predicted and observed. Combination therapies using the selective PR modulator/antagonist (SPRM) CDB4124 in combination with tamoxifen elicited 70% cytotoxic tumor regression of T47D tumor xenografts, whereas individual therapies inhibited tumor growth without net regression. Our findings demonstrate that PR redirects ER chromatin binding to antagonize estrogen signaling and that SPRMs can potentiate responses to antiestrogens, suggesting that cotargeting of ER and PR in ER+/PR+ breast cancers should be explored. PMID:27386569

The successes and future prospects of the linear antisense RNA amplification methodology.

PubMed

Li, Jifen; Eberwine, James

2018-05-01

It has been over a quarter of a century since the introduction of the linear RNA amplification methodology known as antisense RNA (aRNA) amplification. Whereas most molecular biology techniques are rapidly replaced owing to the fast-moving nature of development in the field, the aRNA procedure has become a base that can be built upon through varied uses of the technology. The technique was originally developed to assess RNA populations from small amounts of starting material, including single cells, but over time its use has evolved to include the detection of various cellular entities such as proteins, RNA-binding-protein-associated cargoes, and genomic DNA. In this Perspective we detail the linear aRNA amplification procedure and its use in assessing various components of a cell's chemical phenotype. This procedure is particularly useful in efforts to multiplex the simultaneous detection of various cellular processes. These efforts are necessary to identify the quantitative chemical phenotype of cells that underlies cellular function.

Patient-derived iPSCs show premature neural differentiation and neuron-type specific phenotypes relevant to neurodevelopment

PubMed Central

Yeh, Erika; Dao, Dang Q.; Wu, Zhi Y.; Kandalam, Santoshi M.; Camacho, Federico M.; Tom, Curtis; Zhang, Wandong; Krencik, Robert; Rauen, Katherine A.; Ullian, Erik M.; Weiss, Lauren A.

2017-01-01

Ras/MAPK pathway signaling is a major participant in neurodevelopment, and evidence suggests that BRAF, a key Ras signal mediator, influences human behavior. We studied the role of the mutation BRAFQ257R, the most common cause of cardiofaciocutaneous syndrome (CFC), in an induced pluripotent stem cell (iPSC)-derived model of human neurodevelopment. In iPSC-derived neuronal cultures from CFC subjects, we observed decreased p-AKT and p-ERK1/2 compared to controls, as well as a depleted neural progenitor pool and rapid neuronal maturation. Pharmacological PI3K/AKT pathway manipulation recapitulated cellular phenotypes in control cells and attenuated them in CFC cells. CFC cultures displayed altered cellular subtype ratios and increased intrinsic excitability. Moreover, in CFC cells, Ras/MAPK pathway activation and morphological abnormalities exhibited cell subtype-specific differences. Our results highlight the importance of exploring specific cellular subtypes and of using iPSC models to reveal relevant human-specific neurodevelopmental events. PMID:29158583

Lytic and latent antigens of the human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus induce T-cell responses with similar functional properties and memory phenotypes.

PubMed

Bihl, Florian; Narayan, Murli; Chisholm, John V; Henry, Leah M; Suscovich, Todd J; Brown, Elizabeth E; Welzel, Tania M; Kaufmann, Daniel E; Zaman, Tauheed M; Dollard, Sheila; Martin, Jeff N; Wang, Fred; Scadden, David T; Kaye, Kenneth M; Brander, Christian

2007-05-01

The cellular immunity against Kaposi's sarcoma-associated herpesvirus (KSHV) is poorly characterized and has not been compared to T-cell responses against other human herpesviruses. Here, novel and dominant targets of KSHV-specific cellular immunity are identified and compared to T cells specific for lytic and latent antigens in a second human gammaherpesvirus, Epstein-Barr virus. The data identify a novel HLA-B57- and HLA-B58-restricted epitope in the Orf57 protein and show consistently close parallels in immune phenotypes and functional response patterns between cells targeting lytic or latent KSHV- and EBV-encoded antigens, suggesting common mechanisms in the induction of these responses.

A method to measure cellular adhesion utilizing a polymer micro-cantilever

NASA Astrophysics Data System (ADS)

Gaitas, Angelo; Malhotra, Ricky; Pienta, Kenneth

2013-09-01

In the present study we engineered a micro-machined polyimide cantilever with an embedded sensing element to investigate cellular adhesion, in terms of its relative ability to stick to a cross-linker, 3,3'-dithiobis[sulfosuccinimidylpropionate], coated on the cantilever surface. To achieve this objective, we investigated adhesive properties of three human prostate cancer cell lines, namely, a bone metastasis derived human prostate cancer cell line (PC3), a brain metastasis derived human prostate cancer cell line (DU145), and a subclone of PC3 (PC3-EMT14). We found that PC3-EMT14, which displays a mesenchymal phenotype, has the least adhesion compared to PC3 and DU145, which exhibit an epithelial phenotype.

The impact of cellular senescence in skin ageing: A notion of mosaic and therapeutic strategies.

PubMed

Toutfaire, Marie; Bauwens, Emilie; Debacq-Chainiaux, Florence

2017-10-15

Cellular senescence is now recognized as one of the nine hallmarks of ageing. Recent data show the involvement of senescent cells in tissue ageing and some age-related diseases. Skin represents an ideal model for the study of ageing. Indeed, skin ageing varies between individuals depending on their chronological age but also on their exposure to various exogenous factors (mainly ultraviolet rays). If senescence traits can be detected with ageing in the skin, the senescent phenotype varies among the various skin cell types. Moreover, the origin of cellular senescence in the skin is still unknown, and multiple origins are possible. This reflects the mosaic of skin ageing. Senescent cells can interfere with their microenvironment, either via the direct secretion of factors (the senescence-associated secretory phenotype) or via other methods of communication, such as extracellular vesicles. Knowledge regarding the impact of cellular senescence on skin ageing could be integrated into dermatology research, especially to limit the appearance of senescent cells after photo(chemo)therapy or in age-related skin diseases. Therapeutic approaches include the clearance of senescent cells via the use of senolytics or via the cooperation with the immune system. Copyright © 2017 Elsevier Inc. All rights reserved.

Engineering microbial phenotypes through rewiring of genetic networks

PubMed Central

Rodrigues, Rui T.L.; Lee, Sangjin; Haines, Matthew

2017-01-01

Abstract The ability to program cellular behaviour is a major goal of synthetic biology, with applications in health, agriculture and chemicals production. Despite efforts to build ‘orthogonal’ systems, interactions between engineered genetic circuits and the endogenous regulatory network of a host cell can have a significant impact on desired functionality. We have developed a strategy to rewire the endogenous cellular regulatory network of yeast to enhance compatibility with synthetic protein and metabolite production. We found that introducing novel connections in the cellular regulatory network enabled us to increase the production of heterologous proteins and metabolites. This strategy is demonstrated in yeast strains that show significantly enhanced heterologous protein expression and higher titers of terpenoid production. Specifically, we found that the addition of transcriptional regulation between free radical induced signalling and nitrogen regulation provided robust improvement of protein production. Assessment of rewired networks revealed the importance of key topological features such as high betweenness centrality. The generation of rewired transcriptional networks, selection for specific phenotypes, and analysis of resulting library members is a powerful tool for engineering cellular behavior and may enable improved integration of heterologous protein and metabolite pathways. PMID:28369627

Antibiotic efficacy is linked to bacterial cellular respiration

PubMed Central

Lobritz, Michael A.; Belenky, Peter; Porter, Caroline B. M.; Gutierrez, Arnaud; Yang, Jason H.; Schwarz, Eric G.; Dwyer, Daniel J.; Khalil, Ahmad S.; Collins, James J.

2015-01-01

Bacteriostatic and bactericidal antibiotic treatments result in two fundamentally different phenotypic outcomes—the inhibition of bacterial growth or, alternatively, cell death. Most antibiotics inhibit processes that are major consumers of cellular energy output, suggesting that antibiotic treatment may have important downstream consequences on bacterial metabolism. We hypothesized that the specific metabolic effects of bacteriostatic and bactericidal antibiotics contribute to their overall efficacy. We leveraged the opposing phenotypes of bacteriostatic and bactericidal drugs in combination to investigate their activity. Growth inhibition from bacteriostatic antibiotics was associated with suppressed cellular respiration whereas cell death from most bactericidal antibiotics was associated with accelerated respiration. In combination, suppression of cellular respiration by the bacteriostatic antibiotic was the dominant effect, blocking bactericidal killing. Global metabolic profiling of bacteriostatic antibiotic treatment revealed that accumulation of metabolites involved in specific drug target activity was linked to the buildup of energy metabolites that feed the electron transport chain. Inhibition of cellular respiration by knockout of the cytochrome oxidases was sufficient to attenuate bactericidal lethality whereas acceleration of basal respiration by genetically uncoupling ATP synthesis from electron transport resulted in potentiation of the killing effect of bactericidal antibiotics. This work identifies a link between antibiotic-induced cellular respiration and bactericidal lethality and demonstrates that bactericidal activity can be arrested by attenuated respiration and potentiated by accelerated respiration. Our data collectively show that antibiotics perturb the metabolic state of bacteria and that the metabolic state of bacteria impacts antibiotic efficacy. PMID:26100898

Metabolic Host Responses to Malarial Infection during the Intraerythrocytic Developmental Cycle

DTIC Science & Technology

2016-08-08

by reproducing the experimentally determined 1) stage-specific production of biomass components and their precursors in the parasite and 2) metabolite...uptake, allow for the prediction of cellular growth ( biomass accumulation) and other phenotypic functions related to metabolism [9]. For example...our group to capture stage-specific growth phenotypes and biomass metabolite production [15]. Among these metabolic descriptions, only the network

Nondestructive cryomicro-CT imaging enables structural and molecular analysis of human lung tissue.

PubMed

Vasilescu, Dragoş M; Phillion, André B; Tanabe, Naoya; Kinose, Daisuke; Paige, David F; Kantrowitz, Jacob J; Liu, Gang; Liu, Hanqiao; Fishbane, Nick; Verleden, Stijn E; Vanaudenaerde, Bart M; Lenburg, Marc; Stevenson, Christopher S; Spira, Avrum; Cooper, Joel D; Hackett, Tillie-Louise; Hogg, James C

2017-01-01

Micro-computed tomography (CT) enables three-dimensional (3D) imaging of complex soft tissue structures, but current protocols used to achieve this goal preclude cellular and molecular phenotyping of the tissue. Here we describe a radiolucent cryostage that permits micro-CT imaging of unfixed frozen human lung samples at an isotropic voxel size of (11 µm) 3 under conditions where the sample is maintained frozen at -30°C during imaging. The cryostage was tested for thermal stability to maintain samples frozen up to 8 h. This report describes the methods used to choose the materials required for cryostage construction and demonstrates that whole genome mRNA integrity and expression are not compromised by exposure to micro-CT radiation and that the tissue can be used for immunohistochemistry. The new cryostage provides a novel method enabling integration of 3D tissue structure with cellular and molecular analysis to facilitate the identification of molecular determinants of disease. The described micro-CT cryostage provides a novel way to study the three-dimensional lung structure preserved without the effects of fixatives while enabling subsequent studies of the cellular matrix composition and gene expression. This approach will, for the first time, enable researchers to study structural changes of lung tissues that occur with disease and correlate them with changes in gene or protein signatures. Copyright © 2017 the American Physiological Society.

The light gene of Drosophila melanogaster encodes a homologue of VPS41, a yeast gene involved in cellular-protein trafficking.

PubMed

Warner, T S; Sinclair, D A; Fitzpatrick, K A; Singh, M; Devlin, R H; Honda, B M

1998-04-01

Mutations in a number of genes affect eye colour in Drosophila melanogaster; some of these "eye-colour" genes have been shown to be involved in various aspects of cellular transport processes. In addition, combinations of viable mutant alleles of some of these genes, such as carnation (car) combined with either light (lt) or deep-orange (dor) mutants, show lethal interactions. Recently, dor was shown to be homologous to the yeast gene PEP3 (VPS18), which is known to be involved in intracellular trafficking. We have undertaken to extend our earlier work on the lt gene, in order to examine in more detail its expression pattern and to characterize its gene product via sequencing of a cloned cDNA. The gene appears to be expressed at relatively high levels in all stages and tissues examined, and shows strong homology to VPS41, a gene involved in cellular-protein trafficking in yeast and higher eukaryotes. Further genetic experiments also point to a role for lt in transport processes: we describe lethal interactions between viable alleles of lt and dor, as well as phenotypic interactions (reductions in eye pigment) between allels of lt and another eye-colour gene, garnet (g), whose gene product has close homology to a subunit of the human adaptor complex, AP-3.

Neurodegeneration in ataxia-telangiectasia: Multiple roles of ATM kinase in cellular homeostasis.

PubMed

Choy, Kay Rui; Watters, Dianne J

2018-01-01

Ataxia-telangiectasia (A-T) is characterized by neuronal degeneration, cancer, diabetes, immune deficiency, and increased sensitivity to ionizing radiation. A-T is attributed to the deficiency of the protein kinase coded by the ATM (ataxia-telangiectasia mutated) gene. ATM is a sensor of DNA double-strand breaks (DSBs) and signals to cell cycle checkpoints and the DNA repair machinery. ATM phosphorylates numerous substrates and activates many cell-signaling pathways. There has been considerable debate about whether a defective DNA damage response is causative of the neurological aspects of the disease. In proliferating cells, ATM is localized mainly in the nucleus; however, in postmitotic cells such as neurons, ATM is mostly cytoplasmic. Recent studies reveal an increasing number of roles for ATM in the cytoplasm, including activation by oxidative stress. ATM associates with organelles including mitochondria and peroxisomes, both sources of reactive oxygen species (ROS), which have been implicated in neurodegenerative diseases and aging. ATM is also associated with synaptic vesicles and has a role in regulating cellular homeostasis and autophagy. The cytoplasmic roles of ATM provide a new perspective on the neurodegenerative process in A-T. This review will examine the expanding roles of ATM in cellular homeostasis and relate these functions to the complex A-T phenotype. Developmental Dynamics 247:33-46, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Focus on the emerging new fields of network physiology and network medicine

NASA Astrophysics Data System (ADS)

Ivanov, Plamen Ch; Liu, Kang K. L.; Bartsch, Ronny P.

2016-10-01

Despite the vast progress and achievements in systems biology and integrative physiology in the last decades, there is still a significant gap in understanding the mechanisms through which (i) genomic, proteomic and metabolic factors and signaling pathways impact vertical processes across cells, tissues and organs leading to the expression of different disease phenotypes and influence the functional and clinical associations between diseases, and (ii) how diverse physiological systems and organs coordinate their functions over a broad range of space and time scales and horizontally integrate to generate distinct physiologic states at the organism level. Two emerging fields, network medicine and network physiology, aim to address these fundamental questions. Novel concepts and approaches derived from recent advances in network theory, coupled dynamical systems, statistical and computational physics show promise to provide new insights into the complexity of physiological structure and function in health and disease, bridging the genetic and sub-cellular level with inter-cellular interactions and communications among integrated organ systems and sub-systems. These advances form first building blocks in the methodological formalism and theoretical framework necessary to address fundamental problems and challenges in physiology and medicine. This ‘focus on’ issue contains 26 articles representing state-of-the-art contributions covering diverse systems from the sub-cellular to the organism level where physicists have key role in laying the foundations of these new fields.

A Multidisciplinary, Open Access Platform for Research on Biomolecules.

PubMed

Bähler, Jürg

2011-08-22

I am pleased to introduce Biomolecules, a new journal to report on all aspects of science that focuses on biologically derived substances, from small molecules to complex polymers. Some examples are lipids, carbohydrates, vitamins, hormones, amino acids, nucleotides, peptides, RNA and polysaccharides, but this list is far from exhaustive. Research on biomolecules encompasses multiple fascinating questions. How are biomolecules synthesized and modified? What are their structures and interactions with other biomolecules? How do biomolecules function in biological processes, at the level of organelles, cells, organs, organisms, or even ecosystems? How do biomolecules affect either the organism that produces them or other organisms of the same or different species? How are biomolecules shaped by evolution, and how in turn do they affect cellular phenotypes? What is the systems-level contribution of biomolecules to biological function? [...].

Considering the Lives of Microbes in Microbial Communities.

PubMed

Shank, Elizabeth A

2018-01-01

Over the last decades, sequencing technologies have transformed our ability to investigate the composition and functional capacity of microbial communities. Even so, critical questions remain about these complex systems that cannot be addressed by the bulk, community-averaged data typically provided by sequencing methods. In this Perspective, I propose that future advances in microbiome research will emerge from considering "the lives of microbes": we need to create methods to explicitly interrogate how microbes exist and interact in native-setting-like microenvironments. This approach includes developing approaches that expose the phenotypic heterogeneity of microbes; exploring the effects of coculture cues on cellular differentiation and metabolite production; and designing visualization systems that capture features of native microbial environments while permitting the nondestructive observation of microbial interactions over space and time with single-cell resolution.

Technological advances for interrogating the human kinome.

PubMed

Baharani, Akanksha; Trost, Brett; Kusalik, Anthony; Napper, Scott

2017-02-08

There is increasing appreciation among researchers and clinicians of the value of investigating biology and pathobiology at the level of cellular kinase (kinome) activity. Kinome analysis provides valuable opportunity to gain insights into complex biology (including disease pathology), identify biomarkers of critical phenotypes (including disease prognosis and evaluation of therapeutic efficacy), and identify targets for therapeutic intervention through kinase inhibitors. The growing interest in kinome analysis has fueled efforts to develop and optimize technologies that enable characterization of phosphorylation-mediated signaling events in a cost-effective, high-throughput manner. In this review, we highlight recent advances to the central technologies currently available for kinome profiling and offer our perspectives on the key challenges remaining to be addressed. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Murine Models of Systemic Lupus Erythematosus

PubMed Central

Perry, Daniel; Sang, Allison; Yin, Yiming; Zheng, Ying-Yi; Morel, Laurence

2011-01-01

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disorder. The study of diverse mouse models of lupus has provided clues to the etiology of SLE. Spontaneous mouse models of lupus have led to identification of numerous susceptibility loci from which several candidate genes have emerged. Meanwhile, induced models of lupus have provided insight into the role of environmental factors in lupus pathogenesis as well as provided a better understanding of cellular mechanisms involved in the onset and progression of disease. The SLE-like phenotypes present in these models have also served to screen numerous potential SLE therapies. Due to the complex nature of SLE, it is necessary to understand the effect specific targeted therapies have on immune homeostasis. Furthermore, knowledge gained from mouse models will provide novel therapy targets for the treatment of SLE. PMID:21403825

Resolving TYK2 locus genotype-to-phenotype differences in autoimmunity.

PubMed

Dendrou, Calliope A; Cortes, Adrian; Shipman, Lydia; Evans, Hayley G; Attfield, Kathrine E; Jostins, Luke; Barber, Thomas; Kaur, Gurman; Kuttikkatte, Subita Balaram; Leach, Oliver A; Desel, Christiane; Faergeman, Soren L; Cheeseman, Jane; Neville, Matt J; Sawcer, Stephen; Compston, Alastair; Johnson, Adam R; Everett, Christine; Bell, John I; Karpe, Fredrik; Ultsch, Mark; Eigenbrot, Charles; McVean, Gil; Fugger, Lars

2016-11-02

Thousands of genetic variants have been identified, which contribute to the development of complex diseases, but determining how to elucidate their biological consequences for translation into clinical benefit is challenging. Conflicting evidence regarding the functional impact of genetic variants in the tyrosine kinase 2 (TYK2) gene, which is differentially associated with common autoimmune diseases, currently obscures the potential of TYK2 as a therapeutic target. We aimed to resolve this conflict by performing genetic meta-analysis across disorders; subsequent molecular, cellular, in vivo, and structural functional follow-up; and epidemiological studies. Our data revealed a protective homozygous effect that defined a signaling optimum between autoimmunity and immunodeficiency and identified TYK2 as a potential drug target for certain common autoimmune disorders. Copyright © 2016, American Association for the Advancement of Science.

Exometabolomics and MSI: deconstructing how cells interact to transform their small molecule environment.

PubMed

Silva, Leslie P; Northen, Trent R

2015-08-01

Metabolism is at the heart of many biotechnologies from biofuels to medical diagnostics. Metabolomic methods that provide glimpses into cellular metabolism have rapidly developed into a critical component of the biotechnological development process. Most metabolomics methods have focused on what is happening inside the cell. Equally important are the biochemical transformations of the cell, and their effect on other cells and their environment; the exometabolome. Exometabolomics is therefore gaining popularity as a robust approach for obtaining rich phenotypic data, and being used in bioprocessing and biofuel development. Mass spectrometry imaging approaches, including several nanotechnologies, provide complimentary information by localizing metabolic processes within complex biological matrices. Together, the two technologies can provide new insights into the metabolism and interactions of cells. Published by Elsevier Ltd.

The choreography of neuroinflammation in Huntington’s disease

PubMed Central

Crotti, Andrea; Glass, Christopher K.

2016-01-01

Currently, the concept of ‘neuroinflammation’ includes inflammation associated with neurodegenerative diseases, in which there is little or no infiltration of blood-derived immune cells into the brain. The roles of brain-resident and peripheral immune cells in these inflammatory settings are poorly understood, and it is unclear whether neuroinflammation results from immune reaction to neuronal dysfunction/degeneration, and/or represents cell-autonomous phenotypes of dysfunctional immune cells. Here, we review recent studies examining these questions in the context of Huntington’s disease (HD), where mutant Huntingtin (HTT) is expressed in both neurons and glia. Insights into the cellular and molecular mechanisms underlying neuroinflammation in HD may provide a better understanding of inflammation in more complex neurodegenerative disorders, and of the contribution of the neuroinflammatory component to neurodegenerative disease pathogenesis. PMID:26001312

Ad E1A 243R oncoprotein promotes association of proto-oncogene product MYC with the NuA4/Tip60 complex via the E1A N-terminal repression domain.

PubMed

Zhao, Ling-Jun; Loewenstein, Paul M; Green, Maurice

2016-12-01

The adenovirus E1A 243R oncoprotein targets TRRAP, a scaffold protein that assembles histone acetyltransferase (HAT) complexes, such as the NuA4/Tip60 complex which mediates transcriptional activity of the proto-oncogene MYC and helps determine the cancer cell phenotype. How E1A transforms cells through TRRAP remains obscure. We performed proteomic analysis with the N-terminal transcriptional repression domain of E1A 243R (E1A 1-80) and showed that E1A 1-80 interacts with TRRAP, p400, and three other members of the NuA4 complex - DMAP1, RUVBL1 and RUVBL2 - not previously shown to associate with E1A 243R. E1A 1-80 interacts with these NuA4 components and MYC through the E1A TRRAP-targeting domain. E1A 243R association with the NuA4 complex was demonstrated by co-immunoprecipitation and analysis with DMAP1, Tip60, and MYC. Significantly, E1A 243R promotes association of MYC/MAX with the NuA4/Tip60 complex, implicating the importance of the MYC/NuA4 pathway in cellular transformation by both MYC and E1A. Copyright © 2016 Elsevier Inc. All rights reserved.

The fetal programming of telomere biology hypothesis: an update

PubMed Central

Entringer, Sonja; Buss, Claudia; Wadhwa, Pathik D.

2018-01-01

Research on mechanisms underlying fetal programming of health and disease risk has focused primarily on processes that are specific to cell types, organs or phenotypes of interest. However, the observation that developmental conditions concomitantly influence a diverse set of phenotypes, the majority of which are implicated in age-related disorders, raises the possibility that such developmental conditions may additionally exert effects via a common underlying mechanism that involves cellular/molecular ageing–related processes. In this context, we submit that telomere biology represents a process of particular interest in humans because, firstly, this system represents among the most salient antecedent cellular phenotypes for common age-related disorders; secondly, its initial (newborn) setting appears to be particularly important for its long-term effects; and thirdly, its initial setting appears to be plastic and under developmental regulation. We propose that the effects of suboptimal intrauterine conditions on the initial setting of telomere length and telomerase expression/activity capacity may be mediated by the programming actions of stress-related maternal–placental–fetal oxidative, immune, endocrine and metabolic pathways in a manner that may ultimately accelerate cellular dysfunction, ageing and disease susceptibility over the lifespan. This perspectives paper provides an overview of each of the elements underlying this hypothesis, with an emphasis on recent developments, findings and future directions. This article is part of the theme issue ‘Understanding diversity in telomere dynamics’. PMID:29335381

The fetal programming of telomere biology hypothesis: an update.

PubMed

Entringer, Sonja; de Punder, Karin; Buss, Claudia; Wadhwa, Pathik D

2018-03-05

Research on mechanisms underlying fetal programming of health and disease risk has focused primarily on processes that are specific to cell types, organs or phenotypes of interest. However, the observation that developmental conditions concomitantly influence a diverse set of phenotypes, the majority of which are implicated in age-related disorders, raises the possibility that such developmental conditions may additionally exert effects via a common underlying mechanism that involves cellular/molecular ageing-related processes. In this context, we submit that telomere biology represents a process of particular interest in humans because, firstly, this system represents among the most salient antecedent cellular phenotypes for common age-related disorders; secondly, its initial (newborn) setting appears to be particularly important for its long-term effects; and thirdly, its initial setting appears to be plastic and under developmental regulation. We propose that the effects of suboptimal intrauterine conditions on the initial setting of telomere length and telomerase expression/activity capacity may be mediated by the programming actions of stress-related maternal-placental-fetal oxidative, immune, endocrine and metabolic pathways in a manner that may ultimately accelerate cellular dysfunction, ageing and disease susceptibility over the lifespan. This perspectives paper provides an overview of each of the elements underlying this hypothesis, with an emphasis on recent developments, findings and future directions.This article is part of the theme issue 'Understanding diversity in telomere dynamics'. © 2018 The Author(s).

A genome-wide CRISPR library for high-throughput genetic screening in Drosophila cells.

PubMed

Bassett, Andrew R; Kong, Lesheng; Liu, Ji-Long

2015-06-20

The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines, enabling screening for cellular phenotypes resulting from genetic aberrations. Drosophila cells have proven to be highly effective in identifying genes involved in cellular processes through similar screens using partial knockdown by RNAi. This is in part due to the lower degree of redundancy between genes in this organism, whilst still maintaining highly conserved gene networks and orthologs of many human disease-causing genes. The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques, but allows analysis over longer periods of time which can be critical for certain phenotypes. In this study, we have designed and built a genome-wide CRISPR library covering 13,501 genes, among which 8989 genes are targeted by three or more independent single guide RNAs (sgRNAs). Moreover, we describe strategies to monitor the population of guide RNAs by high throughput sequencing (HTS). We hope that this library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes, and as a source of guide RNA designs for future studies. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

How long bones grow children: Mechanistic paths to variation in human height growth.

PubMed

Lampl, Michelle; Schoen, Meriah

2017-03-01

Eveleth and Tanner's descriptive documentation of worldwide variability in human growth provided evidence of the interaction between genetics and environment during development that has been foundational to the science of human growth. There remains a need, however, to describe the mechanistic foundations of variability in human height growth patterns. A review of research documenting cellular activities at the endochondral growth plate aims to show how the unique microenvironment and cell functions during the sequential phases of the chondrocyte lifecycle affect long bone elongation, a fundamental source of height growth. There are critical junctures within the chondrocytic differentiation cascade at which environmental influences are integrated and have the ability to influence progression to the hypertrophic chondrocyte phase, the primary driver of long bone elongation. Phenotypic differences in height growth patterns reflect variability in amplitude and frequency of discretely timed hypertrophic cellular expansion events, the cellular basis of saltation and stasis growth biology. Final height is a summary of the dynamic processes carried out by the growth plate cellular machinery. As these cell-level mechanisms unfold in an individual, time-specific manner, there are many critical points at which a genetic growth program can be enhanced or perturbed. Recognizing both the complexity and fluidity of this adaptive system questions the likelihood of a single, optimal growth pattern and instead identifies a larger bandwidth of saltatory frequencies for "normal" growth. Further inquiry into mechanistic sources of variability acting at critical organizational points of chondrogenesis can provide new opportunities for growth interventions. © 2017 Wiley Periodicals, Inc.

Mapping the cellular and molecular heterogeneity of normal and malignant breast tissues and cultured cell lines

PubMed Central

2010-01-01

Introduction Normal and neoplastic breast tissues are comprised of heterogeneous populations of epithelial cells exhibiting various degrees of maturation and differentiation. While cultured cell lines have been derived from both normal and malignant tissues, it remains unclear to what extent they retain similar levels of differentiation and heterogeneity as that found within breast tissues. Methods We used 12 reduction mammoplasty tissues, 15 primary breast cancer tissues, and 20 human breast epithelial cell lines (16 cancer lines, 4 normal lines) to perform flow cytometry for CD44, CD24, epithelial cell adhesion molecule (EpCAM), and CD49f expression, as well as immunohistochemistry, and in vivo tumor xenograft formation studies to extensively analyze the molecular and cellular characteristics of breast epithelial cell lineages. Results Human breast tissues contain four distinguishable epithelial differentiation states (two luminal phenotypes and two basal phenotypes) that differ on the basis of CD24, EpCAM and CD49f expression. Primary human breast cancer tissues also contain these four cellular states, but in altered proportions compared to normal tissues. In contrast, cultured cancer cell lines are enriched for rare basal and mesenchymal epithelial phenotypes, which are normally present in small numbers within human tissues. Similarly, cultured normal human mammary epithelial cell lines are enriched for rare basal and mesenchymal phenotypes that represent a minor fraction of cells within reduction mammoplasty tissues. Furthermore, although normal human mammary epithelial cell lines exhibit features of bi-potent progenitor cells they are unable to differentiate into mature luminal breast epithelial cells under standard culture conditions. Conclusions As a group breast cancer cell lines represent the heterogeneity of human breast tumors, but individually they exhibit increased lineage-restricted profiles that fall short of truly representing the intratumoral heterogeneity of individual breast tumors. Additionally, normal human mammary epithelial cell lines fail to retain much of the cellular diversity found in human breast tissues and are enriched for differentiation states that are a minority in breast tissues, although they do exhibit features of bi-potent basal progenitor cells. These findings suggest that collections of cell lines representing multiple cell types can be used to model the cellular heterogeneity of tissues. PMID:20964822

Many Saccharomyces cerevisiae Cell Wall Protein Encoding Genes Are Coregulated by Mss11, but Cellular Adhesion Phenotypes Appear Only Flo Protein Dependent.

PubMed

Bester, Michael C; Jacobson, Dan; Bauer, Florian F

2012-01-01

The outer cell wall of the yeast Saccharomyces cerevisiae serves as the interface with the surrounding environment and directly affects cell-cell and cell-surface interactions. Many of these interactions are facilitated by specific adhesins that belong to the Flo protein family. Flo mannoproteins have been implicated in phenotypes such as flocculation, substrate adhesion, biofilm formation, and pseudohyphal growth. Genetic data strongly suggest that individual Flo proteins are responsible for many specific cellular adhesion phenotypes. However, it remains unclear whether such phenotypes are determined solely by the nature of the expressed FLO genes or rather as the result of a combination of FLO gene expression and other cell wall properties and cell wall proteins. Mss11 has been shown to be a central element of FLO1 and FLO11 gene regulation and acts together with the cAMP-PKA-dependent transcription factor Flo8. Here we use genome-wide transcription analysis to identify genes that are directly or indirectly regulated by Mss11. Interestingly, many of these genes encode cell wall mannoproteins, in particular, members of the TIR and DAN families. To examine whether these genes play a role in the adhesion properties associated with Mss11 expression, we assessed deletion mutants of these genes in wild-type and flo11Δ genetic backgrounds. This analysis shows that only FLO genes, in particular FLO1/10/11, appear to significantly impact on such phenotypes. Thus adhesion-related phenotypes are primarily dependent on the balance of FLO gene expression.

Phenotypic plasticity and epithelial-to-mesenchymal transition in the behaviour and therapeutic response of oral squamous cell carcinoma.

PubMed

Vig, Navin; Mackenzie, Ian C; Biddle, Adrian

2015-10-01

It is increasingly recognised that phenotypic plasticity, apparently driven by epigenetic mechanisms, plays a key role in tumour behaviour and markedly influences the important processes of therapeutic survival and metastasis. An important source of plasticity in malignancy is epithelial-to-mesenchymal transition (EMT), a common epigenetically controlled event that results in transition of malignant cells between different phenotypic states that confer motility and enhance survival. In this review, we discuss the importance of phenotypic plasticity and its contribution to cellular heterogeneity in oral squamous cell carcinoma with emphasis on aspects of drug resistance and EMT. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Multi-parameter phenotypic profiling: using cellular effects to characterize small-molecule compounds.

PubMed

Feng, Yan; Mitchison, Timothy J; Bender, Andreas; Young, Daniel W; Tallarico, John A

2009-07-01

Multi-parameter phenotypic profiling of small molecules provides important insights into their mechanisms of action, as well as a systems level understanding of biological pathways and their responses to small molecule treatments. It therefore deserves more attention at an early step in the drug discovery pipeline. Here, we summarize the technologies that are currently in use for phenotypic profiling--including mRNA-, protein- and imaging-based multi-parameter profiling--in the drug discovery context. We think that an earlier integration of phenotypic profiling technologies, combined with effective experimental and in silico target identification approaches, can improve success rates of lead selection and optimization in the drug discovery process.

Genetic control of root growth: from genes to networks

PubMed Central

Slovak, Radka; Ogura, Takehiko; Satbhai, Santosh B.; Ristova, Daniela; Busch, Wolfgang

2016-01-01

Background Roots are essential organs for higher plants. They provide the plant with nutrients and water, anchor the plant in the soil, and can serve as energy storage organs. One remarkable feature of roots is that they are able to adjust their growth to changing environments. This adjustment is possible through mechanisms that modulate a diverse set of root traits such as growth rate, diameter, growth direction and lateral root formation. The basis of these traits and their modulation are at the cellular level, where a multitude of genes and gene networks precisely regulate development in time and space and tune it to environmental conditions. Scope This review first describes the root system and then presents fundamental work that has shed light on the basic regulatory principles of root growth and development. It then considers emerging complexities and how they have been addressed using systems-biology approaches, and then describes and argues for a systems-genetics approach. For reasons of simplicity and conciseness, this review is mostly limited to work from the model plant Arabidopsis thaliana, in which much of the research in root growth regulation at the molecular level has been conducted. Conclusions While forward genetic approaches have identified key regulators and genetic pathways, systems-biology approaches have been successful in shedding light on complex biological processes, for instance molecular mechanisms involving the quantitative interaction of several molecular components, or the interaction of large numbers of genes. However, there are significant limitations in many of these methods for capturing dynamic processes, as well as relating these processes to genotypic and phenotypic variation. The emerging field of systems genetics promises to overcome some of these limitations by linking genotypes to complex phenotypic and molecular data using approaches from different fields, such as genetics, genomics, systems biology and phenomics. PMID:26558398

Fourier transform infrared spectroscopy as a metabolite fingerprinting tool for monitoring the phenotypic changes in complex bacterial communities capable of degrading phenol.

PubMed

Wharfe, Emma S; Jarvis, Roger M; Winder, Catherine L; Whiteley, Andrew S; Goodacre, Royston

2010-12-01

The coking process produces great volumes of wastewater contaminated with pollutants such as cyanides, sulfides and phenolics. Chemical and physical remediation of this wastewater removes the majority of these pollutants; however, these processes do not remove phenol and thiocyanate. The removal of these compounds has been effected during bioremediation with activated sludge containing a complex microbial community. In this investigation we acquired activated sludge from an industrial bioreactor capable of degrading phenol. The sludge was incubated in our laboratory and monitored for its ability to degrade phenol over a 48 h period. Multiple samples were taken across the time-course and analysed by Fourier transform infrared (FT-IR) spectroscopy. FT-IR was used as a whole-organism fingerprinting approach to monitor biochemical changes in the bacterial cells during the degradation of phenol. We also investigated the ability of the activated sludge to degrade phenol following extended periods (2-131 days) of storage in the absence of phenol. A reduction was observed in the ability of the microbial community to degrade phenol and this was accompanied by a detectable biochemical change in the FT-IR fingerprint related to cellular phenotype of the microbial community. In the absence of phenol a decrease in thiocyanate vibrations was observed, reflecting the ability of these communities to degrade this substrate. Actively degrading communities showed an additional new band in their FT-IR spectra that could be attributed to phenol degradation products from the ortho- and meta-cleavage of the aromatic ring. This study demonstrates that FT-IR spectroscopy when combined with chemometric analysis is a very powerful high throughput screening approach for assessing the metabolic capability of complex microbial communities. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

Fine-tuning of actin dynamics by the HSPB8-BAG3 chaperone complex facilitates cytokinesis and contributes to its impact on cell division.

PubMed

Varlet, Alice Anaïs; Fuchs, Margit; Luthold, Carole; Lambert, Herman; Landry, Jacques; Lavoie, Josée N

2017-07-01

The small heat shock protein HSPB8 and its co-chaperone BAG3 are proposed to regulate cytoskeletal proteostasis in response to mechanical signaling in muscle cells. Here, we show that in dividing cells, the HSPB8-BAG3 complex is instrumental to the accurate disassembly of the actin-based contractile ring during cytokinesis, a process required to allow abscission of daughter cells. Silencing of HSPB8 markedly decreased the mitotic levels of BAG3 in HeLa cells, supporting its crucial role in BAG3 mitotic functions. Cells depleted of HSPB8 were delayed in cytokinesis, remained connected via a disorganized intercellular bridge, and exhibited increased incidence of nuclear abnormalities that result from failed cytokinesis (i.e., bi- and multi-nucleation). Such phenotypes were associated with abnormal accumulation of F-actin at the intercellular bridge of daughter cells at telophase. Remarkably, the actin sequestering drug latrunculin A, like the inhibitor of branched actin polymerization CK666, normalized F-actin during cytokinesis and restored proper cell division in HSPB8-depleted cells, implicating deregulated actin dynamics as a cause of abscission failure. Moreover, this HSPB8-dependent phenotype could be corrected by rapamycin, an autophagy-promoting drug, whereas it was mimicked by drugs impairing lysosomal function. Together, the results further support a role for the HSPB8-BAG3 chaperone complex in quality control of actin-based structure dynamics that are put under high tension, notably during cell cytokinesis. They expand a so-far under-appreciated connection between selective autophagy and cellular morphodynamics that guide cell division.

Dissecting the function of Cullin-RING ubiquitin ligase complex genes in planarian regeneration.

PubMed

Strand, Nicholas S; Allen, John M; Ghulam, Mahjoobah; Taylor, Matthew R; Munday, Roma K; Carrillo, Melissa; Movsesyan, Artem; Zayas, Ricardo M

2018-01-15

The ubiquitin system plays a role in nearly every aspect of eukaryotic cell biology. The enzymes responsible for transferring ubiquitin onto specific substrates are the E3 ubiquitin ligases, a large and diverse family of proteins, for which biological roles and target substrates remain largely undefined. Studies using model organisms indicate that ubiquitin signaling mediates key steps in developmental processes and tissue regeneration. Here, we used the freshwater planarian, Schmidtea mediterranea, to investigate the role of Cullin-RING ubiquitin ligase (CRL) complexes in stem cell regulation during regeneration. We identified six S. mediterranea cullin genes, and used RNAi to uncover roles for homologs of Cullin-1, -3 and -4 in planarian regeneration. The cullin-1 RNAi phenotype included defects in blastema formation, organ regeneration, lesions, and lysis. To further investigate the function of cullin-1-mediated cellular processes in planarians, we examined genes encoding the adaptor protein Skp1 and F-box substrate-recognition proteins that are predicted to partner with Cullin-1. RNAi against skp1 resulted in phenotypes similar to cullin-1 RNAi, and an RNAi screen of the F-box genes identified 19 genes that recapitulated aspects of cullin-1 RNAi, including ones that in mammals are involved in stem cell regulation and cancer biology. Our data provides evidence that CRLs play discrete roles in regenerative processes and provide a platform to investigate how CRLs regulate stem cells in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Genes under weaker stabilizing selection increase network evolvability and rapid regulatory adaptation to an environmental shift.

PubMed

Laarits, T; Bordalo, P; Lemos, B

2016-08-01

Regulatory networks play a central role in the modulation of gene expression, the control of cellular differentiation, and the emergence of complex phenotypes. Regulatory networks could constrain or facilitate evolutionary adaptation in gene expression levels. Here, we model the adaptation of regulatory networks and gene expression levels to a shift in the environment that alters the optimal expression level of a single gene. Our analyses show signatures of natural selection on regulatory networks that both constrain and facilitate rapid evolution of gene expression level towards new optima. The analyses are interpreted from the standpoint of neutral expectations and illustrate the challenge to making inferences about network adaptation. Furthermore, we examine the consequence of variable stabilizing selection across genes on the strength and direction of interactions in regulatory networks and in their subsequent adaptation. We observe that directional selection on a highly constrained gene previously under strong stabilizing selection was more efficient when the gene was embedded within a network of partners under relaxed stabilizing selection pressure. The observation leads to the expectation that evolutionarily resilient regulatory networks will contain optimal ratios of genes whose expression is under weak and strong stabilizing selection. Altogether, our results suggest that the variable strengths of stabilizing selection across genes within regulatory networks might itself contribute to the long-term adaptation of complex phenotypes. © 2016 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2016 European Society For Evolutionary Biology.

Fe65 is required for Tip60-directed histone H4 acetylation at DNA strand breaks

PubMed Central

Stante, Maria; Minopoli, Giuseppina; Passaro, Fabiana; Raia, Maddalena; Vecchio, Luigi Del; Russo, Tommaso

2009-01-01

Fe65 is a binding partner of the Alzheimer's β-amyloid precursor protein APP. The possible involvement of this protein in the cellular response to DNA damage was suggested by the observation that Fe65 null mice are more sensitive to genotoxic stress than WT counterpart. Fe65 associated with chromatin under basal conditions and its involvement in DNA damage repair requires this association. A known partner of Fe65 is the histone acetyltransferase Tip60. Considering the crucial role of Tip60 in DNA repair, we explored the hypothesis that the phenotype of Fe65 null cells depended on its interaction with Tip60. We demonstrated that Fe65 knockdown impaired recruitment of Tip60-TRRAP complex to DNA double strand breaks and decreased histone H4 acetylation. Accordingly, the efficiency of DNA repair was decreased upon Fe65 suppression. To explore whether APP has a role in this mechanism, we analyzed a Fe65 mutant unable to bind to APP. This mutant failed to rescue the phenotypes of Fe65 null cells; furthermore, APP/APLP2 suppression results in the impairment of recruitment of Tip60-TRRAP complex to DNA double strand breaks, decreased histone H4 acetylation and repair efficiency. On these bases, we propose that Fe65 and its interaction with APP play an important role in the response to DNA damage by assisting the recruitment of Tip60-TRRAP to DNA damage sites. PMID:19282473

Age-related epigenetic drift and phenotypic plasticity loss: implications in prevention of age-related human diseases

PubMed Central

Li, Yuanyuan; Tollefsbol, Trygve O

2016-01-01

Aging is considered as one of the most important developmental processes in organisms and is closely associated with global deteriorations of epigenetic markers such as aberrant methylomic patterns. This altered epigenomic state, referred to ‘epigenetic drift’, reflects deficient maintenance of epigenetic marks and contributes to impaired cellular and molecular functions in aged cells. Epigenetic drift-induced abnormal changes during aging are scantily repaired by epigenetic modulators. This inflexibility in the aged epigenome may lead to an age-related decline in phenotypic plasticity at the cellular and molecular levels due to epigenetic drift. This perspective aims to provide novel concepts for understanding epigenetic effects on the aging process and to provide insights into epigenetic prevention and therapeutic strategies for age-related human disease. PMID:27882781

Abrogation of E-cadherin-mediated cell-cell contact in mouse embryonic stem cells results in reversible LIF-independent self-renewal.

PubMed

Soncin, Francesca; Mohamet, Lisa; Eckardt, Dominik; Ritson, Sarah; Eastham, Angela M; Bobola, Nicoletta; Russell, Angela; Davies, Steve; Kemler, Rolf; Merry, Catherine L R; Ward, Christopher M

2009-09-01

We have previously demonstrated that differentiation of embryonic stem (ES) cells is associated with downregulation of cell surface E-cadherin. In this study, we assessed the function of E-cadherin in mouse ES cell pluripotency and differentiation. We show that inhibition of E-cadherin-mediated cell-cell contact in ES cells using gene knockout (Ecad(-/-)), RNA interference (EcadRNAi), or a transhomodimerization-inhibiting peptide (CHAVC) results in cellular proliferation and maintenance of an undifferentiated phenotype in fetal bovine serum-supplemented medium in the absence of leukemia inhibitory factor (LIF). Re-expression of E-cadherin in Ecad(-/-), EcadRNAi, and CHAVC-treated ES cells restores cellular dependence to LIF supplementation. Although reversal of the LIF-independent phenotype in Ecad(-/-) ES cells is dependent on the beta-catenin binding domain of E-cadherin, we show that beta-catenin null (betacat(-/-)) ES cells also remain undifferentiated in the absence of LIF. This suggests that LIF-independent self-renewal of Ecad(-/-) ES cells is unlikely to be via beta-catenin signaling. Exposure of Ecad(-/-), EcadRNAi, and CHAVC-treated ES cells to the activin receptor-like kinase inhibitor SB431542 led to differentiation of the cells, which could be prevented by re-expression of E-cadherin. To confirm the role of transforming growth factor beta family signaling in the self-renewal of Ecad(-/-) ES cells, we show that these cells maintain an undifferentiated phenotype when cultured in serum-free medium supplemented with Activin A and Nodal, with fibroblast growth factor 2 required for cellular proliferation. We conclude that transhomodimerization of E-cadherin protein is required for LIF-dependent ES cell self-renewal and that multiple self-renewal signaling networks subsist in ES cells, with activity dependent upon the cellular context.

Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis

PubMed Central

Yotova, Iveta; Quan, Ping; Gaba, Aulona; Leditznig, Nadja; Pateisky, Petra; Kurz, Christine; Tschugguel, Walter

2012-01-01

Endometriosis is a disease characterized by the localization of endometrial tissue outside the uterine cavity. The differences observed in migration of human endometrial stromal cells (hESC) obtained from patients with endometriosis versus healthy controls were proposed to correlate with the abnormal activation of Raf-1/ROCKII signalling pathway. To evaluate the mechanism by which Raf-1 regulates cytoskeleton reorganization and motility, we used primary eutopic (Eu-, n = 16) and ectopic (Ec-, n = 8; isolated from ovarian cysts) hESC of patients with endometriosis and endometriosis-free controls (Co-hESC, n = 14). Raf-1 siRNA knockdown in Co- and Eu-hESC resulted in contraction and decreased migration versus siRNA controls. This phenotype was reversed following the re-expression of Raf-1 in these cells. Lowest Raf-1 levels in Ec-hESC were associated with hyperactivated ROCKII and ezrin/radixin/moesin (E/R/M), impaired migration and a contracted phenotype similar to Raf-1 knockdown in Co- and Eu-hESC. We further show that the mechanism by which Raf-1 mediates migration in hESC includes direct myosin light chain phosphatase (MYPT1) phosphorylation and regulation of the levels of E/R/M, paxillin, MYPT1 and myosin light chain (MLC) phosphorylation indirectly via the hyperactivation of ROCKII kinase. Furthermore, we suggest that in contrast to Co-and Eu-hESC, where the cellular Raf-1 levels regulate the rate of migration, the low cellular Raf-1 content in Ec-hESC, might ensure their restricted migration by preserving the contracted cellular phenotype. In conclusion, our findings suggest that cellular levels of Raf-1 adjust the threshold of hESC migration in endometriosis. PMID:22225925

Capturing the essence of a metabolic network: a flux balance analysis approach.

PubMed

Murabito, Ettore; Simeonidis, Evangelos; Smallbone, Kieran; Swinton, Jonathan

2009-10-07

As genome-scale metabolic reconstructions emerge, tools to manage their size and complexity will be increasingly important. Flux balance analysis (FBA) is a constraint-based approach widely used to study the metabolic capabilities of cellular or subcellular systems. FBA problems are highly underdetermined and many different phenotypes can satisfy any set of constraints through which the metabolic system is represented. Two of the main concerns in FBA are exploring the space of solutions for a given metabolic network and finding a specific phenotype which is representative for a given task such as maximal growth rate. Here, we introduce a recursive algorithm suitable for overcoming both of these concerns. The method proposed is able to find the alternate optimal patterns of active reactions of an FBA problem and identify the minimal subnetwork able to perform a specific task as optimally as the whole. Our method represents an alternative to and an extension of other approaches conceived for exploring the space of solutions of an FBA problem. It may also be particularly helpful in defining a scaffold of reactions upon which to build up a dynamic model, when the important pathways of the system have not yet been well-defined.

A Nonsynonymous Mutation in the Transcriptional Regulator lbh Is Associated with Cichlid Craniofacial Adaptation and Neural Crest Cell Development

PubMed Central

Powder, Kara E.; Cousin, Hélène; McLinden, Gretchen P.; Craig Albertson, R.

2014-01-01

Since the time of Darwin, biologists have sought to understand the origins and maintenance of life’s diversity of form. However, the nature of the exact DNA mutations and molecular mechanisms that result in morphological differences between species remains unclear. Here, we characterize a nonsynonymous mutation in a transcriptional coactivator, limb bud and heart homolog (lbh), which is associated with adaptive variation in the lower jaw of cichlid fishes. Using both zebrafish and Xenopus, we demonstrate that lbh mediates migration of cranial neural crest cells, the cellular source of the craniofacial skeleton. A single amino acid change that is alternatively fixed in cichlids with differing facial morphologies results in discrete shifts in migration patterns of this multipotent cell type that are consistent with both embryological and adult craniofacial phenotypes. Among animals, this polymorphism in lbh represents a rare example of a coding change that is associated with continuous morphological variation. This work offers novel insights into the development and evolution of the craniofacial skeleton, underscores the evolutionary potential of neural crest cells, and extends our understanding of the genetic nature of mutations that underlie divergence in complex phenotypes. PMID:25234704

Evaluation of T-cell activation in the duodenum of dogs with cutaneous food hypersensitivity.

PubMed

Veenhof, Eveline Z; Rutten, Victor P; van Noort, Ronald; Knol, Edward F; Willemse, Ton

2010-04-01

To determine whether skin-related clinical signs in cutaneous food hypersensitivity (CFH) coincide with immune reactivity in the intestine in dogs. 11 dogs with CFH without intestinal clinical signs and 8 healthy control dogs. After a provocation and elimination diet, the duodenal gene expression levels of Th1-, Th2- and Treg-related cytokines and transcription factors were investigated by means of quantitative PCR assay. The presence of CD3(+), CD8(+), CD4(+), CD1c(+), gammadelta T-cell receptor(+), and major histocompatibility complex II(+) cells in duodenal epithelium and lamina propria were determined. The expression of Th1-, Th2-, and Treg-related genes in dogs with CFH and healthy control dogs was similar. Although clinical signs disappeared, there was no effect of the elimination diet on cytokines, transcription factors, or cellular phenotypes. No change in T-cell phenotypes or a distinct Th1, Th2, or Treg profile was detected in the duodenum of dogs with only cutaneous clinical signs of food hypersensitivity. This suggested that the intestinal mucosa is not the primary site of T-cell activation that eventually leads to cutaneous food hypersensitivity.

Liver peroxisomal alanine:glyoxylate aminotransferase and the effects of mutations associated with Primary Hyperoxaluria Type I: An overview.

PubMed

Oppici, Elisa; Montioli, Riccardo; Cellini, Barbara

2015-09-01

Liver peroxisomal alanine:glyoxylate aminotransferase (AGT) (EC 2.6.1.44) catalyses the conversion of l-alanine and glyoxylate to pyruvate and glycine, a reaction that allows glyoxylate detoxification. Inherited mutations on the AGXT gene encoding AGT lead to Primary Hyperoxaluria Type I (PH1), a rare disorder characterized by the deposition of calcium oxalate crystals primarily in the urinary tract. Here we describe the results obtained on the biochemical features of AGT as well as on the molecular and cellular effects of polymorphic and pathogenic mutations. A complex scenario on the molecular pathogenesis of PH1 emerges in which the co-inheritance of polymorphic changes and the condition of homozygosis or compound heterozygosis are two important factors that determine the enzymatic phenotype of PH1 patients. All the reported data represent relevant steps toward the understanding of genotype/phenotype correlations, the prediction of the response of the patients to the available therapies, and the development of new therapeutic approaches. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications. Copyright © 2015 Elsevier B.V. All rights reserved.

AKT-ions with a TWIST between EMT and MET.

PubMed

Tang, Huifang; Massi, Daniela; Hemmings, Brian A; Mandalà, Mario; Hu, Zhengqiang; Wicki, Andreas; Xue, Gongda

2016-09-20

The transcription factor Twist is an important regulator of cranial suture during embryogenesis. Closure of the neural tube is achieved via Twist-triggered cellular transition from an epithelial to mesenchymal phenotype, a process known as epithelial-mesenchymal transition (EMT), characterized by a remarkable increase in cell motility. In the absence of Twist activity, EMT and associated phenotypic changes in cell morphology and motility can also be induced, albeit moderately, by other transcription factor families, including Snail and Zeb. Aberrant EMT triggered by Twist in human mammary tumour cells was first reported to drive metastasis to the lung in a metastatic breast cancer model. Subsequent analysis of many types of carcinoma demonstrated overexpression of these unique EMT transcription factors, which statistically correlated with worse outcome, indicating their potential as biomarkers in the clinic. However, the mechanisms underlying their activation remain unclear. Interestingly, increasing evidence indicates they are selectively activated by distinct intracellular kinases, thereby acting as downstream effectors facilitating transduction of cytoplasmic signals into nucleus and reprogramming EMT and mesenchymal-epithelial transition (MET) transcription to control cell plasticity. Understanding these relationships and emerging data indicating differential phosphorylation of Twist leads to complex and even paradoxical functionalities, will be vital to unlocking their potential in clinical settings.

Inflammation, leukocytes and menstruation.

PubMed

Evans, Jemma; Salamonsen, Lois A

2012-12-01

Menstruation has many of the features of an inflammatory process. The complexity and sequence of inflammatory-type events leading to the final tissue breakdown and bleeding are slowly being unravelled. Progesterone has anti-inflammatory properties, and its rapidly declining levels (along with those of estrogen) in the late secretory phase of each non-conception cycle, initiates a sequence of interdependent events of an inflammatory nature involving local inter-cellular interactions within the endometrium. Intracellular responses to loss of progesterone (in decidualized stromal, vascular and epithelial cells) lead to decreased prostaglandin metabolism and loss of protection from reactive oxygen species (ROS). Increased ROS results in release of NFκB from suppression with activation of target gene transcription and increased synthesis of pro-inflammatory prostaglandins, cytokines, chemokines and matrix metalloproteinases (MMP). The resultant leukocyte recruitment, with changing phenotypes and activation, provide further degradative enzymes and MMP activators, which together with a hypoxic environment induced by prostaglandin actions, lead to the tissue breakdown and bleeding characteristic of menstruation. In parallel, at sites where shedding is complete, microenvironmentally-induced changes in phenotypes of neutrophils and macrophages from pro- to anti-inflammatory, in addition to induction of growth factors, contribute to the very rapid re-epithelialization and restoration of tissue integrity.

Long noncoding RNA NORAD regulates transforming growth factor -β signaling and epithelial-to-mesenchymal transition-like phenotype.

PubMed

Kawasaki, Natsumi; Miwa, Toshiki; Hokari, Satoshi; Sakurai, Tsubasa; Ohmori, Kazuho; Miyauchi, Kensuke; Miyazono, Kohei; Koinuma, Daizo

2018-05-02

Long noncoding RNAs are involved in a variety of cellular functions. In particular, an increasing number of studies have revealed the functions of long noncoding RNAs in various cancers; however, their precise roles and mechanisms of action remain to be elucidated. NORAD, a cytoplasmic long noncoding RNA, is upregulated by irradiation and functions as a potential oncogenic factor by binding and inhibiting Pumilio proteins (PUM1/PUM2). Here, we show that NORAD upregulates transforming growth factor-β (TGF-β) signaling and regulates TGF-β-induced epithelial-to-mesenchymal transition (EMT)-like phenotype, which is a critical step in the progression of lung adenocarcinoma, A549 cells. However, PUM1 does not appear to be involved in this process. We thus focused on importin β1 as a binding partner of NORAD and found that knock down of NORAD partially inhibits the physical interaction of importin β1 with Smad3, inhibiting the nuclear accumulation of Smad complexes in response to TGF-β. Our findings may provide a new mechanism underlying the function of NORAD in cancer cells. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Phenotypic feature quantification of patient derived 3D cancer spheroids in fluorescence microscopy image

NASA Astrophysics Data System (ADS)

Kang, Mi-Sun; Rhee, Seon-Min; Seo, Ji-Hyun; Kim, Myoung-Hee

2017-03-01

Patients' responses to a drug differ at the cellular level. Here, we present an image-based cell phenotypic feature quantification method for predicting the responses of patient-derived glioblastoma cells to a particular drug. We used high-content imaging to understand the features of patient-derived cancer cells. A 3D spheroid culture formation resembles the in vivo environment more closely than 2D adherent cultures do, and it allows for the observation of cellular aggregate characteristics. However, cell analysis at the individual level is more challenging. In this paper, we demonstrate image-based phenotypic screening of the nuclei of patient-derived cancer cells. We first stitched the images of each well of the 384-well plate with the same state. We then used intensity information to detect the colonies. The nuclear intensity and morphological characteristics were used for the segmentation of individual nuclei. Next, we calculated the position of each nucleus that is appeal of the spatial pattern of cells in the well environment. Finally, we compared the results obtained using 3D spheroid culture cells with those obtained using 2D adherent culture cells from the same patient being treated with the same drugs. This technique could be applied for image-based phenotypic screening of cells to determine the patient's response to the drug.

Helicase-inactivating mutations as a basis for dominant negative phenotypes

PubMed Central

Wu, Yuliang

2010-01-01

There is ample evidence from studies of both unicellular and multicellular organisms that helicase-inactivating mutations lead to cellular dysfunction and disease phenotypes. In this review, we will discuss the mechanisms underlying the basis for abnormal phenotypes linked to mutations in genes encoding DNA helicases. Recent evidence demonstrates that a clinically relevant patient missense mutation in Fanconi Anemia Complementation Group J exerts detrimental effects on the biochemical activities of the FANC J helicase, and these molecular defects are responsible for aberrant genomic stability and a poor DNA damage response. The ability of FANC J to use the energy from AT P hydrolysis to produce the force required to unwind duplex or G-quadruplex DNA structures or destabilize protein bound to DNA is required for its DNA repair functions in vivo. Strikingly, helicase-inactivating mutations can exert a spectrum of dominant negative phenotypes, indicating that expression of the mutant helicase protein potentially interferes with normal DNA metabolism and has an effect on basic cellular processes such as DNA replication, the DNA damage response and protein trafficking. This review emphasizes that future studies of clinically relevant mutations in helicase genes will be important to understand the molecular pathologies of the associated diseases and their impact on heterozygote carriers. PMID:20980836

Ploidy-Regulated Variation in Biofilm-Related Phenotypes in Natural Isolates of Saccharomyces cerevisiae

PubMed Central

Hope, Elyse A.; Dunham, Maitreya J.

2014-01-01

The ability of yeast to form biofilms contributes to better survival under stressful conditions. We see the impact of yeast biofilms and “flocs” (clumps) in human health and industry, where forming clumps enables yeast to act as a natural filter in brewing and forming biofilms enables yeast to remain virulent in cases of fungal infection. Despite the importance of biofilms in yeast natural isolates, the majority of our knowledge about yeast biofilm genetics comes from work with a few tractable laboratory strains. A new collection of sequenced natural isolates from the Saccharomyces Genome Resequencing Project enabled us to examine the breadth of biofilm-related phenotypes in geographically, ecologically, and genetically diverse strains of Saccharomyces cerevisiae. We present a panel of 31 haploid and 24 diploid strains for which we have characterized six biofilm-related phenotypes: complex colony morphology, complex mat formation, flocculation, agar invasion, polystyrene adhesion, and psuedohyphal growth. Our results show that there is extensive phenotypic variation between and within strains, and that these six phenotypes are primarily uncorrelated or weakly correlated, with the notable exception of complex colony and complex mat formation. We also show that the phenotypic strength of these strains varies significantly depending on ploidy, and the diploid strains demonstrate both decreased and increased phenotypic strength with respect to their haploid counterparts. This is a more complex view of the impact of ploidy on biofilm-related phenotypes than previous work with laboratory strains has suggested, demonstrating the importance and enormous potential of working with natural isolates of yeast. PMID:25060625

Ploidy-regulated variation in biofilm-related phenotypes in natural isolates of Saccharomyces cerevisiae.

PubMed

Hope, Elyse A; Dunham, Maitreya J

2014-07-24

The ability of yeast to form biofilms contributes to better survival under stressful conditions. We see the impact of yeast biofilms and "flocs" (clumps) in human health and industry, where forming clumps enables yeast to act as a natural filter in brewing and forming biofilms enables yeast to remain virulent in cases of fungal infection. Despite the importance of biofilms in yeast natural isolates, the majority of our knowledge about yeast biofilm genetics comes from work with a few tractable laboratory strains. A new collection of sequenced natural isolates from the Saccharomyces Genome Resequencing Project enabled us to examine the breadth of biofilm-related phenotypes in geographically, ecologically, and genetically diverse strains of Saccharomyces cerevisiae. We present a panel of 31 haploid and 24 diploid strains for which we have characterized six biofilm-related phenotypes: complex colony morphology, complex mat formation, flocculation, agar invasion, polystyrene adhesion, and psuedohyphal growth. Our results show that there is extensive phenotypic variation between and within strains, and that these six phenotypes are primarily uncorrelated or weakly correlated, with the notable exception of complex colony and complex mat formation. We also show that the phenotypic strength of these strains varies significantly depending on ploidy, and the diploid strains demonstrate both decreased and increased phenotypic strength with respect to their haploid counterparts. This is a more complex view of the impact of ploidy on biofilm-related phenotypes than previous work with laboratory strains has suggested, demonstrating the importance and enormous potential of working with natural isolates of yeast. Copyright © 2014 Hope and Dunham.

From integrative genomics to systems genetics in the rat to link genotypes to phenotypes

PubMed Central

Moreno-Moral, Aida

2016-01-01

ABSTRACT Complementary to traditional gene mapping approaches used to identify the hereditary components of complex diseases, integrative genomics and systems genetics have emerged as powerful strategies to decipher the key genetic drivers of molecular pathways that underlie disease. Broadly speaking, integrative genomics aims to link cellular-level traits (such as mRNA expression) to the genome to identify their genetic determinants. With the characterization of several cellular-level traits within the same system, the integrative genomics approach evolved into a more comprehensive study design, called systems genetics, which aims to unravel the complex biological networks and pathways involved in disease, and in turn map their genetic control points. The first fully integrated systems genetics study was carried out in rats, and the results, which revealed conserved trans-acting genetic regulation of a pro-inflammatory network relevant to type 1 diabetes, were translated to humans. Many studies using different organisms subsequently stemmed from this example. The aim of this Review is to describe the most recent advances in the fields of integrative genomics and systems genetics applied in the rat, with a focus on studies of complex diseases ranging from inflammatory to cardiometabolic disorders. We aim to provide the genetics community with a comprehensive insight into how the systems genetics approach came to life, starting from the first integrative genomics strategies [such as expression quantitative trait loci (eQTLs) mapping] and concluding with the most sophisticated gene network-based analyses in multiple systems and disease states. Although not limited to studies that have been directly translated to humans, we will focus particularly on the successful investigations in the rat that have led to primary discoveries of genes and pathways relevant to human disease. PMID:27736746

Growth condition dependency is the major cause of non-responsiveness upon genetic perturbation

PubMed Central

Amini, Saman; Holstege, Frank C. P.

2017-01-01

Investigating the role and interplay between individual proteins in biological processes is often performed by assessing the functional consequences of gene inactivation or removal. Depending on the sensitivity of the assay used for determining phenotype, between 66% (growth) and 53% (gene expression) of Saccharomyces cerevisiae gene deletion strains show no defect when analyzed under a single condition. Although it is well known that this non-responsive behavior is caused by different types of redundancy mechanisms or by growth condition/cell type dependency, it is not known what the relative contribution of these different causes is. Understanding the underlying causes of and their relative contribution to non-responsive behavior upon genetic perturbation is extremely important for designing efficient strategies aimed at elucidating gene function and unraveling complex cellular systems. Here, we provide a systematic classification of the underlying causes of and their relative contribution to non-responsive behavior upon gene deletion. The overall contribution of redundancy to non-responsive behavior is estimated at 29%, of which approximately 17% is due to homology-based redundancy and 12% is due to pathway-based redundancy. The major determinant of non-responsiveness is condition dependency (71%). For approximately 14% of protein complexes, just-in-time assembly can be put forward as a potential mechanistic explanation for how proteins can be regulated in a condition dependent manner. Taken together, the results underscore the large contribution of growth condition requirement to non-responsive behavior, which needs to be taken into account for strategies aimed at determining gene function. The classification provided here, can also be further harnessed in systematic analyses of complex cellular systems. PMID:28257504

From integrative genomics to systems genetics in the rat to link genotypes to phenotypes.

PubMed

Moreno-Moral, Aida; Petretto, Enrico

2016-10-01

Complementary to traditional gene mapping approaches used to identify the hereditary components of complex diseases, integrative genomics and systems genetics have emerged as powerful strategies to decipher the key genetic drivers of molecular pathways that underlie disease. Broadly speaking, integrative genomics aims to link cellular-level traits (such as mRNA expression) to the genome to identify their genetic determinants. With the characterization of several cellular-level traits within the same system, the integrative genomics approach evolved into a more comprehensive study design, called systems genetics, which aims to unravel the complex biological networks and pathways involved in disease, and in turn map their genetic control points. The first fully integrated systems genetics study was carried out in rats, and the results, which revealed conserved trans-acting genetic regulation of a pro-inflammatory network relevant to type 1 diabetes, were translated to humans. Many studies using different organisms subsequently stemmed from this example. The aim of this Review is to describe the most recent advances in the fields of integrative genomics and systems genetics applied in the rat, with a focus on studies of complex diseases ranging from inflammatory to cardiometabolic disorders. We aim to provide the genetics community with a comprehensive insight into how the systems genetics approach came to life, starting from the first integrative genomics strategies [such as expression quantitative trait loci (eQTLs) mapping] and concluding with the most sophisticated gene network-based analyses in multiple systems and disease states. Although not limited to studies that have been directly translated to humans, we will focus particularly on the successful investigations in the rat that have led to primary discoveries of genes and pathways relevant to human disease. © 2016. Published by The Company of Biologists Ltd.

The phenotypic equilibrium of cancer cells: From average-level stability to path-wise convergence.

PubMed

Niu, Yuanling; Wang, Yue; Zhou, Da

2015-12-07

The phenotypic equilibrium, i.e. heterogeneous population of cancer cells tending to a fixed equilibrium of phenotypic proportions, has received much attention in cancer biology very recently. In the previous literature, some theoretical models were used to predict the experimental phenomena of the phenotypic equilibrium, which were often explained by different concepts of stabilities of the models. Here we present a stochastic multi-phenotype branching model by integrating conventional cellular hierarchy with phenotypic plasticity mechanisms of cancer cells. Based on our model, it is shown that: (i) our model can serve as a framework to unify the previous models for the phenotypic equilibrium, and then harmonizes the different kinds of average-level stabilities proposed in these models; and (ii) path-wise convergence of our model provides a deeper understanding to the phenotypic equilibrium from stochastic point of view. That is, the emergence of the phenotypic equilibrium is rooted in the stochastic nature of (almost) every sample path, the average-level stability just follows from it by averaging stochastic samples. Copyright © 2015 Elsevier Ltd. All rights reserved.

The penis: a new target and source of estrogen in male reproduction.

PubMed

Mowa, C N; Jesmin, S; Miyauchi, T

2006-01-01

In the past decade, interest and knowledge in the role of estrogen in male reproduction and fertility has gained significant momentum. More recently, the cellular distribution and activity of estrogen receptors (alpha and beta)(ER) and aromatase (estrogen synthesis) has been reported in the penis, making the penis the latest "frontier" in the study of estrogen in male reproduction. ER and aromatase are broadly and abundantly expressed in various penile compartments and cell types (erectile tissues, urethral epithelia, vascular and neuronal cells), suggesting the complexity and significance of the estrogen-ER system in penile events. Unraveling this complexity is important and will require utilization of the various resources that are now at our disposal including, animal models and human lacking or deficient in ER and aromatase and the use of advanced and sensitive techniques. Some of the obvious areas that require our attention include: 1) a comprehensive mapping of ER-alpha and -beta cellular expression in the different penile compartments and subpopulations of cells, 2) delineation of the specific roles of estrogen in the different subpopulations of cells, 3) establishing the relationship of the estrogen-ER system with the androgen-androgen receptor system, if any, and 4) characterizing the specific penile phenotypes in human and animals lacking or deficient in estrogen and ER. Some data generated thus far, although preliminary, appear to challenge the long held dogma that, overall, androgens have a regulatory monopoly of penile development and function.

A microfluidic co-culture system to monitor tumor-stromal interactions on a chip

PubMed Central

Menon, Nishanth V.; Cao, Bin; Lim, Mayasari; Kang, Yuejun

2014-01-01

The living cells are arranged in a complex natural environment wherein they interact with extracellular matrix and other neighboring cells. Cell-cell interactions, especially those between distinct phenotypes, have attracted particular interest due to the significant physiological relevance they can reveal for both fundamental and applied biomedical research. To study cell-cell interactions, it is necessary to develop co-culture systems, where different cell types can be cultured within the same confined space. Although the current advancement in lab-on-a-chip technology has allowed the creation of in vitro models to mimic the complexity of in vivo environment, it is still rather challenging to create such co-culture systems for easy control of different colonies of cells. In this paper, we have demonstrated a straightforward method for the development of an on-chip co-culture system. It involves a series of steps to selectively change the surface property for discriminative cell seeding and to induce cellular interaction in a co-culture region. Bone marrow stromal cells (HS5) and a liver tumor cell line (HuH7) have been used to demonstrate this co-culture model. The cell migration and cellular interaction have been analyzed using microscopy and biochemical assays. This co-culture system could be used as a disease model to obtain biological insight of pathological progression, as well as a tool to evaluate the efficacy of different drugs for pharmaceutical studies. PMID:25553194

A functional screen for copper homeostasis genes identifies a pharmacologically tractable cellular system

PubMed Central

2014-01-01

Background Copper is essential for the survival of aerobic organisms. If copper is not properly regulated in the body however, it can be extremely cytotoxic and genetic mutations that compromise copper homeostasis result in severe clinical phenotypes. Understanding how cells maintain optimal copper levels is therefore highly relevant to human health. Results We found that addition of copper (Cu) to culture medium leads to increased respiratory growth of yeast, a phenotype which we then systematically and quantitatively measured in 5050 homozygous diploid deletion strains. Cu’s positive effect on respiratory growth was quantitatively reduced in deletion strains representing 73 different genes, the function of which identify increased iron uptake as a cause of the increase in growth rate. Conversely, these effects were enhanced in strains representing 93 genes. Many of these strains exhibited respiratory defects that were specifically rescued by supplementing the growth medium with Cu. Among the genes identified are known and direct regulators of copper homeostasis, genes required to maintain low vacuolar pH, and genes where evidence supporting a functional link with Cu has been heretofore lacking. Roughly half of the genes are conserved in man, and several of these are associated with Mendelian disorders, including the Cu-imbalance syndromes Menkes and Wilson’s disease. We additionally demonstrate that pharmacological agents, including the approved drug disulfiram, can rescue Cu-deficiencies of both environmental and genetic origin. Conclusions A functional screen in yeast has expanded the list of genes required for Cu-dependent fitness, revealing a complex cellular system with implications for human health. Respiratory fitness defects arising from perturbations in this system can be corrected with pharmacological agents that increase intracellular copper concentrations. PMID:24708151

Disease-causing mitochondrial heteroplasmy segregated within induced pluripotent stem cell clones derived from a patient with MELAS.

PubMed

Folmes, Clifford D L; Martinez-Fernandez, Almudena; Perales-Clemente, Ester; Li, Xing; McDonald, Amber; Oglesbee, Devin; Hrstka, Sybil C; Perez-Terzic, Carmen; Terzic, Andre; Nelson, Timothy J

2013-07-01

Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA), known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multilineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts, the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA, resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. On comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared with isogenic clones with high heteroplasmy. Thus, mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny, and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases. Copyright © 2013 AlphaMed Press.

Whole blood genome-wide expression profiling and network analysis suggest MELAS master regulators.

PubMed

Mende, Susanne; Royer, Loic; Herr, Alexander; Schmiedel, Janet; Deschauer, Marcus; Klopstock, Thomas; Kostic, Vladimir S; Schroeder, Michael; Reichmann, Heinz; Storch, Alexander

2011-07-01

The heteroplasmic mitochondrial DNA (mtDNA) mutation A3243G causes the mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome as one of the most frequent mitochondrial diseases. The process of reconfiguration of nuclear gene expression profile to accommodate cellular processes to the functional status of mitochondria might be a key to MELAS disease manifestation and could contribute to its diverse phenotypic presentation. To determine master regulatory protein networks and disease-modifying genes in MELAS syndrome. Analyses of whole blood transcriptomes from 10 MELAS patients using a novel strategy by combining classic Affymetrix oligonucleotide microarray profiling with regulatory and protein interaction network analyses. Hierarchical cluster analysis elucidated that the relative abundance of mutant mtDNA molecules is decisive for the nuclear gene expression response. Further analyses confirmed not only transcription factors already known to be involved in mitochondrial diseases (such as TFAM), but also detected the hypoxia-inducible factor 1 complex, nuclear factor Y and cAMP responsive element-binding protein-related transcription factors as novel master regulators for reconfiguration of nuclear gene expression in response to the MELAS mutation. Correlation analyses of gene alterations and clinico-genetic data detected significant correlations between A3243G-induced nuclear gene expression changes and mutant mtDNA load as well as disease characteristics. These potential disease-modifying genes influencing the expression of the MELAS phenotype are mainly related to clusters primarily unrelated to cellular energy metabolism, but important for nucleic acid and protein metabolism, and signal transduction. Our data thus provide a framework to search for new pathogenetic concepts and potential therapeutic approaches to treat the MELAS syndrome.

Androgenic signaling systems and their role in behavioral evolution.

PubMed

Fuxjager, Matthew J; Schuppe, Eric R

2018-06-05

Sex steroids mediate the organization and activation of masculine reproductive phenotypes in diverse vertebrate taxa. However, the effects of sex steroid action in this context vary tremendously, in that steroid action influences reproductive physiology and behavior in markedly different ways (even among closely related species). This leads to the idea that the mechanisms underlying sex steroid action similarly differ across vertebrates in a manner that supports diversification of important sexual traits. Here, we highlight the Evolutionary Potential Hypothesis as a framework for understanding how androgen-dependent reproductive behavior evolves. This idea posits that the cellular mechanisms underlying androgenic action can independently evolve within a given target tissue to adjust the hormone's functional effects. The result is a seemingly endless number of permutations in androgenic signaling pathways that can be mapped onto the incredible diversity of reproductive phenotypes. One reason this hypothesis is important is because it shifts current thinking about the evolution of steroid-dependent traits away from an emphasis on circulating steroid levels and toward a focus on molecular mechanisms of hormone action. To this end, we also provide new empirical data suggesting that certain cellular modulators of androgen action-namely, the co-factors that dynamically adjust transcritpional effects of steroid action either up or down-are also substrates on which evolution can act. We then close the review with a detailed look at a case study in the golden-collared manakin (Manacus vitellinus). Work in this tropical bird shows how androgenic signaling systems are modified in specific parts of the skeletal muscle system to enhance motor performance necessary to produce acrobatic courtship displays. Altogether, this paper seeks to develop a platform to better understand how steroid action influences the evolution of complex animal behavior. Copyright © 2018 Elsevier Ltd. All rights reserved.

The Drosophila ETV5 Homologue Ets96B: Molecular Link between Obesity and Bipolar Disorder.

PubMed

Williams, Michael J; Klockars, Anica; Eriksson, Anders; Voisin, Sarah; Dnyansagar, Rohit; Wiemerslage, Lyle; Kasagiannis, Anna; Akram, Mehwish; Kheder, Sania; Ambrosi, Valerie; Hallqvist, Emilie; Fredriksson, Robert; Schiöth, Helgi B

2016-06-01

Several reports suggest obesity and bipolar disorder (BD) share some physiological and behavioural similarities. For instance, obese individuals are more impulsive and have heightened reward responsiveness, phenotypes associated with BD, while bipolar patients become obese at a higher rate and earlier age than people without BD; however, the molecular mechanisms of such an association remain obscure. Here we demonstrate, using whole transcriptome analysis, that Drosophila Ets96B, homologue of obesity-linked gene ETV5, regulates cellular systems associated with obesity and BD. Consistent with a role in obesity and BD, loss of nervous system Ets96B during development increases triacylglyceride concentration, while inducing a heightened startle-response, as well as increasing hyperactivity and reducing sleep. Of notable interest, mouse Etv5 and Drosophila Ets96B are expressed in dopaminergic-rich regions, and loss of Ets96B specifically in dopaminergic neurons recapitulates the metabolic and behavioural phenotypes. Moreover, our data indicate Ets96B inhibits dopaminergic-specific neuroprotective systems. Additionally, we reveal that multiple SNPs in human ETV5 link to body mass index (BMI) and BD, providing further evidence for ETV5 as an important and novel molecular intermediate between obesity and BD. We identify a novel molecular link between obesity and bipolar disorder. The Drosophila ETV5 homologue Ets96B regulates the expression of cellular systems with links to obesity and behaviour, including the expression of a conserved endoplasmic reticulum molecular chaperone complex known to be neuroprotective. Finally, a connection between the obesity-linked gene ETV5 and bipolar disorder emphasizes a functional relationship between obesity and BD at the molecular level.

TNFα Levels and Macrophages Expression Reflect an Inflammatory Potential of Trigeminal Ganglia in a Mouse Model of Familial Hemiplegic Migraine

PubMed Central

Franceschini, Alessia; Vilotti, Sandra; Ferrari, Michel D.; van den Maagdenberg, Arn M. J. M.; Nistri, Andrea; Fabbretti, Elsa

2013-01-01

Latent changes in trigeminal ganglion structure and function resembling inflammatory conditions may predispose to acute attacks of migraine pain. Here, we investigated whether, in trigeminal sensory ganglia, cytokines such as TNFα might contribute to a local inflammatory phenotype of a transgenic knock-in (KI) mouse model of familial hemiplegic migraine type-1 (FHM-1). To this end, macrophage occurrence and cytokine expression in trigeminal ganglia were compared between wild type (WT) and R192Q mutant CaV2.1 Ca2+ channel (R192Q KI) mice, a genetic model of FHM-1. Cellular and molecular characterization was performed using a combination of confocal immunohistochemistry and cytokine assays. With respect to WT, R192Q KI trigeminal ganglia were enriched in activated macrophages as suggested by their morphology and immunoreactivity to the markers Iba1, CD11b, and ED1. R192Q KI trigeminal ganglia constitutively expressed higher mRNA levels of IL1β, IL6, IL10 and TNFα cytokines and the MCP-1 chemokine. Consistent with the report that TNFα is a major factor to sensitize trigeminal ganglia, we observed that, following an inflammatory reaction evoked by LPS injection, TNFα expression and macrophage occurrence were significantly higher in R192Q KI ganglia with respect to WT ganglia. Our data suggest that, in KI trigeminal ganglia, the complex cellular and molecular environment could support a new tissue phenotype compatible with a neuroinflammatory profile. We propose that, in FHM patients, this condition might contribute to trigeminal pain pathophysiology through release of soluble mediators, including TNFα, that may modulate the crosstalk between sensory neurons and resident glia, underlying the process of neuronal sensitisation. PMID:23326332

A split motor domain in a cytoplasmic dynein

PubMed Central

Straube, Anne; Enard, Wolfgang; Berner, Alexandra; Wedlich-Söldner, Roland; Kahmann, Regine; Steinberg, Gero

2001-01-01

The heavy chain of dynein forms a globular motor domain that tightly couples the ATP-cleavage region and the microtubule-binding site to transform chemical energy into motion along the cytoskeleton. Here we show that, in the fungus Ustilago maydis, two genes, dyn1 and dyn2, encode the dynein heavy chain. The putative ATPase region is provided by dyn1, while dyn2 includes the predicted microtubule-binding site. Both genes are located on different chromosomes, are transcribed into independent mRNAs and are translated into separate polypeptides. Both Dyn1 and Dyn2 co-immunoprecipitated and co-localized within growing cells, and Dyn1–Dyn2 fusion proteins partially rescued mutant phenotypes, suggesting that both polypeptides interact to form a complex. In cell extracts the Dyn1–Dyn2 complex dissociated, and microtubule affinity purification indicated that Dyn1 or associated polypeptides bind microtubules independently of Dyn2. Both Dyn1 and Dyn2 were essential for cell survival, and conditional mutants revealed a common role in nuclear migration, cell morphogenesis and microtubule organization, indicating that the Dyn1–Dyn2 complex serves multiple cellular functions. PMID:11566874

Functional interdependence at the chromatin level between the MKK6/p38 and IGF1/PI3K/AKT pathways during muscle differentiation.

PubMed

Serra, Carlo; Palacios, Daniela; Mozzetta, Chiara; Forcales, Sonia V; Morantte, Ianessa; Ripani, Meri; Jones, David R; Du, Keyong; Jhala, Ulupi S; Simone, Cristiano; Puri, Pier Lorenzo

2007-10-26

During muscle regeneration, the mechanism integrating environmental cues at the chromatin of muscle progenitors is unknown. We show that inflammation-activated MKK6-p38 and insulin growth factor 1 (IGF1)-induced PI3K/AKT pathways converge on the chromatin of muscle genes to target distinct components of the muscle transcriptosome. p38 alpha/beta kinases recruit the SWI/SNF chromatin-remodeling complex; AKT1 and 2 promote the association of MyoD with p300 and PCAF acetyltransferases, via direct phosphorylation of p300. Pharmacological or genetic interference with either pathway led to partial assembly of discrete chromatin-bound complexes, which reflected two reversible and distinct cellular phenotypes. Remarkably, PI3K/AKT blockade was permissive for chromatin recruitment of MEF2-SWI/SNF complex, whose remodeling activity was compromised in the absence of MyoD and acetyltransferases. The functional interdependence between p38 and IGF1/PI3K/AKT pathways was further established by the evidence that blockade of AKT chromatin targets was sufficient to prevent the activation of the myogenic program triggered by deliberate activation of p38 signaling.

Functional interdependence at the chromatin level between the MKK6/p38 and IGF1/Pi3K/AKT pathways during muscle differentiation

PubMed Central

Carlo, Serra; Daniela, Palacios; Chiara, Mozzetta; Sonia, Forcales; Ianessa, Morantte; Meri, Ripani; Jones David, R.; Keyong, Du; Jhala Ulupi, S.; Cristiano, Simone; Lorenzo, Puri Pier

2009-01-01

During muscle regeneration, the mechanism integrating environmental cues at the chromatin of muscle progenitors is unknown. We show that inflammation-activated MKK6-p38 and IGF1-induced Pi3K/AKT pathways converge on the chromatin of muscle genes to target distinct components of the muscle transcriptosome. p38 α/β kinases recruit the SWI/SNF chromatin-remodeling complex; AKT 1 and 2 promote the association of MyoD with p300 and PCAF acetyltransferases, via direct phosphorylation of p300. Pharmacological or genetic interference with either pathway led to partial assembly of discrete chromatin-bound complexes, which reflected two reversible and distinct cellular phenotypes. Remarkably, Pi3K/AKT blockade was permissive for chromatin recruitment of MEF2-SWI/SNF complex, whose remodeling activity was compromised in the absence of MyoD and acetyltransferases. The functional interdependence between p38 and IGF1/Pi3K/AKT pathways was further established by the evidence that blockade of AKT chromatin targets was sufficient to prevent the activation of the myogenic program triggered by deliberate activation of p38 signaling PMID:17964260

Systems Biology Analysis Merging Phenotype, Metabolomic and Genomic Data Identifies Non-SMC Condensin I Complex, Subunit G (NCAPG) and Cellular Maintenance Processes as Major Contributors to Genetic Variability in Bovine Feed Efficiency

PubMed Central

Widmann, Philipp; Reverter, Antonio; Weikard, Rosemarie; Suhre, Karsten; Hammon, Harald M.; Albrecht, Elke; Kuehn, Christa

2015-01-01

Feed efficiency is a paramount factor for livestock economy. Previous studies had indicated a substantial heritability of several feed efficiency traits. In our study, we investigated the genetic background of residual feed intake, a commonly used parameter of feed efficiency, in a cattle resource population generated from crossing dairy and beef cattle. Starting from a whole genome association analysis, we subsequently performed combined phenotype-metabolome-genome analysis taking a systems biology approach by inferring gene networks based on partial correlation and information theory approaches. Our data about biological processes enriched with genes from the feed efficiency network suggest that genetic variation in feed efficiency is driven by genetic modulation of basic processes relevant to general cellular functions. When looking at the predicted upstream regulators from the feed efficiency network, the Tumor Protein P53 (TP53) and Transforming Growth Factor beta 1 (TGFB1) genes stood out regarding significance of overlap and number of target molecules in the data set. These results further support the hypothesis that TP53 is a major upstream regulator for genetic variation of feed efficiency. Furthermore, our data revealed a significant effect of both, the Non-SMC Condensin I Complex, Subunit G (NCAPG) I442M (rs109570900) and the Growth /differentiation factor 8 (GDF8) Q204X (rs110344317) loci, on residual feed intake and feed conversion. For both loci, the growth promoting allele at the onset of puberty was associated with a negative, but favorable effect on residual feed intake. The elevated energy demand for increased growth triggered by the NCAPG 442M allele is obviously not fully compensated for by an increased efficiency in converting feed into body tissue. As a consequence, the individuals carrying the NCAPG 442M allele had an additional demand for energy uptake that is reflected by the association of the allele with increased daily energy intake as observed in our study. PMID:25875852

Effect of Phospholipidosis on the Cellular Pharmacokinetics of ChloroquineS⃞

PubMed Central

Zheng, Nan; Zhang, Xinyuan

2011-01-01

In vivo, the weakly basic, lipophilic drug chloroquine (CQ) accumulates in the kidney to concentrations more than a thousand-fold greater than those in plasma. To study the cellular pharmacokinetics of chloroquine in cells derived from the distal tubule, Madin-Darby canine kidney cells were incubated with CQ under various conditions. CQ progressively accumulated without exhibiting steady-state behavior. Experiments failed to yield evidence that known active transport mechanisms mediated CQ uptake at the plasma membrane. CQ induced a phospholipidosis-like phenotype, characterized by the appearance of numerous multivesicular and multilamellar bodies (MLBs/MVBs) within the lumen of expanded cytoplasmic vesicles. Other induced phenotypic changes including changes in the volume and pH of acidic organelles were measured, and the integrated effects of all these changes were computationally modeled to establish their impact on intracellular CQ mass accumulation. Based on the passive transport behavior of CQ, the measured phenotypic changes fully accounted for the continuous, nonsteady-state CQ accumulation kinetics. Consistent with the simulation results, Raman confocal microscopy of live cells confirmed that CQ became highly concentrated within induced, expanded cytoplasmic vesicles that contained multiple MLBs/MVBs. Progressive CQ accumulation was increased by sucrose, a compound that stimulated the phospholipidosis-like phenotype, and was decreased by bafilomycin A1, a compound that inhibited this phenotype. Thus, phospholipidosis-associated changes in organelle structure and intracellular membrane content can exert a major influence on the local bioaccumulation and biodistribution of drugs. PMID:21156819

Interpreting phenotypic antibiotic tolerance and persister cells as evolution via epigenetic inheritance.

PubMed

Day, Troy

2016-04-01

Epigenetic inheritance is the transmission of nongenetic material such as gene expression levels, RNA and other biomolecules from parents to offspring. There is a growing realization that such forms of inheritance can play an important role in evolution. Bacteria represent a prime example of epigenetic inheritance because a large array of cellular components is transmitted to offspring, in addition to genetic material. Interestingly, there is an extensive and growing empirical literature showing that many bacteria can form 'persister' cells that are phenotypically resistant or tolerant to antibiotics, but most of these results are not interpreted within the context of epigenetic inheritance. Instead, persister cells are usually viewed as a genetically encoded bet-hedging strategy that has evolved in response to a fluctuating environment. Here I show, using a relatively simple model, that many of these empirical findings can be more simply understood as arising from a combination of epigenetic inheritance and cellular noise. I therefore suggest that phenotypic drug tolerance in bacteria might represent one of the best-studied examples of evolution under epigenetic inheritance. © 2016 John Wiley & Sons Ltd.

Cancer cell metabolism and the modulating effects of nitric oxide.

PubMed

Chang, Ching-Fang; Diers, Anne R; Hogg, Neil

2015-02-01

Altered metabolic phenotype has been recognized as a hallmark of tumor cells for many years, but this aspect of the cancer phenotype has come into greater focus in recent years. NOS2 (inducible nitric oxide synthase of iNOS) has been implicated as a component in many aggressive tumor phenotypes, including melanoma, glioblastoma, and breast cancer. Nitric oxide has been well established as a modulator of cellular bioenergetics pathways, in many ways similar to the alteration of cellular metabolism observed in aggressive tumors. In this review we attempt to bring these concepts together with the general hypothesis that one function of NOS2 and NO in cancer is to modulate metabolic processes to facilitate increased tumor aggression. There are many mechanisms by which NO can modulate tumor metabolism, including direct inhibition of respiration, alterations in mitochondrial mass, oxidative inhibition of bioenergetic enzymes, and the stimulation of secondary signaling pathways. Here we review metabolic alterations in the context of cancer cells and discuss the role of NO as a potential mediator of these changes. Copyright © 2015. Published by Elsevier Inc.

Roles of GSK3 in metabolic shift toward abnormal anabolism in cell senescence.

PubMed

Kim, You-Mie; Seo, Yong-Hak; Park, Chan-Bae; Yoon, Soo-Han; Yoon, Gyesoon

2010-07-01

Diverse metabolic alterations, including mitochondrial dysfunction, have often been reported as characteristic phenotypes of senescent cells. However, the overall consequence of senescent metabolic features, how they develop, and how they are linked to other senescent phenotypes, such as enlarged cell volume, increased granularity, and oxidative stress, is not clear. We investigated the potential roles of glycogen synthase kinase 3 (GSK3), a multifunctional kinase, in the development of the metabolic phenotypes in cell senescence. The inactivation of GSK3 via phosphorylation is commonly observed in diverse cell senescences. Furthermore, subcytotoxic concentration of GSK3 inhibitor was sufficient to induce cellular senescence, accompanied by augmented anabolism, such as enhanced protein synthesis, and increased glycogenesis and lipogenesis, in addition to mitochondrial dysfunction. Anabolism was accomplished through glycogen synthase, eIF2B, and SREBP1. These metabolic features seem to contribute to an increase in cellular mass by increasing glycogen granules, protein mass, and organelles. Taken together, our results suggest that GSK3 is one of the key modulators of metabolic alteration, leading the cells to senescence.

Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping

PubMed Central

Treweek, Jennifer B; Deverman, Benjamin E; Greenbaum, Alon; Lignell, Antti; Xiao, Cheng; Cai, Long; Ladinsky, Mark S; Bjorkman, Pamela J; Fowlkes, Charless C; Gradinaru, Viviana

2016-01-01

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks. PMID:26492141

Dynamic Microenvironment Induces Phenotypic Plasticity of Esophageal Cancer Cells Under Flow

NASA Astrophysics Data System (ADS)

Calibasi Kocal, Gizem; Güven, Sinan; Foygel, Kira; Goldman, Aaron; Chen, Pu; Sengupta, Shiladitya; Paulmurugan, Ramasamy; Baskin, Yasemin; Demirci, Utkan

2016-12-01

Cancer microenvironment is a remarkably heterogeneous composition of cellular and non-cellular components, regulated by both external and intrinsic physical and chemical stimuli. Physical alterations driven by increased proliferation of neoplastic cells and angiogenesis in the cancer microenvironment result in the exposure of the cancer cells to elevated levels of flow-based shear stress. We developed a dynamic microfluidic cell culture platform utilizing eshopagael cancer cells as model cells to investigate the phenotypic changes of cancer cells upon exposure to fluid shear stress. We report the epithelial to hybrid epithelial/mesenchymal transition as a result of decreasing E-Cadherin and increasing N-Cadherin and vimentin expressions, higher clonogenicity and ALDH positive expression of cancer cells cultured in a dynamic microfluidic chip under laminar flow compared to the static culture condition. We also sought regulation of chemotherapeutics in cancer microenvironment towards phenotypic control of cancer cells. Such in vitro microfluidic system could potentially be used to monitor how the interstitial fluid dynamics affect cancer microenvironment and plasticity on a simple, highly controllable and inexpensive bioengineered platform.

Cancer Cell Metabolism and the Modulating Effects of Nitric Oxide

PubMed Central

Chang, Ching-Fang; Diers, Anne R.; Hogg, Neil

2016-01-01

Altered metabolic phenotype has been recognized as a hallmark of tumor cells for many years, but this aspect of the cancer phenotype has come into greater focus in recent years. NOS2 (inducible nitric oxide synthase of iNOS) has been implicated as a component in many aggressive tumor phenotypes, including melanoma, glioblastoma and breast cancer. Nitric oxide has been well established as a modulator of cellular bioenergetics pathways, in many ways similar to the alteration of cellular metabolism observed in aggressive tumors. In this review we attempt to bring these concepts together with the general hypothesis that one function of NOS2 and NO in cancer is to modulate metabolic processes to facilitate increased tumor aggression. There are many mechanisms by which NO can modulate tumor metabolism, including direct inhibition of respiration, alterations in mitochondrial mass, oxidative inhibition of bioenergetic enzymes, and the stimulation of secondary signaling pathways. Here we review metabolic alterations in the context of cancer cells and discuss the role of NO as a potential mediator of these changes. PMID:25464273

GLUT3 gene expression is critical for embryonic growth, brain development and survival.

PubMed

Carayannopoulos, Mary O; Xiong, Fuxia; Jensen, Penny; Rios-Galdamez, Yesenia; Huang, Haigen; Lin, Shuo; Devaskar, Sherin U

2014-04-01

Glucose is the primary energy source for eukaryotic cells and the predominant substrate for the brain. GLUT3 is essential for trans-placental glucose transport and highly expressed in the mammalian brain. To further elucidate the role of GLUT3 in embryonic development, we utilized the vertebrate whole animal model system of Danio rerio as a tractable system for defining the cellular and molecular mechanisms altered by impaired glucose transport and metabolism related to perturbed expression of GLUT3. The comparable orthologue of human GLUT3 was identified and the expression of this gene abrogated during early embryonic development. In a dose-dependent manner embryonic brain development was disrupted resulting in a phenotype of aberrant brain organogenesis, associated with embryonic growth restriction and increased cellular apoptosis. Rescue of the morphant phenotype was achieved by providing exogenous GLUT3 mRNA. We conclude that GLUT3 is critically important for brain organogenesis and embryonic growth. Disruption of GLUT3 is responsible for the phenotypic spectrum of embryonic growth restriction to demise and neural apoptosis with microcephaly. Copyright © 2014 Elsevier Inc. All rights reserved.

Histone methylation and aging: Lessons learned from model systems

PubMed Central

McCauley, Brenna S.; Dang, Weiwei

2014-01-01

Aging induces myriad cellular and, ultimately, physiological changes that cause a decline in an organism's functional capabilities. Although the aging process and pathways that regulate it have been extensively studied, only in the last decade have we begun to appreciate that dynamic histone methylation may contribute to this process. In this review, we discuss recent work implicating histone methylation in aging. Loss of certain histone methyltransferases and demethylases changes lifespan in invertebrates, and alterations in histone methylation in aged organisms regulate lifespan and aging phenotypes, including oxidative stress-induced hormesis in yeast, insulin signaling in Caenorhabiditis elegans and mammals, and the senescence-associated secretory phenotype in mammals. In all cases where histone methylation has been shown to impact aging and aging phenotypes, it does so by regulating transcription, suggesting that this is a major mechanism of its action in this context. Histone methylation additionally regulates or is regulated by other cellular pathways that contribute to or combat aging. Given the numerous processes that regulate aging and histone methylation, and are in turn regulated by them, the role of histone methylation in aging is almost certainly underappreciated. PMID:24859460

Disruption of Src Is Associated with Phenotypes Related to Williams-Beuren Syndrome and Altered Cellular Localization of TFII-I1,2

PubMed Central

Ivakine, Evgueni A.; Lam, Emily; Deurloo, Marielle; Dida, Joana; Zirngibl, Ralph A.

2015-01-01

Abstract Src is a nonreceptor protein tyrosine kinase that is expressed widely throughout the central nervous system and is involved in diverse biological functions. Mice homozygous for a spontaneous mutation in Src (Src thl/thl) exhibited hypersociability and hyperactivity along with impairments in visuospatial, amygdala-dependent, and motor learning as well as an increased startle response to loud tones. The phenotype of Src thl/thl mice showed significant overlap with Williams-Beuren syndrome (WBS), a disorder caused by the deletion of several genes, including General Transcription Factor 2-I (GTF2I). Src phosphorylation regulates the movement of GTF2I protein (TFII-I) between the nucleus, where it is a transcriptional activator, and the cytoplasm, where it regulates trafficking of transient receptor potential cation channel, subfamily C, member 3 (TRPC3) subunits to the plasma membrane. Here, we demonstrate altered cellular localization of both TFII-I and TRPC3 in the Src mutants, suggesting that disruption of Src can phenocopy behavioral phenotypes observed in WBS through its regulation of TFII-I. PMID:26464974

GLUT3 Gene Expression is Critical for Embryonic Growth, Brain Development and Survival

PubMed Central

Carayannopoulos, Mary O.; Xiong, Fuxia; Jensen, Penny; Rios-Galdamez, Yesenia; Huang, Haigen; Lin, Shuo; Devaskar, Sherin U.

2015-01-01

Glucose is the primary energy source for eukaryotic cells and the predominant substrate for the brain. GLUT3 is essential for trans-placental glucose transport and highly expressed in the mammalian brain. To further elucidate the role of GLUT3 in embryonic development, we utilized the vertebrate whole animal model system of Danio rerio as a tractable system for defining the cellular and molecular mechanisms altered by impaired glucose transport and metabolism related to perturbed expression of GLUT3. The comparable orthologue of human GLUT3 was identified and the expression of this gene abrogated during early embryonic development. In a dose-dependent manner embryonic brain development was disrupted resulting in a phenotype of aberrant brain organogenesis, associated with embryonic growth restriction and increased cellular apoptosis. Rescue of the morphant phenotype was achieved by providing exogenous GLUT3 mRNA. We conclude that GLUT3 is critically important for brain organogenesis and embryonic growth. Disruption of GLUT3 is responsible for the phenotypic spectrum of embryonic growth restriction to demise and neural apoptosis with microcephaly. PMID:24529979

The two Dictyostelium discoideum autophagy 8 proteins have distinct autophagic functions.

PubMed

Meßling, Susanne; Matthias, Jan; Xiong, Qiuhong; Fischer, Sarah; Eichinger, Ludwig

2017-06-01

Autophagy is a highly conserved cellular degradation pathway which is crucial for various cellular processes. The autophagic process is subdivided in the initiation, autophagosome maturation and lysosomal degradation phases and involves more than forty core and accessory autophagy-related (ATG) proteins. Autophagy 8 (ATG8, in mammals LC3) is a well-established marker of autophagy and is linked to the autophagic membrane from initiation until fusion with the lysosome. We generated single and double knock-out mutants of the two Dictyostelium paralogues, ATG8a and ATG8b, as well as strains that expressed RFP-ATG8a and/or GFP-ATG8b, RFP-ATG8b, RFP-GFP-ATG8a or RFP-GFP-ATG8b in different knock-out mutants. The ATG8b¯ mutant displayed only subtle phenotypic changes in comparison to AX2 wild-type cells. In contrast, deletion of ATG8a resulted in a complex phenotype with delayed development, reduced growth, phagocytosis and cell viability, an increase in ubiquitinylated proteins and a concomitant decrease in proteasomal activity. The phenotype of the ATG8a¯/b¯ strain was, except for cell viability, in all aforementioned aspects more severe, showing that both proteins function in parallel during most analysed cellular processes. Immunofluorescence analysis of knock-out strains expressing either RFP-GFP-ATG8a or RFP-GFP-ATG8b suggests a crucial function for ATG8b in autophagosome-lysosome fusion. Quantitative analysis of strains expressing RFP-ATG8a, RFP-ATG8b, or RFP-ATG8a and GFP-ATG8b revealed that ATG8b generally localised to small and large vesicles, whereas ATG8a preferentially co-localised with ATG8b on large vesicles, indicating that ATG8b associated with nascent autophagosomes before ATG8a, which is supported by previous results (Matthias et al., 2016). Deconvoluted confocal fluorescence images showed that ATG8b localised around ATG8a and was presumably mainly present on the outer membrane of the autophagosome while ATG8a appears to be mainly associated with the inner membrane. In summary, our data show that ATG8a and ATG8b have distinct functions and are involved in canonical as well as non-canonical autophagy. The data further suggest that ATG8b predominantly acts as adapter for the autophagy machinery at the outer and ATG8a as cargo receptor at the inner membrane of the autophagosome. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

The UL24 protein of herpes simplex virus 1 affects the sub-cellular distribution of viral glycoproteins involved in fusion

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

Ben Abdeljelil, Nawel; Rochette, Pierre-Alexandre; Pearson, Angela, E-mail: angela.pearson@iaf.inrs.ca

2013-09-15

Mutations in UL24 of herpes simplex virus type 1 can lead to a syncytial phenotype. We hypothesized that UL24 affects the sub-cellular distribution of viral glycoproteins involved in fusion. In non-immortalized human foreskin fibroblasts (HFFs) we detected viral glycoproteins B (gB), gD, gH and gL present in extended blotches throughout the cytoplasm with limited nuclear membrane staining; however, in HFFs infected with a UL24-deficient virus (UL24X), staining for the viral glycoproteins appeared as long, thin streaks running across the cell. Interestingly, there was a decrease in co-localized staining of gB and gD with F-actin at late times in UL24X-infected HFFs.more » Treatment with chemical agents that perturbed the actin cytoskeleton hindered the formation of UL24X-induced syncytia in these cells. These data support a model whereby the UL24 syncytial phenotype results from a mislocalization of viral glycoproteins late in infection. - Highlights: • UL24 affects the sub-cellular distribution of viral glycoproteins required for fusion. • Sub-cellular distribution of viral glycoproteins varies in cell-type dependent manner. • Drugs targeting actin microfilaments affect formation of UL24-related syncytia in HFFs.« less

Senescent intervertebral disc cells exhibit perturbed matrix homeostasis phenotype.

PubMed

Ngo, Kevin; Patil, Prashanti; McGowan, Sara J; Niedernhofer, Laura J; Robbins, Paul D; Kang, James; Sowa, Gwendolyn; Vo, Nam

2017-09-01

Aging greatly increases the risk for intervertebral disc degeneration (IDD) as a result of proteoglycan loss due to reduced synthesis and enhanced degradation of the disc matrix proteoglycan (PG). How disc matrix PG homeostasis becomes perturbed with age is not known. The goal of this study is to determine whether cellular senescence is a source of this perturbation. We demonstrated that disc cellular senescence is dramatically increased in the DNA repair-deficient Ercc1 -/Δ mouse model of human progeria. In these accelerated aging mice, increased disc cellular senescence is closely associated with the rapid loss of disc PG. We also directly examine PG homeostasis in oxidative damage-induced senescent human cells using an in vitro cell culture model system. Senescence of human disc cells treated with hydrogen peroxide was confirmed by growth arrest, senescence-associated β-galactosidase activity, γH2AX foci, and acquisition of senescence-associated secretory phenotype. Senescent human disc cells also exhibited perturbed matrix PG homeostasis as evidenced by their decreased capacity to synthesize new matrix PG and enhanced degradation of aggrecan, a major matrix PG. of the disc. Our in vivo and in vitro findings altogether suggest that disc cellular senescence is an important driver of PG matrix homeostatic perturbation and PG loss. Published by Elsevier B.V.

Simultaneous capture and in situ analysis of circulating tumor cells using multiple hybrid nanoparticles.

PubMed

Lee, Hun Joo; Cho, Hyeon-Yeol; Oh, Jin Ho; Namkoong, Kak; Lee, Jeong Gun; Park, Jong-Myeon; Lee, Soo Suk; Huh, Nam; Choi, Jeong-Woo

2013-09-15

Using hybrid nanoparticles (HNPs), we demonstrate simultaneous capture, in situ protein expression analysis, and cellular phenotype identification of circulating tumor cells (CTCs). Each HNP consists of three parts: (i) antibodies that bind specifically to a known biomarker for CTCs, (ii) a quantum dot that emits fluorescence signals, and (iii) biotinylated DNA that allows capture and release of CTC-HNP complex to an in-house developed capture & recovery chip (CRC). To evaluate our approach, cells representative of different breast cancer subtypes (MCF-7: luminal; SK-BR-3: HER2; and MDA-MB-231: basal-like) were captured onto CRC and expressions of EpCAM, HER2, and EGFR were detected concurrently. The average capture efficiency of CTCs was 87.5% with identification accuracy of 92.4%. Subsequently, by cleaving the DNA portion with restriction enzymes, captured cells were released at efficiencies of 86.1%. Further studies showed that these recovered cells are viable and can proliferate in vitro. Using HNPs, it is possible to count, analyze in situ protein expression, and culture CTCs, all from the same set of cells, enabling a wide range of molecular- and cellular-based studies using CTCs. Copyright © 2013 Elsevier B.V. All rights reserved.

Customizing microarrays for neuroscience drug discovery.

PubMed

Girgenti, Matthew J; Newton, Samuel S

2007-08-01

Microarray-based gene profiling has become the centerpiece of gene expression studies in the biological sciences. The ability to now interrogate the entire genome using a single chip demonstrates the progress in technology and instrumentation that has been made over the last two decades. Although this unbiased approach provides researchers with an immense quantity of data, obtaining meaningful insight is not possible without intensive data analysis and processing. Custom developed arrays have emerged as a viable and attractive alternative that can take advantage of this robust technology and tailor it to suit the needs and requirements of individual investigations. The ability to simplify data analysis, reduce noise and carefully optimize experimental conditions makes it a suitable tool that can be effectively utilized in neuroscience drug discovery efforts. Furthermore, incorporating recent advancements in fine focusing gene profiling to include specific cellular phenotypes can help resolve the complex cellular heterogeneity of the brain. This review surveys the use of microarray technology in neuroscience paying special attention to customized arrays and their potential in drug discovery. Novel applications of microarrays and ancillary techniques, such as laser microdissection, FAC sorting and RNA amplification, have also been discussed. The notion that a hypothesis-driven approach can be integrated into drug development programs is highlighted.

How to Train a Cell–Cutting-Edge Molecular Tools

PubMed Central

Czapiński, Jakub; Kiełbus, Michał; Kałafut, Joanna; Kos, Michał; Stepulak, Andrzej; Rivero-Müller, Adolfo

2017-01-01

In biological systems, the formation of molecular complexes is the currency for all cellular processes. Traditionally, functional experimentation was targeted to single molecular players in order to understand its effects in a cell or animal phenotype. In the last few years, we have been experiencing rapid progress in the development of ground-breaking molecular biology tools that affect the metabolic, structural, morphological, and (epi)genetic instructions of cells by chemical, optical (optogenetic) and mechanical inputs. Such precise dissection of cellular processes is not only essential for a better understanding of biological systems, but will also allow us to better diagnose and fix common dysfunctions. Here, we present several of these emerging and innovative techniques by providing the reader with elegant examples on how these tools have been implemented in cells, and, in some cases, organisms, to unravel molecular processes in minute detail. We also discuss their advantages and disadvantages with particular focus on their translation to multicellular organisms for in vivo spatiotemporal regulation. We envision that further developments of these tools will not only help solve the processes of life, but will give rise to novel clinical and industrial applications. PMID:28344971

Mutant power: using mutant allele collections for yeast functional genomics.

PubMed

Norman, Kaitlyn L; Kumar, Anuj

2016-03-01

The budding yeast has long served as a model eukaryote for the functional genomic analysis of highly conserved signaling pathways, cellular processes and mechanisms underlying human disease. The collection of reagents available for genomics in yeast is extensive, encompassing a growing diversity of mutant collections beyond gene deletion sets in the standard wild-type S288C genetic background. We review here three main types of mutant allele collections: transposon mutagen collections, essential gene collections and overexpression libraries. Each collection provides unique and identifiable alleles that can be utilized in genome-wide, high-throughput studies. These genomic reagents are particularly informative in identifying synthetic phenotypes and functions associated with essential genes, including those modeled most effectively in complex genetic backgrounds. Several examples of genomic studies in filamentous/pseudohyphal backgrounds are provided here to illustrate this point. Additionally, the limitations of each approach are examined. Collectively, these mutant allele collections in Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans promise insights toward an advanced understanding of eukaryotic molecular and cellular biology. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Archimedes' principle for characterisation of recombinant whole cell biocatalysts.

PubMed

Schmitt, Steven; Walser, Marcel; Rehmann, Michael; Oesterle, Sabine; Panke, Sven; Held, Martin

2018-02-14

The ability of whole cells to catalyse multistep reactions, often yielding synthetically demanding compounds later used by industrial biotech or pharma, makes them an indispensable tool of synthetic chemistry. The complex reaction network employed by cellular catalysts and the still only moderate predictive power of modelling approaches leaves this tool challenging to engineer. Frequently, large libraries of semi-rationally generated variants are sampled in high-throughput mode in order to then identify improved catalysts. We present a method for space- and time-efficient processing of very large libraries (10 7 ) of recombinant cellular catalysts, in which the phenotypic characterisation and the isolation of positive variants for the entire library is done within one minute in a single, highly parallelized operation. Specifically, product formation in nanolitre-sized cultivation vessels is sensed and translated into the formation of catalase as a reporter protein. Exposure to hydrogen peroxide leads to oxygen gas formation and thus to a density shift of the cultivation vessel. Exploiting Archimedes' principle, this density shift and the resulting upward buoyancy force can be used for batch-wise library sampling. We demonstrate the potential of the method for both, screening and selection protocols, and envision a wide applicability of the system for biosensor-based assays.

IFN-γ signaling maintains skin pigmentation homeostasis through regulation of melanosome maturation

PubMed Central

Natarajan, Vivek T.; Ganju, Parul; Singh, Archana; Vijayan, Vinaya; Kirty, Kritika; Yadav, Shalini; Puntambekar, Shraddha; Bajaj, Sonali; Dani, Prachi P.; Kar, Hemanta K.; Gadgil, Chetan J.; Natarajan, Krishnamurthy; Rani, Rajni; Gokhale, Rajesh S.

2014-01-01

Cellular homeostasis is an outcome of complex interacting processes with nonlinear feedbacks that can span distinct spatial and temporal dimensions. Skin tanning is one such dynamic response that maintains genome integrity of epidermal cells. Although pathways underlying hyperpigmentation cascade are recognized, negative feedback regulatory loops that can dampen the activated melanogenesis process are not completely understood. In this study, we delineate a regulatory role of IFN-γ in skin pigmentation biology. We show that IFN-γ signaling impedes maturation of the key organelle melanosome by concerted regulation of several pigmentation genes. Withdrawal of IFN-γ signal spontaneously restores normal cellular programming. This effect in melanocytes is mediated by IFN regulatory factor-1 and is not dependent on the central regulator microphthalmia-associated transcription factor. Chronic IFN-γ signaling shows a clear hypopigmentation phenotype in both mouse and human skin. Interestingly, IFN-γ KO mice display a delayed recovery response to restore basal state of epidermal pigmentation after UV-induced tanning. Together, our studies delineate a new spatiotemporal role of the IFN-γ signaling network in skin pigmentation homeostasis, which could have implications in various cutaneous depigmentary and malignant disorders. PMID:24474804

Cellular Models: HD Patient-Derived Pluripotent Stem Cells.

PubMed

Geater, Charlene; Hernandez, Sarah; Thompson, Leslie; Mattis, Virginia B

2018-01-01

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by expanded polyglutamine (polyQ)-encoding repeats in the Huntingtin (HTT) gene. Traditionally, HD cellular models consisted of either patient cells not affected by disease or rodent neurons expressing expanded polyQ repeats in HTT. As these models can be limited in their disease manifestation or proper genetic context, respectively, human HD pluripotent stem cells (PSCs) are currently under investigation as a way to model disease in patient-derived neurons and other neural cell types. This chapter reviews embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) models of disease, including published differentiation paradigms for neurons and their associated phenotypes, as well as current challenges to the field such as validation of the PSCs and PSC-derived cells. Highlighted are potential future technical advances to HD PSC modeling, including transdifferentiation, complex in vitro multiorgan/system reconstruction, and personalized medicine. Using a human HD patient model of the central nervous system, hopefully one day researchers can tease out the consequences of mutant HTT (mHTT) expression on specific cell types within the brain in order to identify and test novel therapies for disease.

Multistructural biomimetic substrates for controlled cellular differentiation

NASA Astrophysics Data System (ADS)

Orza, Anamaria I.; Mihu, Carmen; Soritau, Olga; Diudea, Mircea; Florea, Adrian; Matei, Horea; Balici, Stefana; Mudalige, Thilak; Kanarpardy, Ganesh K.; Biris, Alexandru S.

2014-02-01

Multidimensional scaffolds are considered to be ideal candidates for regenerative medicine and tissue engineering based on their potential to provide an excellent microenvironment and direct the fate of the cultured cells. More recently, the use of stem cells in medicine has opened a new technological opportunity for controlled tissue formation. However, the mechanism through which the substrate directs the differentiation of stem cells is still rather unclear. Data concerning its specific surface chemistry, topology, and its signaling ability need to be further understood and analyzed. In our study, atomic force microscopy was used to study the stiffness, roughness, and topology of the collagen (Coll) and metallized collagen (MC) substrates, proposed as an excellent substrate for regenerative medicine. The importance of signaling molecules was studied by constructing a new hybrid signaling substrate that contains both collagen and laminin extracellular matrix (ECM) proteins. The cellular response—such as attachment capability, proliferation and cardiac and neuronal phenotype expression on the metallized and non-metallized hybrid substrates (collagen + laminin)—was studied using MTT viability assay and immunohistochemistry studies. Our findings indicate that such hybrid materials could play an important role in the regeneration of complex tissues.

The Potential of iPSCs for the Treatment of Premature Aging Disorders

PubMed Central

Compagnucci, Claudia; Bertini, Enrico

2017-01-01

Premature aging disorders including Hutchinson-Gilford progeria syndrome (HGPS) and Werner syndrome, are a group of rare monogenic diseases leading to reduced lifespan of the patients. Importantly, these disorders mimic several features of physiological aging. Despite the interest on the study of these diseases, the underlying biological mechanisms remain unknown and no treatment is available. Recent studies on HGPS (due to mutations of the LMNA gene encoding for the nucleoskeletal proteins lamin A/C) have reported disruptions in cellular and molecular mechanisms modulating genomic stability and stem cell populations, thus giving the nuclear lamina a relevant function in nuclear organization, epigenetic regulation and in the maintenance of the stem cell pool. In this context, modeling premature aging with induced pluripotent stem cells (iPSCs) offers the possibility to study these disorders during self-renewal and differentiation into relevant cell types. iPSCs generated by cellular reprogramming from adult somatic cells allows researchers to understand pathophysiological mechanisms and enables the performance of drug screenings. Moreover, the recent development of precision genome editing offers the possibility to study the complex mechanisms underlying senescence and the possibility to correct disease phenotypes, paving the way for future therapeutic interventions. PMID:29112121

Feedback between p21 and reactive oxygen production is necessary for cell senescence

PubMed Central

Passos, João F; Nelson, Glyn; Wang, Chunfang; Richter, Torsten; Simillion, Cedric; Proctor, Carole J; Miwa, Satomi; Olijslagers, Sharon; Hallinan, Jennifer; Wipat, Anil; Saretzki, Gabriele; Rudolph, Karl Lenhard; Kirkwood, Tom B L; von Zglinicki, Thomas

2010-01-01

Cellular senescence—the permanent arrest of cycling in normally proliferating cells such as fibroblasts—contributes both to age-related loss of mammalian tissue homeostasis and acts as a tumour suppressor mechanism. The pathways leading to establishment of senescence are proving to be more complex than was previously envisaged. Combining in-silico interactome analysis and functional target gene inhibition, stochastic modelling and live cell microscopy, we show here that there exists a dynamic feedback loop that is triggered by a DNA damage response (DDR) and, which after a delay of several days, locks the cell into an actively maintained state of ‘deep' cellular senescence. The essential feature of the loop is that long-term activation of the checkpoint gene CDKN1A (p21) induces mitochondrial dysfunction and production of reactive oxygen species (ROS) through serial signalling through GADD45-MAPK14(p38MAPK)-GRB2-TGFBR2-TGFβ. These ROS in turn replenish short-lived DNA damage foci and maintain an ongoing DDR. We show that this loop is both necessary and sufficient for the stability of growth arrest during the establishment of the senescent phenotype. PMID:20160708

Motor neurons and glia exhibit specific individualized responses to TDP-43 expression in a Drosophila model of amyotrophic lateral sclerosis.

PubMed

Estes, Patricia S; Daniel, Scott G; McCallum, Abigail P; Boehringer, Ashley V; Sukhina, Alona S; Zwick, Rebecca A; Zarnescu, Daniela C

2013-05-01

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by complex neuronal and glial phenotypes. Recently, RNA-based mechanisms have been linked to ALS via RNA-binding proteins such as TDP-43, which has been studied in vivo using models ranging from yeast to rodents. We have developed a Drosophila model of ALS based on TDP-43 that recapitulates several aspects of pathology, including motor neuron loss, locomotor dysfunction and reduced survival. Here we report the phenotypic consequences of expressing wild-type and four different ALS-linked TDP-43 mutations in neurons and glia. We show that TDP-43-driven neurodegeneration phenotypes are dose- and age-dependent. In motor neurons, TDP-43 appears restricted to nuclei, which are significantly misshapen due to mutant but not wild-type protein expression. In glia and in the developing neuroepithelium, TDP-43 associates with cytoplasmic puncta. TDP-43-containing RNA granules are motile in cultured motor neurons, although wild-type and mutant variants exhibit different kinetic properties. At the neuromuscular junction, the expression of TDP-43 in motor neurons versus glia leads to seemingly opposite synaptic phenotypes that, surprisingly, translate into comparable locomotor defects. Finally, we explore sleep as a behavioral readout of TDP-43 expression and find evidence of sleep fragmentation consistent with hyperexcitability, a suggested mechanism in ALS. These findings support the notion that although motor neurons and glia are both involved in ALS pathology, at the cellular level they can exhibit different responses to TDP-43. In addition, our data suggest that individual TDP-43 alleles utilize distinct molecular mechanisms, which will be important for developing therapeutic strategies.

Systematic identification of an integrative network module during senescence from time-series gene expression.

PubMed

Park, Chihyun; Yun, So Jeong; Ryu, Sung Jin; Lee, Soyoung; Lee, Young-Sam; Yoon, Youngmi; Park, Sang Chul

2017-03-15

Cellular senescence irreversibly arrests growth of human diploid cells. In addition, recent studies have indicated that senescence is a multi-step evolving process related to important complex biological processes. Most studies analyzed only the genes and their functions representing each senescence phase without considering gene-level interactions and continuously perturbed genes. It is necessary to reveal the genotypic mechanism inferred by affected genes and their interaction underlying the senescence process. We suggested a novel computational approach to identify an integrative network which profiles an underlying genotypic signature from time-series gene expression data. The relatively perturbed genes were selected for each time point based on the proposed scoring measure denominated as perturbation scores. Then, the selected genes were integrated with protein-protein interactions to construct time point specific network. From these constructed networks, the conserved edges across time point were extracted for the common network and statistical test was performed to demonstrate that the network could explain the phenotypic alteration. As a result, it was confirmed that the difference of average perturbation scores of common networks at both two time points could explain the phenotypic alteration. We also performed functional enrichment on the common network and identified high association with phenotypic alteration. Remarkably, we observed that the identified cell cycle specific common network played an important role in replicative senescence as a key regulator. Heretofore, the network analysis from time series gene expression data has been focused on what topological structure was changed over time point. Conversely, we focused on the conserved structure but its context was changed in course of time and showed it was available to explain the phenotypic changes. We expect that the proposed method will help to elucidate the biological mechanism unrevealed by the existing approaches.

Molecular Genetics of Ubiquinone Biosynthesis in Animals

PubMed Central

Wang, Ying; Hekimi, Siegfried

2014-01-01

Ubiquinone (UQ), also known as coenzyme Q (CoQ), is a redox-active lipid present in all cellular membranes where it functions in a variety of cellular processes. The best known functions of UQ are to act as a mobile electron carrier in the mitochondrial respiratory chain and to serve as a lipid soluble antioxidant in cellular membranes. All eukaryotic cells synthesize their own UQ. Most of the current knowledge on the UQ biosynthetic pathway was obtained by studying Escherichia coli and S. cerevisiae UQ-deficient mutants. The orthologues of all the genes known from yeast studies to be involved in UQ biosynthesis have subsequently been found in higher organisms. Animal mutants with different genetic defects in UQ biosynthesis display very different phenotypes, despite the fact that in all these mutants the same biosynthetic pathway is affected. This review summarizes the present knowledge of the eukaryotic biosynthesis of UQ, with focus on the biosynthetic genes identified in animals, including C. elegans, rodents and humans. Moreover, we review the phenotypes of mutants in these genes and discuss the functional consequences of UQ deficiency in general. PMID:23190198

The effect of hydrodynamic conditions on the phenotype of Pseudomonas fluorescens biofilms.

PubMed

Simões, Manuel; Pereira, Maria O; Sillankorva, Sanna; Azeredo, Joana; Vieira, Maria J

2007-01-01

This study investigated the phenotypic characteristics of monoculture P. fluorescens biofilms grown under turbulent and laminar flow, using flow cells reactors with stainless steel substrata. The cellular physiology and the overall biofilm activity, structure and composition were characterized, and compared, within hydrodynamically distinct conditions. The results indicate that turbulent flow-generated biofilm cells were significantly less extensive, with decreased metabolic activity and a lower protein and polysaccharides composition per cell than those from laminar flow-generated biofilms. The effect of flow regime did not cause significantly different outer membrane protein expression. From the analysis of biofilm activity, structure and composition, turbulent flow-generated biofilms were metabolically more active, had twice more mass per cm(2), and higher cellular density and protein content (mainly cellular) than laminar flow-generated biofilms. Conversely, laminar flow-generated biofilms presented higher total and matrix polysaccharide contents. Direct visualisation and scanning electron microscopy analysis showed that these different flows generate structurally different biofilms, corroborating the quantitative results. The combination of applied methods provided useful information regarding a broad spectrum of biofilm parameters, which can contribute to control and model biofilm processes.

New concept: cellular senescence in pathophysiology of cholangiocarcinoma.

PubMed

Sasaki, Motoko; Nakanuma, Yasuni

2016-01-01

Cholangiocarcinoma, a malignant tumor arising in the hepatobiliary system, presents with poor prognosis because of difficulty in its early detection/diagnosis. Recent progress revealed that cellular senescence may be involved in the pathophysiology of cholangiocarcinoma. Cellular senescence is defined as permanent growth arrest caused by several cellular injuries, such as oncogenic mutations and oxidative stress. "Oncogene-induced" and/or stress-induced senescence may occur in the process of multi-step cholangiocarcinogenesis, and overexpression of a polycomb group protein EZH2 may play a role in the escape from, and/or bypassing of, senescence. Furthermore, senescent cells may play important roles in tumor development and progression via the production of senescence-associated secretory phenotypes. Cellular senescence may be a new target for the prevention, early diagnosis, and therapy of cholangiocarcinoma in the near future.

KPNA2 is a nuclear export protein that contributes to aberrant localisation of key proteins and poor prognosis of breast cancer.

PubMed

Alshareeda, A T; Negm, O H; Green, A R; Nolan, C C; Tighe, P; Albarakati, N; Sultana, R; Madhusudan, S; Ellis, I O; Rakha, E A

2015-06-09

It is recognised that modulations of the nuclear import of macromolecules have a role in changing cellular phenotypes and carcinogenesis. We and others have noticed that aberrant subcellular localisation of DNA damage response (DDR) proteins in breast cancer (BC) is associated with loss-of-function phenotype. This study aims to investigate the biological and clinical significance of the nucleocytoplasmic transport protein karyopherin α-2 (KPNA2), and its role in controlling DDR proteins subcellular localisation in BC. A large (n=1494) and well-characterised series of early-stage invasive BC with a long-term follow-up was assessed for KPNA2 protein by using immunohistochemistry. KPNA2 expression was associated with the subcellular localisation of key DDR proteins that showed cytoplasmic expression including BRCA1, RAD51, SMC6L1, γH2AX, BARD1, UBC9, PIAS1 and CHK1. High level of KPNA2 was associated not only with cytoplasmic localisation of these proteins but also with their low/negative nuclear expression. Positive KPNA2 expression was associated with negative oestrogen receptor and triple-negative phenotype. Survival analysis showed that KPNA2 was associated with poor outcome (P<0.0001), but this effect was not independent of other prognostic variables. This study provides further evidence for the complexity of DDR mechanism in BC, and that KNPA2 has a role in the aberrant subcellular localisation of DDR proteins with subsequent impaired function.

Structural and functional analysis of the repressor complex in the Notch signaling pathway of Drosophila melanogaster

PubMed Central

Maier, Dieter; Kurth, Patricia; Schulz, Adriana; Russell, Andrew; Yuan, Zhenyu; Gruber, Kim; Kovall, Rhett A.; Preiss, Anette

2011-01-01

In metazoans, the highly conserved Notch pathway drives cellular specification. On receptor activation, the intracellular domain of Notch assembles a transcriptional activator complex that includes the DNA-binding protein CSL, a composite of human C-promoter binding factor 1, Suppressor of Hairless of Drosophila melanogaster [Su(H)], and lin-12 and Glp-1 phenotype of Caenorhabditis elegans. In the absence of ligand, CSL represses Notch target genes. However, despite the structural similarity of CSL orthologues, repression appears largely diverse between organisms. Here we analyze the Notch repressor complex in Drosophila, consisting of the fly CSL protein, Su(H), and the corepressor Hairless, which recruits general repressor proteins. We show that the C-terminal domain of Su(H) is necessary and sufficient for forming a high-affinity complex with Hairless. Mutations in Su(H) that affect interactions with Notch and Mastermind have no effect on Hairless binding. Nonetheless, we demonstrate that Notch and Hairless compete for CSL in vitro and in cell culture. In addition, we identify a site in Hairless that is crucial for binding Su(H) and subsequently show that this Hairless mutant is strongly impaired, failing to properly assemble the repressor complex in vivo. Finally, we demonstrate Hairless-mediated inhibition of Notch signaling in a cell culture assay, which hints at a potentially similar repression mechanism in mammals that might be exploited for therapeutic purposes. PMID:21737682

Human somatic cells acquire the plasticity to generate embryoid-like metamorphosis via the actin cytoskeleton in injured tissues.

PubMed

Diaz, Jairo A; Murillo, Mauricio F; Mendoza, Jhonan A; Barreto, Ana M; Poveda, Lina S; Sanchez, Lina K; Poveda, Laura C; Mora, Katherine T

2016-01-01

Emergent biological responses develop via unknown processes dependent on physical collision. In hypoxia, when the tissue architecture collapses but the geometric core is stable, actin cytoskeleton filament components emerge, revealing a hidden internal order that identifies how each molecule is reassembled into the original mold, using one common connection, i.e., a fractal self-similarity that guides the system from the beginning in reverse metamorphosis, with spontaneous self-assembly of past forms that mimics an embryoid phenotype. We captured this hidden collective filamentous assemblage in progress: Hypoxic deformed cells enter into intercellular collisions, generate migratory ejected filaments, and produce self-assembly of triangular chiral hexagon complexes; this dynamic geometry guides the microenvironment scaffold in which this biological process is incubated, recapitulating embryonic morphogenesis. In all injured tissues, especially in damaged skeletal (striated) muscle cells, visibly hypertrophic intercalated actin-myosin filaments are organized in zebra stripe pattern along the anterior-posterior axis in the interior of the cell, generating cephalic-caudal polarity segmentation, with a high selective level of immunopositivity for Actin, Alpha Skeletal Muscle antibody and for Neuron-Specific Enolase expression of ectodermal differentiation. The function of actin filaments in emergent responses to tissue injury is to reconstitute, reactivate and orchestrate cellular metamorphosis, involving the re-expression of fetal genes, providing evidence of the reverse flow of genetic information within a biological system. The resultant embryoid phenotype emerges as a microscopic fractal template copy of the organization of the whole body, likely allowing the modification and reprogramming of the phenotype of the tumor in which these structures develop, as well as establishing a reverse primordial microscopic mold to collectively re-form cellular building blocks to regenerate injured tissues. Tumorigenesis mimics a self-organizing process of early embryo development. All malignant tumors produce fetal proteins, we now know from which these proteins proceed. Embryoid-like metamorphosis phenomena would represent the anatomical and functional entity of the injury stem cell niche. The sufficiently fast identification, isolation, culture, and expansion of these self-organized structures or genetically derived products could, in our opinion, be used to develop new therapeutic strategies against cancer and in regenerative medicine.

Human somatic cells acquire the plasticity to generate embryoid-like metamorphosis via the actin cytoskeleton in injured tissues

PubMed Central

Diaz, Jairo A; Murillo, Mauricio F; Mendoza, Jhonan A; Barreto, Ana M; Poveda, Lina S; Sanchez, Lina K; Poveda, Laura C; Mora, Katherine T

2016-01-01

Emergent biological responses develop via unknown processes dependent on physical collision. In hypoxia, when the tissue architecture collapses but the geometric core is stable, actin cytoskeleton filament components emerge, revealing a hidden internal order that identifies how each molecule is reassembled into the original mold, using one common connection, i.e., a fractal self-similarity that guides the system from the beginning in reverse metamorphosis, with spontaneous self-assembly of past forms that mimics an embryoid phenotype. We captured this hidden collective filamentous assemblage in progress: Hypoxic deformed cells enter into intercellular collisions, generate migratory ejected filaments, and produce self-assembly of triangular chiral hexagon complexes; this dynamic geometry guides the microenvironment scaffold in which this biological process is incubated, recapitulating embryonic morphogenesis. In all injured tissues, especially in damaged skeletal (striated) muscle cells, visibly hypertrophic intercalated actin-myosin filaments are organized in zebra stripe pattern along the anterior-posterior axis in the interior of the cell, generating cephalic-caudal polarity segmentation, with a high selective level of immunopositivity for Actin, Alpha Skeletal Muscle antibody and for Neuron-Specific Enolase expression of ectodermal differentiation. The function of actin filaments in emergent responses to tissue injury is to reconstitute, reactivate and orchestrate cellular metamorphosis, involving the re-expression of fetal genes, providing evidence of the reverse flow of genetic information within a biological system. The resultant embryoid phenotype emerges as a microscopic fractal template copy of the organization of the whole body, likely allowing the modification and reprogramming of the phenotype of the tumor in which these structures develop, as well as establishing a reverse primordial microscopic mold to collectively re-form cellular building blocks to regenerate injured tissues. Tumorigenesis mimics a self-organizing process of early embryo development. All malignant tumors produce fetal proteins, we now know from which these proteins proceed. Embryoid-like metamorphosis phenomena would represent the anatomical and functional entity of the injury stem cell niche. The sufficiently fast identification, isolation, culture, and expansion of these self-organized structures or genetically derived products could, in our opinion, be used to develop new therapeutic strategies against cancer and in regenerative medicine. PMID:27725917

Yeast Phenomics: An Experimental Approach for Modeling Gene Interaction Networks that Buffer Disease

PubMed Central

Hartman, John L.; Stisher, Chandler; Outlaw, Darryl A.; Guo, Jingyu; Shah, Najaf A.; Tian, Dehua; Santos, Sean M.; Rodgers, John W.; White, Richard A.

2015-01-01

The genome project increased appreciation of genetic complexity underlying disease phenotypes: many genes contribute each phenotype and each gene contributes multiple phenotypes. The aspiration of predicting common disease in individuals has evolved from seeking primary loci to marginal risk assignments based on many genes. Genetic interaction, defined as contributions to a phenotype that are dependent upon particular digenic allele combinations, could improve prediction of phenotype from complex genotype, but it is difficult to study in human populations. High throughput, systematic analysis of S. cerevisiae gene knockouts or knockdowns in the context of disease-relevant phenotypic perturbations provides a tractable experimental approach to derive gene interaction networks, in order to deduce by cross-species gene homology how phenotype is buffered against disease-risk genotypes. Yeast gene interaction network analysis to date has revealed biology more complex than previously imagined. This has motivated the development of more powerful yeast cell array phenotyping methods to globally model the role of gene interaction networks in modulating phenotypes (which we call yeast phenomic analysis). The article illustrates yeast phenomic technology, which is applied here to quantify gene X media interaction at higher resolution and supports use of a human-like media for future applications of yeast phenomics for modeling human disease. PMID:25668739

Ascites-induced shift along epithelial-mesenchymal spectrum in ovarian cancer cells: enhancement of their invasive behavior partly dependant on αv integrins.

PubMed

Carduner, L; Leroy-Dudal, J; Picot, C R; Gallet, O; Carreiras, F; Kellouche, S

2014-08-01

At least one-third of patients with epithelial ovarian cancer (OC) present ascites at diagnosis and almost all have ascites at recurrence. The presence of ascites, which acts as a dynamic reservoir of active molecules and cellular components, correlates with the OC peritoneal metastasis and is associated with poor prognosis. Since epithelial-mesenchymal transition (EMT) is involved in different phases of OC progression, we have investigated the effect of the unique ascitic tumor microenvironment on the EMT status and the behavior of OC cells. The exposure of three OC cell lines to ascites leads to changes in cellular morphologies. Within ascites, OC cells harboring an initial intermediate epithelial phenotype are characterized by marked dislocation of epithelial markers (E-cadherin, ZO-1 staining) while OC cells initially harboring an intermediate mesenchymal phenotype strengthen their mesenchymal markers (N-cadherin, vimentin). Ascites differentially triggers a dissemination phenotype related to the initial cell features by either allowing the proliferation and the formation of spheroids and the extension of colonies for cells that present an initial epithelial intermediate phenotype, or favoring the migration of cells with a mesenchymal intermediate phenotype. In an ascitic microenvironment, a redeployment of αv integrins into cells was observed and the ascites-induced accentuation of the two different invasive phenotypes (i.e. spheroids formation or migration) was shown to involve αv integrins. Thus, ascites induces a shift toward an unstable intermediate state of the epithelial-mesenchymal spectrum and confers a more aggressive cell behavior that takes on a different pathway based on the initial epithelial-mesenchymal cell features.

Notch3 Maintains Luminal Phenotype and Suppresses Tumorigenesis and Metastasis of Breast Cancer via Trans-Activating Estrogen Receptor-α.

PubMed

Dou, Xiao-Wei; Liang, Yuan-Ke; Lin, Hao-Yu; Wei, Xiao-Long; Zhang, Yong-Qu; Bai, Jing-Wen; Chen, Chun-Fa; Chen, Min; Du, Cai-Wen; Li, Yao-Chen; Tian, Jie; Man, Kwan; Zhang, Guo-Jun

2017-01-01

The luminal A phenotype is the most common breast cancer subtype and is characterized by estrogen receptor α expression (ERα). Identification of the key regulator that governs the luminal phenotype of breast cancer will clarify the pathogenic mechanism and provide novel therapeutic strategies for this subtype of cancer. ERα signaling pathway sustains the epithelial phenotype and inhibits the epithelial-mesenchymal transition (EMT) of breast cancer. In this study, we demonstrate that Notch3 positively associates with ERα in both breast cancer cell lines and human breast cancer tissues. We found that overexpression of Notch3 intra-cellular domain, a Notch3 active form (N3ICD), in ERα negative breast cancer cells re-activated ERα, while knock-down of Notch3 reduced ERα transcript and proteins, with alteration of down-stream genes, suggesting its ability to regulate ERα. Mechanistically, our results show that Notch3 specifically binds to the CSL binding element of the ERα promoter and activates ERα expression. Moreover, Notch3 suppressed EMT, while suppression of Notch3 promoted EMT in cellular assay. Overexpressing N3ICD in triple-negative breast cancer suppressed tumorigenesis and metastasis in vivo . Conversely, depletion of Notch3 in luminal breast cancer promoted metastasis in vivo . Furthermore, Notch3 transcripts were significantly associated with prolonged relapse-free survival in breast cancer, in particular in ERα positive breast cancer patients. Our observations demonstrate that Notch3 governs the luminal phenotype via trans-activating ERα expression in breast cancer. These findings delineate the role of a Notch3/ERα axis in maintaining the luminal phenotype and inhibiting tumorigenesis and metastasis in breast cancer, providing a novel strategy to re-sensitize ERα negative or low-expressing breast cancers to hormone therapy.

Notch3 Maintains Luminal Phenotype and Suppresses Tumorigenesis and Metastasis of Breast Cancer via Trans-Activating Estrogen Receptor-α

PubMed Central

Dou, Xiao-Wei; Liang, Yuan-Ke; Lin, Hao-Yu; Wei, Xiao-Long; Zhang, Yong-Qu; Bai, Jing-Wen; Chen, Chun-Fa; Chen, Min; Du, Cai-Wen; Li, Yao-Chen; Tian, Jie; Man, Kwan; Zhang, Guo-Jun

2017-01-01

The luminal A phenotype is the most common breast cancer subtype and is characterized by estrogen receptor α expression (ERα). Identification of the key regulator that governs the luminal phenotype of breast cancer will clarify the pathogenic mechanism and provide novel therapeutic strategies for this subtype of cancer. ERα signaling pathway sustains the epithelial phenotype and inhibits the epithelial-mesenchymal transition (EMT) of breast cancer. In this study, we demonstrate that Notch3 positively associates with ERα in both breast cancer cell lines and human breast cancer tissues. We found that overexpression of Notch3 intra-cellular domain, a Notch3 active form (N3ICD), in ERα negative breast cancer cells re-activated ERα, while knock-down of Notch3 reduced ERα transcript and proteins, with alteration of down-stream genes, suggesting its ability to regulate ERα. Mechanistically, our results show that Notch3 specifically binds to the CSL binding element of the ERα promoter and activates ERα expression. Moreover, Notch3 suppressed EMT, while suppression of Notch3 promoted EMT in cellular assay. Overexpressing N3ICD in triple-negative breast cancer suppressed tumorigenesis and metastasis in vivo. Conversely, depletion of Notch3 in luminal breast cancer promoted metastasis in vivo. Furthermore, Notch3 transcripts were significantly associated with prolonged relapse-free survival in breast cancer, in particular in ERα positive breast cancer patients. Our observations demonstrate that Notch3 governs the luminal phenotype via trans-activating ERα expression in breast cancer. These findings delineate the role of a Notch3/ERα axis in maintaining the luminal phenotype and inhibiting tumorigenesis and metastasis in breast cancer, providing a novel strategy to re-sensitize ERα negative or low-expressing breast cancers to hormone therapy. PMID:29109797

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

Slug is upregulated during wound healing and regulates cellular phenotypes in corneal epithelial cells.

PubMed

Aomatsu, Keiichi; Arao, Tokuzo; Abe, Kosuke; Kodama, Aya; Sugioka, Koji; Matsumoto, Kazuko; Kudo, Kanae; Kimura, Hideharu; Fujita, Yoshihiko; Hayashi, Hidetoshi; Nagai, Tomoyuki; Shimomura, Yoshikazu; Nishio, Kazuto

2012-02-16

The involvement of the epithelial mesenchymal transition (EMT) in the process of corneal wound healing remains largely unclear. The purpose of the present study was to gain insight into Slug expression and corneal wound healing. Slug expression during wound healing in the murine cornea was evaluated using fluorescence staining in vivo. Slug or Snail was stably introduced into human corneal epithelial cells (HCECs). These stable transfectants were evaluated for the induction of the EMT, cellular growth, migration activity, and expression changes in differentiation-related molecules. Slug, but not Snail, was clearly expressed in the nuclei of corneal epithelial cells in basal lesion of the corneal epithelium during wound healing in vivo. The overexpression of Slug or Snail induced an EMT-like cellular morphology and cadherin switching in HCECs, indicating that these transcription factors were able to mediate the typical EMT in HCECs. The overexpression of Slug or Snail suppressed cellular proliferation but enhanced the migration activity. Furthermore, ABCG2, TP63, and keratin 19, which are known as stemness-related molecules, were downregulated in these transfectants. It was found that Slug is upregulated during corneal wound healing in vivo. The overexpression of Slug mediated a change in the cellular phenotype affecting proliferation, migration, and expression levels of differentiation-related molecules. This is the first evidence that Slug is regulated during the process of corneal wound healing in the corneal epithelium in vivo, providing a novel insight into the EMT and Slug expression in corneal wound healing.

Phenotypic variations in chondrocyte subpopulations and their response to in vitro culture and external stimuli.

PubMed

Coates, Emily E; Fisher, John P

2010-11-01

Articular cartilage defects have limited capacity to self-repair, and cost society up to 60 billion dollars annually in both medical treatments and loss of working days. Recent developments in cartilage tissue engineering have resulted in many new products coming to market or entering clinical trials. However, there is a distinct lack of treatments which aim to recreate the complex zonal organization of articular cartilage. Cartilage tissue withstands repetitive strains throughout an individual's lifetime and provides frictionless movement between joints. The structure and composition of its intricately organized extracellular matrix varies with tissue depth to provide optimal resistance to loading, ensure ease of movement, and integrate with the subchondral bone. Each tissue zone is specially designed to resist the load it experiences, and maximize the tissue properties needed for its location. It is unlikely that a homogenous solution to tissue repair will be able to optimally restore the function of such a heterogeneous tissue. For zonal engineering of articular cartilage to become practical, maintenance of phenotypically stable zonal cell populations must be achieved. The chondrocyte phenotype varies considerably by zone, and it is the activity of these cells that help achieve the structural organization of the tissue. This review provides an examination of literature which has studied variations in cellular phenotype between cartilage zones. By doing so, we have identified critical differences between cell populations and highlighted areas of research which show potential in the field. Current research has made the morphological and metabolic variations between these cell populations clear, but an ideal way of maintaining these differences in vitro culture is yet to be established. Combinations of delivered growth factors, mechanical loading, and layered three-dimensional culture systems all show potential for achieving this goal. Furthermore, differentiation of progenitor cell populations into chondrocyte subpopulations may also hold promise for achieving large numbers of zonal chondrocytes. Success of the field lies in establishing methods of retaining phenotypically stable cell populations for in vitro culture.

Physiological and glycomic characterization of N-acetylglucosaminyltransferase-IVa and -IVb double deficient mice

PubMed Central

Takamatsu, Shinji; Antonopoulos, Aristotelis; Ohtsubo, Kazuaki; Ditto, David; Chiba, Yasunori; Le, Dzung T.; Morris, Howard R.; Haslam, Stuart M.; Dell, Anne; Marth, Jamey D.; Taniguchi, Naoyuki

2010-01-01

N-Acetylglucosaminyltransferase-IV (GnT-IV) has two isoenzymes, GnT-IVa and GnT-IVb, which initiate the GlcNAcβ1-4 branch synthesis on the Manα1-3 arm of the N-glycan core thereby increasing N-glycan branch complexity and conferring endogenous lectin binding epitopes. To elucidate the physiological significance of GnT-IV, we engineered and characterized GnT-IVb-deficient mice and further generated GnT-IVa/-IVb double deficient mice. In wild-type mice, GnT-IVa expression is restricted to gastrointestinal tissues, whereas GnT-IVb is broadly expressed among organs. GnT-IVb deficiency induced aberrant GnT-IVa expression corresponding to the GnT-IVb distribution pattern that might be attributed to increased Ets-1, which conceivably activates the Mgat4a promoter, and thereafter preserved apparent GnT-IV activity. The compensative GnT-IVa expression might contribute to amelioration of the GnT-IVb-deficient phenotype. GnT-IVb deficiency showed mild phenotypic alterations in hematopoietic cell populations and hemostasis. GnT-IVa/-IVb double deficiency completely abolished GnT-IV activity that resulted in the disappearance of the GlcNAcβ1-4 branch on the Manα1-3 arm that was confirmed by MALDI-TOF MS and GC-MS linkage analyses. Comprehensive glycomic analyses revealed that the abundance of terminal moieties was preserved in GnT-IVa/-IVb double deficiency that was due to the elevated expression of glycosyltransferases regarding synthesis of terminal moieties. Thereby, this may maintain the expression of glycan ligands for endogenous lectins and prevent cellular dysfunctions. The fact that the phenotype of GnT-IVa/-IVb double deficiency largely overlapped that of GnT-IVa single deficiency can be attributed to the induced glycomic compensation. This is the first report that mammalian organs have highly organized glycomic compensation systems to preserve N-glycan branch complexity. PMID:20015870

Phenotypic screening in cancer drug discovery - past, present and future.

PubMed

Moffat, John G; Rudolph, Joachim; Bailey, David

2014-08-01

There has been a resurgence of interest in the use of phenotypic screens in drug discovery as an alternative to target-focused approaches. Given that oncology is currently the most active therapeutic area, and also one in which target-focused approaches have been particularly prominent in the past two decades, we investigated the contribution of phenotypic assays to oncology drug discovery by analysing the origins of all new small-molecule cancer drugs approved by the US Food and Drug Administration (FDA) over the past 15 years and those currently in clinical development. Although the majority of these drugs originated from target-based discovery, we identified a significant number whose discovery depended on phenotypic screening approaches. We postulate that the contribution of phenotypic screening to cancer drug discovery has been hampered by a reliance on 'classical' nonspecific drug effects such as cytotoxicity and mitotic arrest, exacerbated by a paucity of mechanistically defined cellular models for therapeutically translatable cancer phenotypes. However, technical and biological advances that enable such mechanistically informed phenotypic models have the potential to empower phenotypic drug discovery in oncology.

Cell surface physiology and outer cell envelope impermeability for hydrophobic substances in Burkholderia multivorans.

PubMed

Ruskoski, Sallie A; Champlin, Franklin R

2017-07-01

The purpose of the present study was to obtain a better understanding of the relationship between cell surface physiology and outer cellular envelope permeability for hydrophobic substances in mucoid and non-mucoid B. multivorans strains, as well as in two capsule-deficient derivatives of a mucoid parental strain. Cell surface hydrophobicity properties were determined using the hydrocarbon adherence method, while outer cell envelope accessibility and permeability for non-polar compounds were measured using hydrophobic antimicrobial agent susceptibility and fluorescent probe assays. Extracellular polysaccharide (EPS) production was assessed by cultivating strains of disparate origin on yeast extract agar (YEA) containing different sugars, while the resultant colonial and cellular morphological parameters were assessed macro- and microscopically, respectively.Results/Key findings. The cell surfaces of all the strains were hydrophilic, impermeable to mechanistically disparate hydrophobic antibacterial agents and inaccessible to the hydrophobic probe N-phenyl-1-napthylamine, regardless of EPS phenotype. Supplementation of basal YEA with eight different sugars enhanced macroscopic EPS expression for all but one non-mucoid strain, with mannose potentiating the greatest effect. Despite acquisition of the mucoid phenotype, non-mucoid strains remained non-capsulated and capsulation of a hyper-mucoid strain and its two non-mucoid derivative strains was unaffected, as judged by microscopic observation. These data support the conclusion that EPS expression and the consistent mucoid phenotype are not necessarily associated with the ability of the outer cell surface to associate with non-polar substances or cellular capsulation.

A Quantitative Systems Pharmacology Approach to Infer Pathways Involved in Complex Disease Phenotypes.

PubMed

Schurdak, Mark E; Pei, Fen; Lezon, Timothy R; Carlisle, Diane; Friedlander, Robert; Taylor, D Lansing; Stern, Andrew M

2018-01-01

Designing effective therapeutic strategies for complex diseases such as cancer and neurodegeneration that involve tissue context-specific interactions among multiple gene products presents a major challenge for precision medicine. Safe and selective pharmacological modulation of individual molecular entities associated with a disease often fails to provide efficacy in the clinic. Thus, development of optimized therapeutic strategies for individual patients with complex diseases requires a more comprehensive, systems-level understanding of disease progression. Quantitative systems pharmacology (QSP) is an approach to drug discovery that integrates computational and experimental methods to understand the molecular pathogenesis of a disease at the systems level more completely. Described here is the chemogenomic component of QSP for the inference of biological pathways involved in the modulation of the disease phenotype. The approach involves testing sets of compounds of diverse mechanisms of action in a disease-relevant phenotypic assay, and using the mechanistic information known for the active compounds, to infer pathways and networks associated with the phenotype. The example used here is for monogenic Huntington's disease (HD), which due to the pleiotropic nature of the mutant phenotype has a complex pathogenesis. The overall approach, however, is applicable to any complex disease.

T cell subtypes and reciprocal inflammatory mediator expression differentiate P. falciparum memory recall responses in asymptomatic and symptomatic malaria patients in southeastern Haiti

PubMed Central

Campo, Joseph J.; Cicéron, Micheline; Raccurt, Christian P.; Beau De Rochars, Valery E. M.

2017-01-01

Asymptomatic Plasmodium falciparum infection is responsible for maintaining malarial disease within human populations in low transmission countries such as Haiti. Investigating differential host immune responses to the parasite as a potential underlying mechanism could help provide insight into this highly complex phenomenon and possibly identify asymptomatic individuals. We performed a cross-sectional analysis of individuals who were diagnosed with malaria in Sud-Est, Haiti by comparing the cellular and humoral responses of both symptomatic and asymptomatic subjects. Plasma samples were analyzed with a P. falciparum protein microarray, which demonstrated serologic reactivity to 3,877 P. falciparum proteins of known serologic reactivity; however, no antigen-antibody reactions delineating asymptomatics from symptomatics were identified. In contrast, differences in cellular responses were observed. Flow cytometric analysis of patient peripheral blood mononuclear cells co-cultured with P. falciparum infected erythrocytes demonstrated a statistically significant increase in the proportion of T regulatory cells (CD4+ CD25+ CD127-), and increases in unique populations of both NKT-like cells (CD3+ CD8+ CD56+) and CD8mid T cells in asymptomatics compared to symptomatics. Also, CD38+/HLA-DR+ expression on γδ T cells, CD8mid (CD56-) T cells, and CD8mid CD56+ NKT-like cells decreased upon exposure to infected erythrocytes in both groups. Cytometric bead analysis of the co-culture supernatants demonstrated an upregulation of monocyte-activating chemokines/cytokines in asymptomatics, while immunomodulatory soluble factors were elevated in symptomatics. Principal component analysis of these expression values revealed a distinct clustering of individual responses within their respective phenotypic groups. This is the first comprehensive investigation of immune responses to P. falciparum in Haiti, and describes unique cell-mediated immune repertoires that delineate individuals into asymptomatic and symptomatic phenotypes. Future investigations using large scale biological data sets analyzing multiple components of adaptive immunity, could collectively define which cellular responses and molecular correlates of disease outcome are malaria region specific, and which are truly generalizable features of asymptomatic Plasmodium immunity, a research goal of critical priority. PMID:28369062

T cell subtypes and reciprocal inflammatory mediator expression differentiate P. falciparum memory recall responses in asymptomatic and symptomatic malaria patients in southeastern Haiti.

PubMed

Lehmann, Jason S; Campo, Joseph J; Cicéron, Micheline; Raccurt, Christian P; Boncy, Jacques; Beau De Rochars, Valery E M; Cannella, Anthony P

2017-01-01

Asymptomatic Plasmodium falciparum infection is responsible for maintaining malarial disease within human populations in low transmission countries such as Haiti. Investigating differential host immune responses to the parasite as a potential underlying mechanism could help provide insight into this highly complex phenomenon and possibly identify asymptomatic individuals. We performed a cross-sectional analysis of individuals who were diagnosed with malaria in Sud-Est, Haiti by comparing the cellular and humoral responses of both symptomatic and asymptomatic subjects. Plasma samples were analyzed with a P. falciparum protein microarray, which demonstrated serologic reactivity to 3,877 P. falciparum proteins of known serologic reactivity; however, no antigen-antibody reactions delineating asymptomatics from symptomatics were identified. In contrast, differences in cellular responses were observed. Flow cytometric analysis of patient peripheral blood mononuclear cells co-cultured with P. falciparum infected erythrocytes demonstrated a statistically significant increase in the proportion of T regulatory cells (CD4+ CD25+ CD127-), and increases in unique populations of both NKT-like cells (CD3+ CD8+ CD56+) and CD8mid T cells in asymptomatics compared to symptomatics. Also, CD38+/HLA-DR+ expression on γδ T cells, CD8mid (CD56-) T cells, and CD8mid CD56+ NKT-like cells decreased upon exposure to infected erythrocytes in both groups. Cytometric bead analysis of the co-culture supernatants demonstrated an upregulation of monocyte-activating chemokines/cytokines in asymptomatics, while immunomodulatory soluble factors were elevated in symptomatics. Principal component analysis of these expression values revealed a distinct clustering of individual responses within their respective phenotypic groups. This is the first comprehensive investigation of immune responses to P. falciparum in Haiti, and describes unique cell-mediated immune repertoires that delineate individuals into asymptomatic and symptomatic phenotypes. Future investigations using large scale biological data sets analyzing multiple components of adaptive immunity, could collectively define which cellular responses and molecular correlates of disease outcome are malaria region specific, and which are truly generalizable features of asymptomatic Plasmodium immunity, a research goal of critical priority.

Microenvironmental Modulation of Decorin and Lumican in Temozolomide-Resistant Glioblastoma and Neuroblastoma Cancer Stem-Like Cells.

PubMed

Farace, Cristiano; Oliver, Jaime Antonio; Melguizo, Consolacion; Alvarez, Pablo; Bandiera, Pasquale; Rama, Ana Rosa; Malaguarnera, Giulia; Ortiz, Raul; Madeddu, Roberto; Prados, Jose

2015-01-01

The presence of cancer stem cells (CSCs) or tumor-initiating cells can lead to cancer recurrence in a permissive cell-microenvironment interplay, promoting invasion in glioblastoma (GBM) and neuroblastoma (NB). Extracellular matrix (ECM) small leucine-rich proteoglycans (SLRPs) play multiple roles in tissue homeostasis by remodeling the extracellular matrix (ECM) components and modulating intracellular signaling pathways. Due to their pan-inhibitory properties against receptor tyrosine kinases (RTKs), SLRPs are reported to exert anticancer effects in vitro and in vivo. However, their roles seem to be tissue-specific and they are also involved in cancer cell migration and drug resistance, paving the way to complex different scenarios. The aim of this study was to determine whether the SLRPs decorin (DCN) and lumican (LUM) are recruited in cell plasticity and microenvironmental adaptation of differentiated cancer cells induced towards stem-like phenotype. Floating neurospheres were generated by applying CSC enrichment medium (neural stem cell serum-free medium, NSC SFM) to the established SF-268 and SK-N-SH cancer cell lines, cellular models of GBM and NB, respectively. In both models, the time-dependent synergistic activation of DCN and LUM was observed. The highest DCN and LUM mRNA/protein expression was detected after cell exposure to NSC SFM for 8/12 days, considering these cells as SLRP-expressing (SLRP+) CSC-like. Ultrastructural imaging showed the cellular heterogeneity of both the GBM and NB neurospheres and identified the inner living cells. Parental cell lines of both GBM and NB grew only in soft agar + NSC SFM, whereas the secondary neurospheres (originated from SLRP+ t8 CSC-like) showed lower proliferation rates than primary neurospheres. Interestingly, the SLRP+ CSC-like from the GBM and NB neurospheres were resistant to temozolomide (TMZ) at concentrations >750 μM. Our results suggest that GBM and NB CSC-like promote the activation of huge quantities of SLRP in response to CSC enrichment, simultaneously acquiring TMZ resistance, cellular heterogeneity, and a quiescent phenotype, suggesting a novel pivotal role for SLRP in drug resistance and cell plasticity of CSC-like, allowing cell survival and ECM/niche modulation potential.

Microenvironmental Modulation of Decorin and Lumican in Temozolomide-Resistant Glioblastoma and Neuroblastoma Cancer Stem-Like Cells

PubMed Central

Melguizo, Consolacion; Alvarez, Pablo; Bandiera, Pasquale; Rama, Ana Rosa; Malaguarnera, Giulia; Ortiz, Raul; Madeddu, Roberto; Prados, Jose

2015-01-01

The presence of cancer stem cells (CSCs) or tumor-initiating cells can lead to cancer recurrence in a permissive cell–microenvironment interplay, promoting invasion in glioblastoma (GBM) and neuroblastoma (NB). Extracellular matrix (ECM) small leucine-rich proteoglycans (SLRPs) play multiple roles in tissue homeostasis by remodeling the extracellular matrix (ECM) components and modulating intracellular signaling pathways. Due to their pan-inhibitory properties against receptor tyrosine kinases (RTKs), SLRPs are reported to exert anticancer effects in vitro and in vivo. However, their roles seem to be tissue-specific and they are also involved in cancer cell migration and drug resistance, paving the way to complex different scenarios. The aim of this study was to determine whether the SLRPs decorin (DCN) and lumican (LUM) are recruited in cell plasticity and microenvironmental adaptation of differentiated cancer cells induced towards stem-like phenotype. Floating neurospheres were generated by applying CSC enrichment medium (neural stem cell serum-free medium, NSC SFM) to the established SF-268 and SK-N-SH cancer cell lines, cellular models of GBM and NB, respectively. In both models, the time-dependent synergistic activation of DCN and LUM was observed. The highest DCN and LUM mRNA/protein expression was detected after cell exposure to NSC SFM for 8/12 days, considering these cells as SLRP-expressing (SLRP+) CSC-like. Ultrastructural imaging showed the cellular heterogeneity of both the GBM and NB neurospheres and identified the inner living cells. Parental cell lines of both GBM and NB grew only in soft agar + NSC SFM, whereas the secondary neurospheres (originated from SLRP+ t8 CSC-like) showed lower proliferation rates than primary neurospheres. Interestingly, the SLRP+ CSC-like from the GBM and NB neurospheres were resistant to temozolomide (TMZ) at concentrations >750 μM. Our results suggest that GBM and NB CSC-like promote the activation of huge quantities of SLRP in response to CSC enrichment, simultaneously acquiring TMZ resistance, cellular heterogeneity, and a quiescent phenotype, suggesting a novel pivotal role for SLRP in drug resistance and cell plasticity of CSC-like, allowing cell survival and ECM/niche modulation potential. PMID:26230845

Autism: A “Critical Period” Disorder?

PubMed Central

LeBlanc, Jocelyn J.; Fagiolini, Michela

2011-01-01

Cortical circuits in the brain are refined by experience during critical periods early in postnatal life. Critical periods are regulated by the balance of excitatory and inhibitory (E/I) neurotransmission in the brain during development. There is now increasing evidence of E/I imbalance in autism, a complex genetic neurodevelopmental disorder diagnosed by abnormal socialization, impaired communication, and repetitive behaviors or restricted interests. The underlying cause is still largely unknown and there is no fully effective treatment or cure. We propose that alteration of the expression and/or timing of critical period circuit refinement in primary sensory brain areas may significantly contribute to autistic phenotypes, including cognitive and behavioral impairments. Dissection of the cellular and molecular mechanisms governing well-established critical periods represents a powerful tool to identify new potential therapeutic targets to restore normal plasticity and function in affected neuronal circuits. PMID:21826280

Innovative Tools and Technology for Analysis of Single Cells and Cell-Cell Interaction.

PubMed

Konry, Tania; Sarkar, Saheli; Sabhachandani, Pooja; Cohen, Noa

2016-07-11

Heterogeneity in single-cell responses and intercellular interactions results from complex regulation of cell-intrinsic and environmental factors. Single-cell analysis allows not only detection of individual cellular characteristics but also correlation of genetic content with phenotypic traits in the same cell. Technological advances in micro- and nanofabrication have benefited single-cell analysis by allowing precise control of the localized microenvironment, cell manipulation, and sensitive detection capabilities. Additionally, microscale techniques permit rapid, high-throughput, multiparametric screening that has become essential for -omics research. This review highlights innovative applications of microscale platforms in genetic, proteomic, and metabolic detection in single cells; cell sorting strategies; and heterotypic cell-cell interaction. We discuss key design aspects of single-cell localization and isolation in microfluidic systems, dynamic and endpoint analyses, and approaches that integrate highly multiplexed detection of various intracellular species.

Mutations in STN1 cause Coats plus syndrome and are associated with genomic and telomere defects

PubMed Central

Simon, Amos J.; Lev, Atar; Zhang, Yong; Weiss, Batia; Rylova, Anna; Eyal, Eran; Kol, Nitzan; Cesarkas, Keren; Rhodes, Michele; Schiby, Ginette; Barshack, Iris; Katz, Shulamit; Reznik-Wolf, Haike; Ribakovsky, Elena; Simon, Carlos; Hazou, Wadi; Katzir, Hagar; Sagie, Shira; Amariglio, Ninette; Rechavi, Gideon

2016-01-01

The analysis of individuals with telomere defects may shed light on the delicate interplay of factors controlling genome stability, premature aging, and cancer. We herein describe two Coats plus patients with telomere and genomic defects; both harbor distinct, novel mutations in STN1, a member of the human CTC1–STN1–TEN1 (CST) complex, thus linking this gene for the first time to a human telomeropathy. We characterized the patients’ phenotype, recapitulated it in a zebrafish model and rescued cellular and clinical aspects by the ectopic expression of wild-type STN1 or by thalidomide treatment. Interestingly, a significant lengthy control of the gastrointestinal bleeding in one of our patients was achieved by thalidomide treatment, exemplifying a successful bed-to-bench-and-back approach. PMID:27432940

Neurobehavioral phenotype in Prader-Willi syndrome.

PubMed

Whittington, Joyce; Holland, Anthony

2010-11-15

The focus of this article is on the lifetime development of people with Prader-Willi syndrome (PWS) and specifically on the neurobehavioral phenotype. We consider studies of this aspect of the phenotype (the "behavioral phenotype" of the syndrome) that have confirmed that there are specific behaviors and psychiatric disorders, the propensities to which are increased in those with PWS, and cannot be accounted for by other variables such as IQ or adaptive behavior. Beginning with a description of what is observed in people with PWS, we review the evolving PWS phenotype and consider how some aspects of the phenotype might be best explained, and how this complex phenotype may relate to the equally complex genotype. We then consider in more detail some of the neurobehavioral aspects of the phenotype listed above that raise the greatest management problems for parents and carers. © 2010 Wiley-Liss, Inc.

From genotype to phenotype: genetics and medical practice in the new millennium.

PubMed Central

Weatherall, D

1999-01-01

The completion of the human genome project will provide a vast amount of information about human genetic diversity. One of the major challenges for the medical sciences will be to relate genotype to phenotype. Over recent years considerable progress has been made in relating the molecular pathology of monogenic diseases to the associated clinical phenotypes. Studies of the inherited disorders of haemoglobin, notably the thalassaemias, have shown how even in these, the simplest of monogenic diseases, there is remarkable complexity with respect to their phenotypic expression. Although studies of other monogenic diseases are less far advanced, it is clear that the same level of complexity will exist. This information provides some indication of the difficulties that will be met when trying to define the genes that are involved in common multigenic disorders and, in particular, in trying to relate disease phenotypes to the complex interactions between many genes and multiple environmental factors. PMID:10670020

Aberrant RNA splicing and mutations in spliceosome complex in acute myeloid leukemia.

PubMed

Zhou, Jianbiao; Chng, Wee-Joo

2017-01-01

The spliceosome, the cellular splicing machinery, regulates RNA splicing of messenger RNA precursors (pre-mRNAs) into maturation of protein coding RNAs. Recurrent mutations and copy number changes in genes encoding spliceosomal proteins and splicing regulatory factors have tumor promoting or suppressive functions in hematological malignancies, as well as some other cancers. Leukemia stem cell (LSC) populations, although rare, are essential contributors of treatment failure and relapse. Recent researches have provided the compelling evidence that link the erratic spicing activity to the LSC phenotype in acute myeloid leukemia (AML). In this article, we describe the diverse roles of aberrant splicing in hematological malignancies, particularly in AML and their contributions to the characteristics of LSC. We review these promising strategies to exploit the addiction of aberrant spliceosomal machinery for anti-leukemic therapy with aim to eradicate LSC. However, given the complexity and plasticity of spliceosome and not fully known functions of splicing in cancer, the challenges facing the development of the therapeutic strategies targeting RAN splicing are highlighted and future directions are discussed too.

Mitochondrial iron-sulfur cluster biogenesis from molecular understanding to clinical disease

PubMed Central

Alfadhel, Majid; Nashabat, Marwan; Ali, Qais Abu; Hundallah, Khalid

2017-01-01

Iron–sulfur clusters (ISCs) are known to play a major role in various protein functions. Located in the mitochondria, cytosol, endoplasmic reticulum and nucleus, they contribute to various core cellular functions. Until recently, only a few human diseases related to mitochondrial ISC biogenesis defects have been described. Such diseases include Friedreich ataxia, combined oxidative phosphorylation deficiency 19, infantile complex II/III deficiency defect, hereditary myopathy with lactic acidosis and mitochondrial muscle myopathy, lipoic acid biosynthesis defects, multiple mitochondrial dysfunctions syndromes and non ketotic hyperglycinemia due to glutaredoxin 5 gene defect. Disorders of mitochondrial import, export and translation, including sideroblastic anemia with ataxia, EVEN-PLUS syndrome and mitochondrial complex I deficiency due to nucleotide-binding protein-like protein gene defect, have also been implicated in ISC biogenesis defects. With advances in next generation sequencing technologies, more disorders related to ISC biogenesis defects are expected to be elucidated. In this article, we aim to shed the light on mitochondrial ISC biogenesis, related proteins and their function, pathophysiology, clinical phenotypes of related disorders, diagnostic approach, and future implications. PMID:28064324

Development and Use of the Lens Epithelial Explant System to Study Lens Differentiation and Cataractogenesis

PubMed Central

West-Mays, Judith A.; Pino, Guiseppe; Lovicu, Frank J.

2010-01-01

Over the last two decades much progress has been made in identifying and characterizing many of the molecules involved in understanding normal lens biology and its pathology. Much of this has been made possible through the establishment and use of the lens epithelial explant system. This simplistic tissue culture model, comprised of a sheet of lens epithelium on its native substratum, has been used effectively to study many cellular processes, including lens epithelial cell proliferation, fiber cell differentiation, cell apoptosis as well as epithelial to mesenchymal transformation of cells. In doing so, a number of key growth factors and cytokines, including members of the FGF, Wnt and TGFβ family have been shown to play essential roles in many of these cellular events. This has led to further studies exploring the signaling pathways downstream of these molecules in the lens, paving the way for the development of a number of in situ models (primarily transgenic mouse lines) to further explore in more detail the nature of these molecular and cellular interactions. To reciprocate, the lens epithelial explant system is increasingly being used to further characterize the nature of many complex phenotypes and pathologies observed in these in situ models, allowing us to selectively isolate and examine the direct impact of an individual molecule on a specific cellular response in lens cells. There is no question that the lens epithelial explant system has served as a powerful tool to further our understanding of lens biology and pathology, and there is no doubt that it will continue to serve in such a capacity, as new developments are realized and putative treatments for aberrant lens cell behaviour are to be trialed. PMID:20006728

A role for the PDZ-binding domain of the coxsackie B virus and adenovirus receptor (CAR) in cell adhesion and growth.

PubMed

Excoffon, Katherine J D Ashbourne; Hruska-Hageman, Alesia; Klotz, Michael; Traver, Geri L; Zabner, Joseph

2004-09-01

The coxsackie and adenovirus receptor (CAR) plays a role in viral infection, maintenance of the junction adhesion complex in polarized epithelia, and modulation of cellular growth properties. As a viral receptor, the C-terminus appears to play no role indicating that the major function of CAR is to tether the virus to the cell. By contrast, the C-terminus is known to play a role in cellular localization and probably has a significant function in CAR-mediated adhesion and cell growth properties. We hypothesized that the CAR PDZ (PSD-95/Disc-large/ZO-1) binding motif interacts with PDZ-domain-containing proteins to modulate the cellular phenotype. CAR was modified by deleting the last four amino acids (CARDeltaGSIV) and evaluated for cell-cell adhesion in polarized primary human airway epithelia and growth characteristics in stably transfected L-cells. Although ablation of the CAR PDZ-binding motif did not affect adenoviral infection, it did have a significant effect both on cell-cell adhesion and on cell growth. Expression of CARDeltaGSIV failed to increase the transepithelial resistance in polarized epithelia to the same degree as wild-type CAR and failed to act as a growth modulator in L-cells. Furthermore, we provide evidence for three new CAR interacting partners, including MAGI-1b, PICK1 and PSD-95. CAR appears to interact with several distinct PDZ-domain-containing proteins and may exert its biological function through these interactions.

Combination of automated high throughput platforms, flow cytometry, and hierarchical clustering to detect cell state.

PubMed

Kitsos, Christine M; Bhamidipati, Phani; Melnikova, Irena; Cash, Ethan P; McNulty, Chris; Furman, Julia; Cima, Michael J; Levinson, Douglas

2007-01-01

This study examined whether hierarchical clustering could be used to detect cell states induced by treatment combinations that were generated through automation and high-throughput (HT) technology. Data-mining techniques were used to analyze the large experimental data sets to determine whether nonlinear, non-obvious responses could be extracted from the data. Unary, binary, and ternary combinations of pharmacological factors (examples of stimuli) were used to induce differentiation of HL-60 cells using a HT automated approach. Cell profiles were analyzed by incorporating hierarchical clustering methods on data collected by flow cytometry. Data-mining techniques were used to explore the combinatorial space for nonlinear, unexpected events. Additional small-scale, follow-up experiments were performed on cellular profiles of interest. Multiple, distinct cellular profiles were detected using hierarchical clustering of expressed cell-surface antigens. Data-mining of this large, complex data set retrieved cases of both factor dominance and cooperativity, as well as atypical cellular profiles. Follow-up experiments found that treatment combinations producing "atypical cell types" made those cells more susceptible to apoptosis. CONCLUSIONS Hierarchical clustering and other data-mining techniques were applied to analyze large data sets from HT flow cytometry. From each sample, the data set was filtered and used to define discrete, usable states that were then related back to their original formulations. Analysis of resultant cell populations induced by a multitude of treatments identified unexpected phenotypes and nonlinear response profiles.

Building New Bridges between In Vitro and In Vivo in Early Drug Discovery: Where Molecular Modeling Meets Systems Biology.

PubMed

Pearlstein, Robert A; McKay, Daniel J J; Hornak, Viktor; Dickson, Callum; Golosov, Andrei; Harrison, Tyler; Velez-Vega, Camilo; Duca, José

2017-01-01

Cellular drug targets exist within networked function-generating systems whose constituent molecular species undergo dynamic interdependent non-equilibrium state transitions in response to specific perturbations (i.e.. inputs). Cellular phenotypic behaviors are manifested through the integrated behaviors of such networks. However, in vitro data are frequently measured and/or interpreted with empirical equilibrium or steady state models (e.g. Hill, Michaelis-Menten, Briggs-Haldane) relevant to isolated target populations. We propose that cells act as analog computers, "solving" sets of coupled "molecular differential equations" (i.e. represented by populations of interacting species)via "integration" of the dynamic state probability distributions among those populations. Disconnects between biochemical and functional/phenotypic assays (cellular/in vivo) may arise with targetcontaining systems that operate far from equilibrium, and/or when coupled contributions (including target-cognate partner binding and drug pharmacokinetics) are neglected in the analysis of biochemical results. The transformation of drug discovery from a trial-and-error endeavor to one based on reliable design criteria depends on improved understanding of the dynamic mechanisms powering cellular function/dysfunction at the systems level. Here, we address the general mechanisms of molecular and cellular function and pharmacological modulation thereof. We outline a first principles theory on the mechanisms by which free energy is stored and transduced into biological function, and by which biological function is modulated by drug-target binding. We propose that cellular function depends on dynamic counter-balanced molecular systems necessitated by the exponential behavior of molecular state transitions under non-equilibrium conditions, including positive versus negative mass action kinetics and solute-induced perturbations to the hydrogen bonds of solvating water versus kT. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Towards improving phenotype representation in OWL

PubMed Central

2012-01-01

Background Phenotype ontologies are used in species-specific databases for the annotation of mutagenesis experiments and to characterize human diseases. The Entity-Quality (EQ) formalism is a means to describe complex phenotypes based on one or more affected entities and a quality. EQ-based definitions have been developed for many phenotype ontologies, including the Human and Mammalian Phenotype ontologies. Methods We analyze formalizations of complex phenotype descriptions in the Web Ontology Language (OWL) that are based on the EQ model, identify several representational challenges and analyze potential solutions to address these challenges. Results In particular, we suggest a novel, role-based approach to represent relational qualities such as concentration of iron in spleen, discuss its ontological foundation in the General Formal Ontology (GFO) and evaluate its representation in OWL and the benefits it can bring to the representation of phenotype annotations. Conclusion Our analysis of OWL-based representations of phenotypes can contribute to improving consistency and expressiveness of formal phenotype descriptions. PMID:23046625

Organ reconstruction: Dream or reality for the future.

PubMed

Stoltz, J-F; Zhang, L; Ye, J S; De Isla, N

2017-01-01

The relevance of research on reconstructed organs is justified by the lack of organs available for transplant and the growing needs for the ageing population. The development of a reconstructed organ involves two parallel complementary steps: de-cellularization of the organ with the need to maintain the structural integrity of the extracellular matrix and vascular network and re-cellularization of the scaffold with stem cells or resident cells.Whole organ engineering for liver, heart, lung or kidneys, is particularly difficult because of the structural complexity of organs and heterogeneity of cells. Rodent, porcine and rhesus monkey organs have been de-cellularized to obtain a scaffold with preserved extracellular matrix and vascular network. As concern the cells for re-cellularization, embryonic, foetal, adult, progenitor stem cells and also iPS have been proposed.Heart construction could be an alternative option for the treatment of cardiac insufficiency. It is based on the use of an extra-cellular matrix coming from an animal's heart and seeded with cells likely to reconstruct a normal cardiac function. Though de-cellularization techniques now seem controlled, the issues posed by the selection of cells capable of generating the various components of cardiac tissue are not settled yet. In addition, the recolonisation of the matrix does not only depend on the phenotype of cells that are used, but it is also impacted by the nature of biochemical signals emitted.Recent researches have shown that it is possible to use decellularized whole liver treated by detergents as scaffold, which keeps the entire network of blood vessels and the integrated extracellular matrix (ECM). Beside of decellularized whole organ scaffold seeding cells selected to repopulate a decellularized liver scaffold are critical for the function of the bioengineered liver. At present, potential cell sources are hepatocyte, and mesenchymal stem cells.Pulmonary regeneration using engineering approaches is complex. In fact, several types of local progenitor cells that contribute to cell repair have been described at different levels of the respiratory tract. Moving towards the alveoles, one finds bronchioalveolar stem cells as well as epithelial cells and pneumocytes. A promising option to increase the donor organ pool is to use allogeneic or xenogeneic decellularized lungs as a scaffold to engineer functional lung tissue ex vivo.The kidney is certainly one of the most difficult organs to reconstruct due to its complex nature and the heterogeneous nature of the cells. There is relatively little research on auto-construction, and experiments have been performed on rats, pigs and monkeys.Nevertheless, before these therapeutic approaches can be applied in clinical practice, many researches are necessary to understand and in particular the behaviour of cells on the decellularized organs as well as the mechanisms of their interaction with the microenvironment. Current knowledges allow optimism for the future but definitive answers can only be given after long term animal studies and controlled clinical studies.

‘Particle genetics’: treating every cell as unique

PubMed Central

Yvert, Gaël

2014-01-01

Genotype-phenotype relations are usually inferred from a deterministic point of view. For example, quantitative trait loci (QTL), which describe regions of the genome associated with a particular phenotype, are based on a mean trait difference between genotype categories. However, living systems comprise huge numbers of cells (the ‘particles’ of biology). Each cell can exhibit substantial phenotypic individuality, which can have dramatic consequences at the organismal level. Now, with technology capable of interrogating individual cells, it is time to consider how genotypes shape the probability laws of single cell traits. The possibility of mapping single cell probabilistic trait loci (PTL), which link genomic regions to probabilities of cellular traits, is a promising step in this direction. This approach requires thinking about phenotypes in probabilistic terms, a concept that statistical physicists have been applying to particles for a century. Here, I describe PTL and discuss their potential to enlarge our understanding of genotype-phenotype relations. PMID:24315431

Genome-wide screen for inositol auxotrophy in Saccharomyces cerevisiae implicates lipid metabolism in stress response signaling.

PubMed

Villa-García, Manuel J; Choi, Myung Sun; Hinz, Flora I; Gaspar, María L; Jesch, Stephen A; Henry, Susan A

2011-02-01

Inositol auxotrophy (Ino(-) phenotype) in budding yeast has classically been associated with misregulation of INO1 and other genes involved in lipid metabolism. To identify all non-essential yeast genes that are necessary for growth in the absence of inositol, we carried out a genome-wide phenotypic screening for deletion mutants exhibiting Ino(-) phenotypes under one or more growth conditions. We report the identification of 419 genes, including 385 genes not previously reported, which exhibit this phenotype when deleted. The identified genes are involved in a wide range of cellular processes, but are particularly enriched in those affecting transcription, protein modification, membrane trafficking, diverse stress responses, and lipid metabolism. Among the Ino(-) mutants involved in stress response, many exhibited phenotypes that are strengthened at elevated temperature and/or when choline is present in the medium. The role of inositol in regulation of lipid metabolism and stress response signaling is discussed.

Some Like It Hot, Some Like It Warm: Phenotyping to Explore Thermotolerance Diversity

PubMed Central

Yeh, Ching-Hui; Kaplinsky, Nicholas J.; Hu, Catherine; Charng, Yee-yung

2012-01-01

Plants have evolved overlapping but distinct cellular responses to different aspects of high temperature stress. These responses include basal thermotolerance, short- and long-term acquired thermotolerance, and thermotolerance to moderately high temperatures. This thermotolerance diversity’ means that multiple phenotypic assays are essential for fully describing the functions of genes involved in heat stress responses. A large number of genes with potential roles in heat stress responses have been identified using genetic screens and genome wide expression studies. We examine the range of phenotypic assays that have been used to characterize thermotolerance phenotypes in both Arabidopsis and crop plants. Three major variables differentiate thermotolerance assays: 1) the heat stress regime used, 2) the developmental stage of the plants being studied, and 3) the actual phenotype which is scored. Consideration of these variables will be essential for deepening our understanding of the molecular genetics of plant thermotolerance. PMID:22920995

RRM2 induces NF-{kappa}B-dependent MMP-9 activation and enhances cellular invasiveness

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

Duxbury, Mark S.; Whang, Edward E.

2007-03-02

Ribonucleotide reductase is a dimeric enzyme that catalyzes conversion of ribonucleotide 5'-diphosphates to their 2'-deoxynucleotide forms, a rate-limiting step in the production of 2'-deoxyribonucleoside 5'-triphosphates required for DNA synthesis. The ribonucleotide reductase M2 subunit (RRM2) is a determinant of malignant cellular behavior in a range of human cancers. We examined the effect of RRM2 overexpression on pancreatic adenocarcinoma cellular invasiveness and nuclear factor-{kappa}B (NF-{kappa}B) transcription factor activity. RRM2 overexpression increases pancreatic adenocarcinoma cellular invasiveness and MMP-9 expression in a NF-{kappa}B-dependent manner. RNA interference (RNAi)-mediated silencing of RRM2 expression attenuates cellular invasiveness and NF-{kappa}B activity. NF-{kappa}B is a key mediator ofmore » the invasive phenotypic changes induced by RRM2 overexpression.« less

HFE gene variants, iron, and lipids: a novel connection in Alzheimer's disease.

PubMed

Ali-Rahmani, Fatima; Schengrund, Cara-Lynne; Connor, James R

2014-01-01

Iron accumulation and associated oxidative stress in the brain have been consistently found in several neurodegenerative diseases. Multiple genetic studies have been undertaken to try to identify a cause of neurodegenerative diseases but direct connections have been rare. In the iron field, variants in the HFE gene that give rise to a protein involved in cellular iron regulation, are associated with iron accumulation in multiple organs including the brain. There is also substantial epidemiological, genetic, and molecular evidence of disruption of cholesterol homeostasis in several neurodegenerative diseases, in particular Alzheimer's disease (AD). Despite the efforts that have been made to identify factors that can trigger the pathological events associated with neurodegenerative diseases they remain mostly unknown. Because molecular phenotypes such as oxidative stress, synaptic failure, neuronal loss, and cognitive decline, characteristics associated with AD, have been shown to result from disruption of a number of pathways, one can easily argue that the phenotype seen may not arise from a linear sequence of events. Therefore, a multi-targeted approach is needed to understand a complex disorder like AD. This can be achieved only when knowledge about interactions between the different pathways and the potential influence of environmental factors on them becomes available. Toward this end, this review discusses what is known about the roles and interactions of iron and cholesterol in neurodegenerative diseases. It highlights the effects of gene variants of HFE (H63D- and C282Y-HFE) on iron and cholesterol metabolism and how they may contribute to understanding the etiology of complex neurodegenerative diseases.

Shared activity patterns arising at genetic susceptibility loci reveal underlying genomic and cellular architecture of human disease.

PubMed

Baillie, J Kenneth; Bretherick, Andrew; Haley, Christopher S; Clohisey, Sara; Gray, Alan; Neyton, Lucile P A; Barrett, Jeffrey; Stahl, Eli A; Tenesa, Albert; Andersson, Robin; Brown, J Ben; Faulkner, Geoffrey J; Lizio, Marina; Schaefer, Ulf; Daub, Carsten; Itoh, Masayoshi; Kondo, Naoto; Lassmann, Timo; Kawai, Jun; Mole, Damian; Bajic, Vladimir B; Heutink, Peter; Rehli, Michael; Kawaji, Hideya; Sandelin, Albin; Suzuki, Harukazu; Satsangi, Jack; Wells, Christine A; Hacohen, Nir; Freeman, Thomas C; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R R; Hume, David A

2018-03-01

Genetic variants underlying complex traits, including disease susceptibility, are enriched within the transcriptional regulatory elements, promoters and enhancers. There is emerging evidence that regulatory elements associated with particular traits or diseases share similar patterns of transcriptional activity. Accordingly, shared transcriptional activity (coexpression) may help prioritise loci associated with a given trait, and help to identify underlying biological processes. Using cap analysis of gene expression (CAGE) profiles of promoter- and enhancer-derived RNAs across 1824 human samples, we have analysed coexpression of RNAs originating from trait-associated regulatory regions using a novel quantitative method (network density analysis; NDA). For most traits studied, phenotype-associated variants in regulatory regions were linked to tightly-coexpressed networks that are likely to share important functional characteristics. Coexpression provides a new signal, independent of phenotype association, to enable fine mapping of causative variants. The NDA coexpression approach identifies new genetic variants associated with specific traits, including an association between the regulation of the OCT1 cation transporter and genetic variants underlying circulating cholesterol levels. NDA strongly implicates particular cell types and tissues in disease pathogenesis. For example, distinct groupings of disease-associated regulatory regions implicate two distinct biological processes in the pathogenesis of ulcerative colitis; a further two separate processes are implicated in Crohn's disease. Thus, our functional analysis of genetic predisposition to disease defines new distinct disease endotypes. We predict that patients with a preponderance of susceptibility variants in each group are likely to respond differently to pharmacological therapy. Together, these findings enable a deeper biological understanding of the causal basis of complex traits.

Epigenetic regulation of osteogenesis: human embryonic palatal mesenchymal cells.

PubMed

Barkhordarian, Andre; Sison, Jay; Cayabyab, Riana; Mahanian, Nicole; Chiappelli, Francesco

2011-01-06

Mesenchymal stem cells (MSCs) provide an appropriate model to study epigenetic changes during osteogenesis and bone regeneration due to their differentiation potential. Since there are no unique markers for MSCs, methods of identification are limited. The complex morphology of human embryonic palatal mesenchyme stem cell (HEPM) requires analysis of fractal dimensions to provide an objective quantification of self-similarity, a statistical transformation of cellular shape and border complexity. We propose the hypothesis of a study to compare and contrast sequential steps of osteogenic differentiation in HEPMs both phenotypically using immunocytochemistry, and morphometrically using fractal analysis from undifferentiated passage 1 (P1) to passage 7 (P7) cells. The proof-of-concept is provided by results we present here that identify and compare the modulation of expression of certain epigenetic biomarkers (alkaline phosphatase, ALP; stromal interaction molecule-1, STRO-1; runt-related transcription factor-2, RUNX2), which are established markers of osteogenesis in bone marrow studies, of osteoblastic/skeletal morphogenesis, and of osteoblast maturation. We show that Osteoinductive medium (OIM) modulates the rate of differentiation of HEPM into Run-2+ cells, the most differentiated subpopulation, followed by ALP+ and STRO-1+ cells. Taken together, our phenotypical and morphometric data demonstrate the feasibility of using HEPM to assess osteogenic differentiation from an early undifferentiated to a differentiated stage. This research model may lay the foundation for future studies aimed at characterizing the epigenetic characteristics of osteoimmunological disorders and dysfunctions (e.g., osteoarthritis, temporomandibular joint disorders), so that proteomic profiling can aid the diagnosis and monitor the prognosis of these and other osteoimmunopathologies.

HFE gene variants, iron, and lipids: a novel connection in Alzheimer’s disease

PubMed Central

Ali-Rahmani, Fatima; Schengrund, Cara-Lynne; Connor, James R.

2014-01-01

Iron accumulation and associated oxidative stress in the brain have been consistently found in several neurodegenerative diseases. Multiple genetic studies have been undertaken to try to identify a cause of neurodegenerative diseases but direct connections have been rare. In the iron field, variants in the HFE gene that give rise to a protein involved in cellular iron regulation, are associated with iron accumulation in multiple organs including the brain. There is also substantial epidemiological, genetic, and molecular evidence of disruption of cholesterol homeostasis in several neurodegenerative diseases, in particular Alzheimer’s disease (AD). Despite the efforts that have been made to identify factors that can trigger the pathological events associated with neurodegenerative diseases they remain mostly unknown. Because molecular phenotypes such as oxidative stress, synaptic failure, neuronal loss, and cognitive decline, characteristics associated with AD, have been shown to result from disruption of a number of pathways, one can easily argue that the phenotype seen may not arise from a linear sequence of events. Therefore, a multi-targeted approach is needed to understand a complex disorder like AD. This can be achieved only when knowledge about interactions between the different pathways and the potential influence of environmental factors on them becomes available. Toward this end, this review discusses what is known about the roles and interactions of iron and cholesterol in neurodegenerative diseases. It highlights the effects of gene variants of HFE (H63D- and C282Y-HFE) on iron and cholesterol metabolism and how they may contribute to understanding the etiology of complex neurodegenerative diseases. PMID:25071582

Biofilm formation and cellulose expression among diverse environmental Pseudomonas isolates.

PubMed

Ude, Susanne; Arnold, Dawn L; Moon, Christina D; Timms-Wilson, Tracey; Spiers, Andrew J

2006-11-01

The ability to form biofilms is seen as an increasingly important colonization strategy among both pathogenic and environmental bacteria. A survey of 185 plant-associated, phytopathogenic, soil and river Pseudomonas isolates resulted in 76% producing biofilms at the air-liquid (A-L) interface after selection in static microcosms. Considerable variation in biofilm phenotype was observed, including waxy aggregations, viscous and floccular masses, and physically cohesive biofilms with continuously varying strengths over 1500-fold. Calcofluor epifluorescent microscopy identified cellulose as the matrix component in biofilms produced by Pseudomonas asplenii, Pseudomonas corrugata, Pseudomonas fluorescens, Pseudomonas marginalis, Pseudomonas putida, Pseudomonas savastanoi and Pseudomonas syringae isolates. Cellulose expression and biofilm formation could be induced by the constitutively active WspR19 mutant of the cyclic-di-GMP-associated, GGDEF domain-containing response regulator involved in the P. fluorescens SBW25 wrinkly spreader phenotype and cellular aggregation in Pseudomonas aeruginosa PA01. WspR19 could also induce P. putida KT2440, which otherwise did not produce a biofilm or express cellulose, as well as Escherichia coli K12 and Salmonella typhimurium LT2, both of which express cellulose yet lack WspR homologues. Statistical analysis of biofilm parameters suggest that biofilm development is a more complex process than that simply described by the production of attachment and matrix components and bacterial growth. This complexity was also seen in multivariate analysis as a species-ecological habitat effect, underscoring the fact that in vitro biofilms are abstractions of those surface and volume colonization processes used by bacteria in their natural environments.

Expanded CAG/CTG Repeat DNA Induces a Checkpoint Response That Impacts Cell Proliferation in Saccharomyces cerevisiae

PubMed Central

Sundararajan, Rangapriya; Freudenreich, Catherine H.

2011-01-01

Repetitive DNA elements are mutational hotspots in the genome, and their instability is linked to various neurological disorders and cancers. Although it is known that expanded trinucleotide repeats can interfere with DNA replication and repair, the cellular response to these events has not been characterized. Here, we demonstrate that an expanded CAG/CTG repeat elicits a DNA damage checkpoint response in budding yeast. Using microcolony and single cell pedigree analysis, we found that cells carrying an expanded CAG repeat frequently experience protracted cell division cycles, persistent arrests, and morphological abnormalities. These phenotypes were further exacerbated by mutations in DSB repair pathways, including homologous recombination and end joining, implicating a DNA damage response. Cell cycle analysis confirmed repeat-dependent S phase delays and G2/M arrests. Furthermore, we demonstrate that the above phenotypes are due to the activation of the DNA damage checkpoint, since expanded CAG repeats induced the phosphorylation of the Rad53 checkpoint kinase in a rad52Δ recombination deficient mutant. Interestingly, cells mutated for the MRX complex (Mre11-Rad50-Xrs2), a central component of DSB repair which is required to repair breaks at CAG repeats, failed to elicit repeat-specific arrests, morphological defects, or Rad53 phosphorylation. We therefore conclude that damage at expanded CAG/CTG repeats is likely sensed by the MRX complex, leading to a checkpoint response. Finally, we show that repeat expansions preferentially occur in cells experiencing growth delays. Activation of DNA damage checkpoints in repeat-containing cells could contribute to the tissue degeneration observed in trinucleotide repeat expansion diseases. PMID:21437275

Genome-Wide Gene Set Analysis for Identification of Pathways Associated with Alcohol Dependence

PubMed Central

Biernacka, Joanna M.; Geske, Jennifer; Jenkins, Gregory D.; Colby, Colin; Rider, David N.; Karpyak, Victor M.; Choi, Doo-Sup; Fridley, Brooke L.

2013-01-01

It is believed that multiple genetic variants with small individual effects contribute to the risk of alcohol dependence. Such polygenic effects are difficult to detect in genome-wide association studies that test for association of the phenotype with each single nucleotide polymorphism (SNP) individually. To overcome this challenge, gene set analysis (GSA) methods that jointly test for the effects of pre-defined groups of genes have been proposed. Rather than testing for association between the phenotype and individual SNPs, these analyses evaluate the global evidence of association with a set of related genes enabling the identification of cellular or molecular pathways or biological processes that play a role in development of the disease. It is hoped that by aggregating the evidence of association for all available SNPs in a group of related genes, these approaches will have enhanced power to detect genetic associations with complex traits. We performed GSA using data from a genome-wide study of 1165 alcohol dependent cases and 1379 controls from the Study of Addiction: Genetics and Environment (SAGE), for all 200 pathways listed in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Results demonstrated a potential role of the “Synthesis and Degradation of Ketone Bodies” pathway. Our results also support the potential involvement of the “Neuroactive Ligand Receptor Interaction” pathway, which has previously been implicated in addictive disorders. These findings demonstrate the utility of GSA in the study of complex disease, and suggest specific directions for further research into the genetic architecture of alcohol dependence. PMID:22717047

Shared activity patterns arising at genetic susceptibility loci reveal underlying genomic and cellular architecture of human disease

PubMed Central

Gray, Alan; Neyton, Lucile P. A.; Barrett, Jeffrey; Stahl, Eli A.; Tenesa, Albert; Andersson, Robin; Brown, J. Ben; Faulkner, Geoffrey J.; Lizio, Marina; Schaefer, Ulf; Daub, Carsten; Kondo, Naoto; Lassmann, Timo; Kawai, Jun; Kawaji, Hideya; Suzuki, Harukazu; Satsangi, Jack; Wells, Christine A.; Hacohen, Nir; Freeman, Thomas C.; Hayashizaki, Yoshihide; Forrest, Alistair R. R.; Hume, David A.

2018-01-01

Genetic variants underlying complex traits, including disease susceptibility, are enriched within the transcriptional regulatory elements, promoters and enhancers. There is emerging evidence that regulatory elements associated with particular traits or diseases share similar patterns of transcriptional activity. Accordingly, shared transcriptional activity (coexpression) may help prioritise loci associated with a given trait, and help to identify underlying biological processes. Using cap analysis of gene expression (CAGE) profiles of promoter- and enhancer-derived RNAs across 1824 human samples, we have analysed coexpression of RNAs originating from trait-associated regulatory regions using a novel quantitative method (network density analysis; NDA). For most traits studied, phenotype-associated variants in regulatory regions were linked to tightly-coexpressed networks that are likely to share important functional characteristics. Coexpression provides a new signal, independent of phenotype association, to enable fine mapping of causative variants. The NDA coexpression approach identifies new genetic variants associated with specific traits, including an association between the regulation of the OCT1 cation transporter and genetic variants underlying circulating cholesterol levels. NDA strongly implicates particular cell types and tissues in disease pathogenesis. For example, distinct groupings of disease-associated regulatory regions implicate two distinct biological processes in the pathogenesis of ulcerative colitis; a further two separate processes are implicated in Crohn’s disease. Thus, our functional analysis of genetic predisposition to disease defines new distinct disease endotypes. We predict that patients with a preponderance of susceptibility variants in each group are likely to respond differently to pharmacological therapy. Together, these findings enable a deeper biological understanding of the causal basis of complex traits. PMID:29494619

Novel throughput phenotyping platforms in plant genetic studies.

PubMed

Montes, Juan M; Melchinger, Albrecht E; Reif, Jochen C

2007-10-01

Unraveling the genetic basis of complex traits in plants is limited by the lack of appropriate phenotyping platforms that enable high-throughput screening of many genotypes in multilocation field trials. Near-infrared spectroscopy on agricultural harvesters and spectral reflectance of plant canopies have recently been reported as promising components of novel phenotyping platforms. Understanding the genetic basis of complex traits is now within reach with the use of these new techniques.

Directed evolution and synthetic biology applications to microbial systems.

PubMed

Bassalo, Marcelo C; Liu, Rongming; Gill, Ryan T

2016-06-01

Biotechnology applications require engineering complex multi-genic traits. The lack of knowledge on the genetic basis of complex phenotypes restricts our ability to rationally engineer them. However, complex phenotypes can be engineered at the systems level, utilizing directed evolution strategies that drive whole biological systems toward desired phenotypes without requiring prior knowledge of the genetic basis of the targeted trait. Recent developments in the synthetic biology field accelerates the directed evolution cycle, facilitating engineering of increasingly complex traits in biological systems. In this review, we summarize some of the most recent advances in directed evolution and synthetic biology that allows engineering of complex traits in microbial systems. Then, we discuss applications that can be achieved through engineering at the systems level. Copyright © 2016 Elsevier Ltd. All rights reserved.

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM).

PubMed

Tang, Anson H L; Lai, Queenie T K; Chung, Bob M F; Lee, Kelvin C M; Mok, Aaron T Y; Yip, G K; Shum, Anderson H C; Wong, Kenneth K Y; Tsia, Kevin K

2017-06-28

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture - information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.

Using ToxCast data to reconstruct dynamic cell state ...

EPA Pesticide Factsheets

AbstractBackground. High-throughput in vitro screening is an important tool for evaluating the potential biological activity of the thousands of existing chemicals in commerce and the hundreds more introduced each year. Among the assay technologies available, high-content imaging (HCI) allows multiplexed measurements of cellular phenotypic changes induced by chemical exposures. For a large chemical inventory having limited concentration-time series data, the deconvolution of cellular response profiles into transitive or irrevocable state trajectories is an important consideration. Objectives. Our goal was to analyze temporal and concentration-related cellular changes measured using HCI to identify the “tipping point” at which the cells did not show recovery towards a normal phenotypic state. Methods. The effects of 976 chemicals (ToxCast Phase I and II) were evaluated using HCI as a function of concentration and time in HepG2 cells over a 72-hr exposure period to concentrations ranging from 0.4- to 200 µM. The cellular endpoints included nuclear p53 accumulation, JNK, markers of oxidative stress, cytoskeletal changes, mitochondrial energization and density, cell viability and cell cycle progression. A novel computational model was developed to interpret dynamic multidimensional system responses as cell-state trajectories. Results. Analysis of cell-state trajectories showed that HepG2 cells were resilient to the effects of 178 chemicals up to the highest co

Understanding the cellular and molecular mechanisms of dominant and recessive inheritance in genetics course.

PubMed

Wanjin, Xing; Morigen, Morigen

2015-01-01

In Mendellian genetics, the dominance and recessiveness are used to describe the functional relationship between two alleles of one gene in a heterozygote. The allele which constitutes a phenotypical character over the other is named dominant and the one functionally masked is called recessive. The definitions thereby led to the creation of Mendel's laws on segregation and independent assortment and subsequent classic genetics. The discrimination of dominance and recessiveness originally is a requirement for Mendel's logical reasoning, but now it should be explained by cellular and molecular principles in the modern genetics. To answer the question raised by students of how the dominance and recessiveness are controlled, we reviewed the recent articles and tried to summarize the cellular and molecular basis of dominant and recessive inheritance. Clearly, understanding the essences of dominant and recessive inheritance requires us to know the dissimilarity of the alleles and their products (RNA and/or proteins), and the way of their function in cells. The alleles spatio-temporally play different roles on offering cells, tissues or organs with discernible phenotypes, namely dominant or recessive. Here, we discuss the changes of allele dominance and recessiveness at the cellular and molecular levels based on the variation of gene structure, gene regulation, function and types of gene products, in order to make students understand gene mutation and function more comprehensively and concretely.

A multi-phenotypic imaging screen to identify bacterial effectors by exogenous expression in a HeLa cell line.

PubMed

Collins, Adam; Huett, Alan

2018-05-15

We present a high-content screen (HCS) for the simultaneous analysis of multiple phenotypes in HeLa cells expressing an autophagy reporter (mcherry-LC3) and one of 224 GFP-fused proteins from the Crohn's Disease (CD)-associated bacterium, Adherent Invasive E. coli (AIEC) strain LF82. Using automated confocal microscopy and image analysis (CellProfiler), we localised GFP fusions within cells, and monitored their effects upon autophagy (an important innate cellular defence mechanism), cellular and nuclear morphology, and the actin cytoskeleton. This data will provide an atlas for the localisation of 224 AIEC proteins within human cells, as well as a dataset to analyse their effects upon many aspects of host cell morphology. We also describe an open-source, automated, image-analysis workflow to identify bacterial effectors and their roles via the perturbations induced in reporter cell lines when candidate effectors are exogenously expressed.

Optimization of industrial microorganisms: recent advances in synthetic dynamic regulators.

PubMed

Min, Byung Eun; Hwang, Hyun Gyu; Lim, Hyun Gyu; Jung, Gyoo Yeol

2017-01-01

Production of biochemicals by industrial fermentation using microorganisms requires maintaining cellular production capacity, because maximal productivity is economically important. High-productivity microbial strains can be developed using static engineering, but these may not maintain maximal productivity throughout the culture period as culture conditions and cell states change dynamically. Additionally, economic reasons limit heterologous protein expression using inducible promoters to prevent metabolic burden for commodity chemical and biofuel production. Recently, synthetic and systems biology has been used to design genetic circuits, precisely controlling gene expression or influencing genetic behavior toward a desired phenotype. Development of dynamic regulators can maintain cellular phenotype in a maximum production state in response to factors including cell concentration, oxygen, temperature, pH, and metabolites. Herein, we introduce dynamic regulators of industrial microorganism optimization and discuss metabolic flux fine control by dynamic regulators in response to metabolites or extracellular stimuli, robust production systems, and auto-induction systems using quorum sensing.

Identification of a Gene That Reverses the Immortal Phenotype of a Subset of Cells and Is a Member of a Novel Family of Transcription Factor-Like Genes†

PubMed Central

Bertram, M. J.; Bérubé, N. G.; Hang-Swanson, X.; Ran, Q.; Leung, J. K.; Bryce, S.; Spurgers, K.; Bick, R. J.; Baldini, A.; Ning, Y.; Clark, L. J.; Parkinson, E. K.; Barrett, J. C.; Smith, J. R.; Pereira-Smith, O. M.

1999-01-01

Based on the dominance of cellular senescence over immortality, immortal human cell lines have been assigned to four complementation groups for indefinite division. Human chromosomes carrying senescence genes have been identified, including chromosome 4. We report the cloning and identification of a gene, mortality factor 4 (MORF 4), which induces a senescent-like phenotype in immortal cell lines assigned to complementation group B with concomitant changes in two markers for senescence. MORF 4 is a member of a novel family of genes with transcription factor-like motifs. We present here the sequences of the seven family members, their chromosomal locations, and a partial characterization of the three members that are expressed. Elucidation of the mechanism of action of these genes should enhance our understanding of growth regulation and cellular aging. PMID:9891081

Unfolding the relationship between secreted molecular chaperones and macrophage activation states

PubMed Central

Henderson, Samantha

2008-01-01

Over the last 20 years, it has emerged that many molecular chaperones and protein-folding catalysts are secreted from cells and function, somewhat in the manner of cytokines, as pleiotropic signals for a variety of cells, with much attention being focused on the macrophage. During the last decade, it has become clear that macrophages respond to bacterial, protozoal, parasitic and host signals to generate phenotypically distinct states of activation. These activation states have been termed ‘classical’ and ‘alternative’ and represent not a simple bifurcation in response to external signals but a range of cellular phenotypes. From an examination of the literature, the hypothesis is propounded that mammalian molecular chaperones are able to induce a wide variety of alternative macrophage activation states, and this may be a system for relating cellular or tissue stress to appropriate macrophage responses to restore homeostatic equilibrium. PMID:18958583

Phenotypic correction of Fanconi anemia cells in the murine bone marrow after carrier cell mediated delivery of lentiviral vector.

PubMed

Chakkaramakkil Verghese, Santhosh; Goloviznina, Natalya A; Kurre, Peter

2016-11-19

Fanconi anemia (FA) is an autosomal-recessive disorder associated with hematopoietic failure and it is a candidate for hematopoietic stem cell (HSC)-directed gene therapy. However, the characteristically reduced HSC numbers found in FA patients, their ineffective mobilization from the marrow, and re-oxygenation damage during ex vivo manipulation have precluded clinical success using conventional in vitro approaches. We previously demonstrated that lentiviral vector (LV) particles reversibly attach to the cell surface where they gain protection from serum complement neutralization. We reasoned that cellular delivery of LV to the bone marrow niche could avoid detrimental losses during FA HSC mobilization and in vitro modification. Here, we demonstrate that a VSV-G pseudotyped lentivector, carrying the FANCC transgene, can be transmitted from carrier to bystander cells. In cell culture and transplantation models of FA, we further demonstrate that LV carrier cells migrate along SDF-1α gradients and transfer vector particles that stably integrate and phenotypically correct the characteristic DNA alkylator sensitivity in murine and human FA-deficient target bystander cells. Altogether, we demonstrate that cellular homing mechanisms can be harnessed for the functional phenotype correction in murine FA hematopoietic cells.

Discovering cancer vulnerabilities using high-throughput micro-RNA screening.

PubMed

Nikolic, Iva; Elsworth, Benjamin; Dodson, Eoin; Wu, Sunny Z; Gould, Cathryn M; Mestdagh, Pieter; Marshall, Glenn M; Horvath, Lisa G; Simpson, Kaylene J; Swarbrick, Alexander

2017-12-15

Micro-RNAs (miRNAs) are potent regulators of gene expression and cellular phenotype. Each miRNA has the potential to target hundreds of transcripts within the cell thus controlling fundamental cellular processes such as survival and proliferation. Here, we exploit this important feature of miRNA networks to discover vulnerabilities in cancer phenotype, and map miRNA-target relationships across different cancer types. More specifically, we report the results of a functional genomics screen of 1280 miRNA mimics and inhibitors in eight cancer cell lines, and its presentation in a sophisticated interactive data portal. This resource represents the most comprehensive survey of miRNA function in oncology, incorporating breast cancer, prostate cancer and neuroblastoma. A user-friendly web portal couples this experimental data with multiple tools for miRNA target prediction, pathway enrichment analysis and visualization. In addition, the database integrates publicly available gene expression and perturbation data enabling tailored and context-specific analysis of miRNA function in a particular disease. As a proof-of-principle, we use the database and its innovative features to uncover novel determinants of the neuroblastoma malignant phenotype. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Molecular deconstruction, detection, and computational prediction of microenvironment-modulated cellular responses to cancer therapeutics

PubMed Central

LaBarge, Mark A; Parvin, Bahram; Lorens, James B

2014-01-01

The field of bioengineering has pioneered the application of new precision fabrication technologies to model the different geometric, physical or molecular components of tissue microenvironments on solid-state substrata. Tissue engineering approaches building on these advances are used to assemble multicellular mimetic-tissues where cells reside within defined spatial contexts. The functional responses of cells in fabricated microenvironments has revealed a rich interplay between the genome and extracellular effectors in determining cellular phenotypes, and in a number of cases has revealed the dominance of microenvironment over genotype. Precision bioengineered substrata are limited to a few aspects, whereas cell/tissue-derived microenvironments have many undefined components. Thus introducing a computational module may serve to integrate these types of platforms to create reasonable models of drug responses in human tissues. This review discusses how combinatorial microenvironment microarrays and other biomimetic microenvironments have revealed emergent properties of cells in particular microenvironmental contexts, the platforms that can measure phenotypic changes within those contexts, and the computational tools that can unify the microenvironment-imposed functional phenotypes with underlying constellations of proteins and genes. Ultimately we propose that a merger of these technologies will enable more accurate pre-clinical drug discovery. PMID:24582543

Genetic analysis of indefinite division in human cells: Evidence for a cell senescence-related gene(s) on human chromosome 4

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

Yi Ning; Ledbetter, D.H.; Smith, J.R.

1991-07-01

Earlier studies had demonstrated that fusion of normal with immortal human cells yielded hybrids having limited division potential. This indicated that the phenotype of limited proliferation (cellular senescence) is dominant and that immortal cells result from recessive changes in normal growth-regulatory genes. In additional studies, the authors exploited the fact that the immortal phenotype is recessive and, by fusing various immortal human cell lines with each other, identified four complementation groups for indefinite division. Assignment of cell lines to specific groups allowed us to take a focused approach to identify the chromosomes and genes involved in growth regulation that havemore » been modified in immortal cells. They report here that introduction of a normal human chromosome 4 into three immortal cell lines (HeLa, J82, T98G) assigned to complementation group B resulted in loss of proliferation and reversal of the immortal phenotype. No effect on the proliferation potential of cell lines representative of the other complementation groups was observed. This result suggests that a gene(s) involved in cellular senescence and normal growth regulation resides on chromosome 4.« less

Antibiotics induce redox-related physiological alterations as part of their lethality

PubMed Central

Dwyer, Daniel J.; Belenky, Peter A.; Yang, Jason H.; MacDonald, I. Cody; Martell, Jeffrey D.; Takahashi, Noriko; Chan, Clement T. Y.; Lobritz, Michael A.; Braff, Dana; Schwarz, Eric G.; Ye, Jonathan D.; Pati, Mekhala; Vercruysse, Maarten; Ralifo, Paul S.; Allison, Kyle R.; Khalil, Ahmad S.; Ting, Alice Y.; Walker, Graham C.; Collins, James J.

2014-01-01

Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species. This notion has been supported by many groups but has been challenged recently. Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular H2O2 sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress. Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H2O2. We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality. PMID:24803433

[Fanconi anemia: genes and function(s) revisited].

PubMed

Papadopoulo, Dora; Moustacchi, Ethel

2005-01-01

Fanconi anemia (FA), a rare inherited disorder, exhibits a complex phenotype including progressive bone marrow failure, congenital malformations and increased risk of cancers, mainly acute myeloid leukaemia. At the cellular level, FA is characterized by hypersensitivity to DNA cross-linking agents and by high frequencies of induced chromosomal aberrations, a property used for diagnosis. FA results from mutations in one of the eleven FANC (FANCA to FANCJ) genes. Nine of them have been identified. In addition, FANCD1 gene has been shown to be identical to BRCA2, one of the two breast cancer susceptibility genes. Seven of the FANC proteins form a complex, which exists in four different forms depending of its subcellular localisation. Four FANC proteins (D1(BRCA2), D2, I and J) are not associated to the complex. The presence of the nuclear form of the FA core complex is necessary for the mono-ubiquitinylation of FANCD2 protein, a modification required for its re-localization to nuclear foci, likely to be sites of DNA repair. A clue towards understanding the molecular function of the FANC genes comes from the recently identified connection of FANC to the BRCA1, ATM, NBS1 and ATR genes. Two of the FANC proteins (A and D2) directly interact with BRCA1, which in turn interacts with the MRE11/RAD50/NBS1 complex, which is one of the key components in the mechanisms involved in the cellular response to DNA double strand breaks (DSB). Moreover, ATM, a protein kinase that plays a central role in the network of DSB signalling, phosphorylates in vitro and in vivo FANCD2 in response to ionising radiations. Moreover, the NBS1 protein and the monoubiquitinated form of FANCD2 seem to act together in response to DNA crosslinking agents. Taken together with the previously reported impaired DSB and DNA interstrand crosslinks repair in FA cells, the connection of FANC genes to the ATM, ATR, NBS1 and BRCA1 links the FANC genes function to the finely orchestrated network involved in the sensing, signalling and repair of DNA replication-blocking lesions.

Proteomics in Heart Failure: Top-down or Bottom-up?

PubMed Central

Gregorich, Zachery R.; Chang, Ying-Hua; Ge, Ying

2014-01-01

Summary The pathophysiology of heart failure (HF) is diverse, owing to multiple etiologies and aberrations in a number of cellular processes. Therefore, it is essential to understand how defects in the molecular pathways that mediate cellular responses to internal and external stressors function as a system to drive the HF phenotype. Mass spectrometry (MS)-based proteomics strategies have great potential for advancing our understanding of disease mechanisms at the systems level because proteins are the effector molecules for all cell functions and, thus, are directly responsible for determining cell phenotype. Two MS-based proteomics strategies exist: peptide-based bottom-up and protein-based top-down proteomics—each with its own unique strengths and weaknesses for interrogating the proteome. In this review, we will discuss the advantages and disadvantages of bottom-up and top-down MS for protein identification, quantification, and the analysis of post-translational modifications, as well as highlight how both of these strategies have contributed to our understanding of the molecular and cellular mechanisms underlying HF. Additionally, the challenges associated with both proteomics approaches will be discussed and insights will be offered regarding the future of MS-based proteomics in HF research. PMID:24619480

The Conserved ATPase Get3/Arr4 Modulates the Activity of Membrane-Associated Proteins in Saccharomyces cerevisiae

PubMed Central

Auld, Kathryn L.; Hitchcock, Amy L.; Doherty, Hugh K.; Frietze, Seth; Huang, Linda S.; Silver, Pamela A.

2006-01-01

The regulation of cellular membrane dynamics is crucial for maintaining proper cell growth and division. The Cdc48-Npl4-Ufd1 complex is required for several regulated membrane-associated processes as part of the ubiquitin–proteasome system, including ER-associated degradation and the control of lipid composition in yeast. In this study we report the results of a genetic screen in Saccharomyces cerevisiae for extragenic suppressors of a temperature-sensitive npl4 allele and the subsequent analysis of one suppressor, GET3/ARR4. The GET3 gene encodes an ATPase with homology to the regulatory component of the bacterial arsenic pump. Mutants of GET3 rescue several phenotypes of the npl4 mutant and transcription of GET3 is coregulated with the proteasome, illustrating a functional relationship between GET3 and NPL4 in the ubiquitin–proteasome system. We have further found that Get3 biochemically interacts with the trans-membrane domain proteins Get1/Mdm39 and Get2/Rmd7 and that Δget3 is able to suppress phenotypes of get1 and get2 mutants, including sporulation defects. In combination, our characterization of GET3 genetic and biochemical interactions with NPL4, GET1, and GET2 implicates Get3 in multiple membrane-dependent pathways. PMID:16816426

Developmental plasticity of mitochondrial function in American alligators, Alligator mississippiensis

PubMed Central

Crossley, Janna; Elsey, Ruth M.; Dzialowski, Edward M.; Shiels, Holly A.; Crossley, Dane A.

2016-01-01

The effect of hypoxia on cellular metabolism is well documented in adult vertebrates, but information is entirely lacking for embryonic organisms. The effect of hypoxia on embryonic physiology is particularly interesting, as metabolic responses during development may have life-long consequences, due to developmental plasticity. To this end, we investigated the effects of chronic developmental hypoxia on cardiac mitochondrial function in embryonic and juvenile American alligators (Alligator mississippiensis). Alligator eggs were incubated in 21% or 10% oxygen from 20 to 90% of embryonic development. Embryos were either harvested at 90% development or allowed to hatch and then reared in 21% oxygen for 3 yr. Ventricular mitochondria were isolated from embryonic/juvenile alligator hearts. Mitochondrial respiration and enzymatic activities of electron transport chain complexes were measured with a microrespirometer and spectrophotometer, respectively. Developmental hypoxia induced growth restriction and increased relative heart mass, and this phenotype persisted into juvenile life. Embryonic mitochondrial function was not affected by developmental hypoxia, but at the juvenile life stage, animals from hypoxic incubations had lower levels of Leak respiration and higher respiratory control ratios, which is indicative of enhanced mitochondrial efficiency. Our results suggest developmental hypoxia can have life-long consequences for alligator morphology and metabolic function. Further investigations are necessary to reveal the adaptive significance of the enhanced mitochondrial efficiency in the hypoxic phenotype. PMID:27707718

Pregnancy associated plasma protein-A links pregnancy and melanoma progression by promoting cellular migration and invasion

PubMed Central

Prithviraj, Prashanth; Anaka, Matthew; McKeown, Sonja J; Permezel, Michael; Walkiewicz, Marzena

2015-01-01

Melanoma is the most common cancer diagnosed in pregnant women and an aggressive course with poorer outcomes is commonly described during pregnancy or shortly after childbirth. The underlying mechanisms for this are not understood. Here, we report that melanoma migration, invasiveness and progression are promoted by pregnancy-associated plasma protein-A (PAPPA), a pregnancy-associated metalloproteinase produced by the placenta that increases the bioavailability of IGF1 by cleaving it from a circulating complex formed with IGFBP4. We show that PAPPA is widely expressed by metastatic melanoma tumors and is elevated in melanoma cells exhibiting mesenchymal, invasive and label-retaining phenotypes. Notably, inhibition of PAPPA significantly reduced invasion and migration of melanoma cells in vitro and in vivo within the embryonic chicken neural tube. PAPPA-enriched pregnancy serum treatment enhanced melanoma motility in vitro. Furthermore, we report that IGF1 can induce the phenotypic and functional effects of epithelial-to-mesenchymal transition (EMT) in melanoma cells. In this study, we establish a clear relationship between a pregnancy-associated protein PAPPA, melanoma and functional effects mediated through IGF1 that provides a plausible mechanism for accelerated melanoma progression during pregnancy. This opens the possibility of targeting the PAPPA/IGF1 axis therapeutically. PMID:25940796

Pregnancy associated plasma protein-A links pregnancy and melanoma progression by promoting cellular migration and invasion.

PubMed

Prithviraj, Prashanth; Anaka, Matthew; McKeown, Sonja J; Permezel, Michael; Walkiewicz, Marzena; Cebon, Jonathan; Behren, Andreas; Jayachandran, Aparna

2015-06-30

Melanoma is the most common cancer diagnosed in pregnant women and an aggressive course with poorer outcomes is commonly described during pregnancy or shortly after childbirth. The underlying mechanisms for this are not understood. Here, we report that melanoma migration, invasiveness and progression are promoted by Pregnancy-Associated Plasma Protein-A (PAPPA), a pregnancy-associated metalloproteinase produced by the placenta that increases the bioavailability of IGF1 by cleaving it from a circulating complex formed with IGFBP4. We show that PAPPA is widely expressed by metastatic melanoma tumors and is elevated in melanoma cells exhibiting mesenchymal, invasive and label-retaining phenotypes. Notably, inhibition of PAPPA significantly reduced invasion and migration of melanoma cells in vitro and in vivo within the embryonic chicken neural tube. PAPPA-enriched pregnancy serum treatment enhanced melanoma motility in vitro. Furthermore, we report that IGF1 can induce the phenotypic and functional effects of epithelial-to-mesenchymal transition (EMT) in melanoma cells. In this study, we establish a clear relationship between a pregnancy-associated protein PAPPA, melanoma and functional effects mediated through IGF1 that provides a plausible mechanism for accelerated melanoma progression during pregnancy. This opens the possibility of targeting the PAPPA/IGF1 axis therapeutically.

Shifting behaviour: epigenetic reprogramming in eusocial insects.

PubMed

Patalano, Solenn; Hore, Timothy A; Reik, Wolf; Sumner, Seirian

2012-06-01

Epigenetic modifications are ancient and widely utilised mechanisms that have been recruited across fungi, plants and animals for diverse but fundamental biological functions, such as cell differentiation. Recently, a functional DNA methylation system was identified in the honeybee, where it appears to underlie queen and worker caste differentiation. This discovery, along with other insights into the epigenetics of social insects, allows provocative analogies to be drawn between insect caste differentiation and cellular differentiation, particularly in mammals. Developing larvae in social insect colonies are totipotent: they retain the ability to specialise as queens or workers, in a similar way to the totipotent cells of early embryos before they differentiate into specific cell lineages. Further, both differentiating cells and insect castes lose phenotypic plasticity by committing to their lineage, losing the ability to be readily reprogrammed. Hence, a comparison of the epigenetic mechanisms underlying lineage differentiation (and reprogramming) between cells and social insects is worthwhile. Here we develop a conceptual model of how loss and regain of phenotypic plasticity might be conserved for individual specialisation in both cells and societies. This framework forges a novel link between two fields of biological research, providing predictions for a unified approach to understanding the molecular mechanisms underlying biological complexity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Identification of developmentally-specific kinotypes and mechanisms of Varroa mite resistance through whole-organism, kinome analysis of honeybee

PubMed Central

Robertson, Albert J.; Trost, Brett; Scruten, Erin; Robertson, Thomas; Mostajeran, Mohammad; Connor, Wayne; Kusalik, Anthony; Griebel, Philip; Napper, Scott

2014-01-01

Recent investigations associate Varroa destructor (Mesostigmata: Varroidae) parasitism and its associated pathogens and agricultural pesticides with negative effects on colony health, resulting in sporadic global declines in domestic honeybee (Apis mellifera) populations. These events have motivated efforts to develop research tools that can offer insight into the causes of declining bee health as well as identify biomarkers to guide breeding programs. Here we report the development of a bee-specific peptide array for characterizing global cellular kinase activity in whole bee extracts. The arrays reveal distinct, developmentally-specific signaling profiles between bees with differential susceptibility to infestation by Varroa mites. Gene ontology analysis of the differentially phosphorylated peptides indicates that the differential susceptibility to Varroa mite infestation does not reflect compromised immunity; rather, there is evidence for mite-mediated immune suppression within the susceptible phenotype that may reduce the ability of these bees to counter secondary viral infections. This hypothesis is supported by the demonstration of more diverse viral infections in mite-infested, susceptible adult bees. The bee-specific peptide arrays are an effective tool for understanding the molecular basis of this complex phenotype as well as for the discovery and utilization of phosphorylation biomarkers for breeding programs. PMID:24904639

Homophilic and heterophilic polycystin 1 interactions regulate E-cadherin recruitment and junction assembly in MDCK cells

PubMed Central

Streets, Andrew J.; Wagner, Bart E.; Harris, Peter C.; Ward, Christopher J.; Ong, Albert C. M.

2009-01-01

Summary Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited human renal disease and is caused by mutations in two genes, PKD1 (85%) and PKD2 (15%). Cyst epithelial cells are characterised by a complex cellular phenotype including changes in proliferation, apoptosis, basement membrane composition and apicobasal polarity. Since polycystin 1 (PC1), the PKD1 protein, has been located in the basolateral membrane of kidney epithelial cells, we hypothesised that it might have a key role in mediating or stabilising cell-cell interactions. In non-ciliated L929 cells, stable or transient surface expression of the PC1 extracellular domain was sufficient to confer an adhesive phenotype and stimulate junction formation. In MDCK cells, we found that PC1 was recruited to the lateral membranes coincident with E-cadherin within 30 minutes after a `calcium switch'. Recruitment of both proteins was significantly delayed when cells were treated with a PC1 blocking antibody raised to the PKD domains. Finally, PC1 and E-cadherin could be coimmunoprecipitated together from MDCK cells. We conclude that PC1 has a key role in initiating junction formation via initial homophilic interactions and facilitates junction assembly and the establishment of apicobasal polarity by E-cadherin recruitment. PMID:19351715

Dynamic binding of replication protein a is required for DNA repair

PubMed Central

Chen, Ran; Subramanyam, Shyamal; Elcock, Adrian H.; Spies, Maria; Wold, Marc S.

2016-01-01

Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for replication, repair and recombination. High-affinity ssDNA-binding by RPA depends on two DNA binding domains in the large subunit of RPA. Mutation of the evolutionarily conserved aromatic residues in these two domains results in a separation-of-function phenotype: aromatic residue mutants support DNA replication but are defective in DNA repair. We used biochemical and single-molecule analyses, and Brownian Dynamics simulations to determine the molecular basis of this phenotype. Our studies demonstrated that RPA binds to ssDNA in at least two modes characterized by different dissociation kinetics. We also showed that the aromatic residues contribute to the formation of the longer-lived state, are required for stable binding to short ssDNA regions and are needed for RPA melting of partially duplex DNA structures. We conclude that stable binding and/or the melting of secondary DNA structures by RPA is required for DNA repair, including RAD51 mediated DNA strand exchange, but is dispensable for DNA replication. It is likely that the binding modes are in equilibrium and reflect dynamics in the RPA–DNA complex. This suggests that dynamic binding of RPA to DNA is necessary for different cellular functions. PMID:27131385

Glyphosate-induced stiffening of HaCaT keratinocytes, a Peak Force Tapping study on living cells.

PubMed

Heu, Celine; Berquand, Alexandre; Elie-Caille, Celine; Nicod, Laurence

2012-04-01

The skin is the first physiological barrier, with a complex constitution, that provides defensive functions against multiple physical and chemical aggressions. Glyphosate is an extensively used herbicide that has been shown to increase the risk of cancer. Moreover there is increasing evidence suggesting that the mechanical phenotype plays an important role in malignant transformation. Atomic force microscopy (AFM) has emerged within the last decade as a powerful tool for providing a nanometer-scale resolution imaging of biological samples. Peak Force Tapping (PFT) is a newly released AFM-based investigation technique allowing extraction of chemical and mechanical properties from a wide range of samples at a relatively high speed and a high resolution. The present work uses the PFT technology to investigate HaCaT keratinocytes, a human epidermal cell line, and offers an original approach to study chemically-induced changes in the cellular mechanical properties under near-physiological conditions. These experiments indicate glyphosate induces cell membrane stiffening, and the appearance of cytoskeleton structures at a subcellular level, for low cytotoxic concentrations whereas cells exposed to IC50 (inhibitory concentration 50%) treatment exhibit control-like mechanical behavior despite obvious membrane damages. Quercetin, a well-known antioxidant, reverses the glyphosate-induced mechanical phenotype. Copyright © 2012 Elsevier Inc. All rights reserved.

Ovarian phagocyte subsets and their distinct tissue distribution patterns.

PubMed

Carlock, Colin; Wu, Jean; Zhou, Cindy; Ross, April; Adams, Henry; Lou, Yahuan

2013-01-01

Ovarian macrophages, which play critical roles in various ovarian events, are probably derived from multiple lineages. Thus, a systemic classification of their subsets is a necessary first step for determination of their functions. Utilizing antibodies to five phagocyte markers, i.e. IA/IE (major histocompatibility complex class II), F4/80, CD11b (Mac-1), CD11c, and CD68, this study investigated subsets of ovarian phagocytes in mice. Three-color immunofluorescence and flow cytometry, together with morphological observation on isolated ovarian cells, demonstrated complicated phenotypes of ovarian phagocytes. Four macrophage and one dendritic cell subset, in addition to many minor phagocyte subsets, were identified. A dendritic cell-like population with a unique phenotype of CD11c(high)IA/IE⁻F4/80⁻ was also frequently observed. A preliminary age-dependent study showed dramatic increases in IA/IE⁺ macrophages and IA/IE⁺ dendritic cells after puberty. Furthermore, immunofluorescences on ovarian sections showed that each subset displayed a distinct tissue distribution pattern. The pattern for each subset may hint to their role in an ovarian function. In addition, partial isolation of ovarian macrophage subset using CD11b antibodies was attempted. Establishment of this isolation method may have provided us a tool for more precise investigation of each subset's functions at the cellular and molecular levels.

Senescence and cancer: an evolving inflammatory paradox

PubMed Central

Ruhland, Megan; Coussens, Lisa M.; Stewart, Sheila

2015-01-01

The senescent phenotype was first describe in 1961 as a phenomenon characterized by the cessation of cellular division. After years of debate as to whether it represented a tissue culture artifact or an important biological process, it is now appreciated that senescence plays an important role in tumorigenesis. Further, senescence is integral to normal biological processes such as embryogenesis and the maintenance of tissue homeostasis. Now with defined roles in development, wound healing, tumor promotion and tumor suppression, it is not surprising that attention has turned to refining our understanding of the mechanisms behind, and consequences of, the induction of senescence. One emerging role for senescence lies in the ability of senescence to orchestrate an inflammatory responses: factors secreted by senescent cells have been identifed in multiple contexts to modulate various aspects of immune response. As with many of the previously described roles for senescence, the type of inflammation established by the senescence phenotype is varied and dependent on context. In this review, we discuss the current state of the field with a focus on the paradoxical outcomes of the senescence-induced inflammatory responses in the context of cancer. A more complete understanding of senescence and an appreciation for its complexities will be important for eventual development of senescence-targeted therapies. PMID:26453912

Microbial forensics: predicting phenotypic characteristics and environmental conditions from large-scale gene expression profiles.

PubMed

Kim, Minseung; Zorraquino, Violeta; Tagkopoulos, Ilias

2015-03-01

A tantalizing question in cellular physiology is whether the cellular state and environmental conditions can be inferred by the expression signature of an organism. To investigate this relationship, we created an extensive normalized gene expression compendium for the bacterium Escherichia coli that was further enriched with meta-information through an iterative learning procedure. We then constructed an ensemble method to predict environmental and cellular state, including strain, growth phase, medium, oxygen level, antibiotic and carbon source presence. Results show that gene expression is an excellent predictor of environmental structure, with multi-class ensemble models achieving balanced accuracy between 70.0% (±3.5%) to 98.3% (±2.3%) for the various characteristics. Interestingly, this performance can be significantly boosted when environmental and strain characteristics are simultaneously considered, as a composite classifier that captures the inter-dependencies of three characteristics (medium, phase and strain) achieved 10.6% (±1.0%) higher performance than any individual models. Contrary to expectations, only 59% of the top informative genes were also identified as differentially expressed under the respective conditions. Functional analysis of the respective genetic signatures implicates a wide spectrum of Gene Ontology terms and KEGG pathways with condition-specific information content, including iron transport, transferases, and enterobactin synthesis. Further experimental phenotypic-to-genotypic mapping that we conducted for knock-out mutants argues for the information content of top-ranked genes. This work demonstrates the degree at which genome-scale transcriptional information can be predictive of latent, heterogeneous and seemingly disparate phenotypic and environmental characteristics, with far-reaching applications.

The Dictyostelium Carmil Protein Links Capping Protein and the Arp2/3 Complex to Type I Myosins through Their Sh3 Domains

PubMed Central

Jung, Goeh; Remmert, Kirsten; Wu, Xufeng; Volosky, Joanne M.; III, John A. Hammer

2001-01-01

Fusion proteins containing the Src homology (SH)3 domains of Dictyostelium myosin IB (myoB) and IC (myoC) bind a 116-kD protein (p116), plus nine other proteins identified as the seven member Arp2/3 complex, and the α and β subunits of capping protein. Immunoprecipitation reactions indicate that myoB and myoC form a complex with p116, Arp2/3, and capping protein in vivo, that the myosins bind to p116 through their SH3 domains, and that capping protein and the Arp2/3 complex in turn bind to p116. Cloning of p116 reveals a protein dominated by leucine-rich repeats and proline-rich sequences, and indicates that it is a homologue of Acan 125. Studies using p116 fusion proteins confirm the location of the myosin I SH3 domain binding site, implicate NH2-terminal sequences in binding capping protein, and show that a region containing a short sequence found in several G-actin binding proteins, as well as an acidic stretch, can activate Arp2/3-dependent actin nucleation. p116 localizes along with the Arp2/3 complex, myoB, and myoC in dynamic actin-rich cellular extensions, including the leading edge of cells undergoing chemotactic migration, and dorsal, cup-like, macropinocytic extensions. Cells lacking p116 exhibit a striking defect in the formation of these macropinocytic structures, a concomitant reduction in the rate of fluid phase pinocytosis, a significant decrease in the efficiency of chemotactic aggregation, and a decrease in cellular F-actin content. These results identify a complex that links key players in the nucleation and termination of actin filament assembly with a ubiquitous barbed end–directed motor, indicate that the protein responsible for the formation of this complex is physiologically important, and suggest that previously reported myosin I mutant phenotypes in Dictyostelium may be due, at least in part, to defects in the assembly state of actin. We propose that p116 and Acan 125, along with homologues identified in Caenorhabditis elegans, Drosophila, mouse, and man, be named CARMIL proteins, for capping protein, Arp2/3, and myosin I linker. PMID:11425877

Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass Spectrometry

NASA Astrophysics Data System (ADS)

Belov, Arseniy M.; Viner, Rosa; Santos, Marcia R.; Horn, David M.; Bern, Marshall; Karger, Barry L.; Ivanov, Alexander R.

2017-12-01

Native mass spectrometry (MS) is a rapidly advancing field in the analysis of proteins, protein complexes, and macromolecular species of various types. The majority of native MS experiments reported to-date has been conducted using direct infusion of purified analytes into a mass spectrometer. In this study, capillary zone electrophoresis (CZE) was coupled online to Orbitrap mass spectrometers using a commercial sheathless interface to enable high-performance separation, identification, and structural characterization of limited amounts of purified proteins and protein complexes, the latter with preserved non-covalent associations under native conditions. The performance of both bare-fused silica and polyacrylamide-coated capillaries was assessed using mixtures of protein standards known to form non-covalent protein-protein and protein-ligand complexes. High-efficiency separation of native complexes is demonstrated using both capillary types, while the polyacrylamide neutral-coated capillary showed better reproducibility and higher efficiency for more complex samples. The platform was then evaluated for the determination of monoclonal antibody aggregation and for analysis of proteomes of limited complexity using a ribosomal isolate from E. coli. Native CZE-MS, using accurate single stage and tandem-MS measurements, enabled identification of proteoforms and non-covalent complexes at femtomole levels. This study demonstrates that native CZE-MS can serve as an orthogonal and complementary technique to conventional native MS methodologies with the advantages of low sample consumption, minimal sample processing and losses, and high throughput and sensitivity. This study presents a novel platform for analysis of ribosomes and other macromolecular complexes and organelles, with the potential for discovery of novel structural features defining cellular phenotypes (e.g., specialized ribosomes). [Figure not available: see fulltext.

Intracellular Localization and Cellular Factors Interaction of HTLV-1 and HTLV-2 Tax Proteins: Similarities and Functional Differences

PubMed Central

Bertazzoni, Umberto; Turci, Marco; Avesani, Francesca; Di Gennaro, Gianfranco; Bidoia, Carlo; Romanelli, Maria Grazia

2011-01-01

Human T-lymphotropic viruses type 1 (HTLV-1) and type 2 (HTLV-2) present very similar genomic structures but HTLV-1 is more pathogenic than HTLV-2. Is this difference due to their transactivating Tax proteins, Tax-1 and Tax-2, which are responsible for viral and cellular gene activation? Do Tax-1 and Tax-2 differ in their cellular localization and in their interaction pattern with cellular factors? In this review, we summarize Tax-1 and Tax-2 structural and phenotypic properties, their interaction with factors involved in signal transduction and their localization-related behavior within the cell. Special attention will be given to the distinctions between Tax-1 and Tax-2 that likely play an important role in their transactivation activity. PMID:21994745

Mitochondria targeting by environmental stressors: Implications for redox cellular signaling.

PubMed

Blajszczak, Chuck; Bonini, Marcelo G

2017-11-01

Mitochondria are cellular powerhouses as well as metabolic and signaling hubs regulating diverse cellular functions, from basic physiology to phenotypic fate determination. It is widely accepted that reactive oxygen species (ROS) generated in mitochondria participate in the regulation of cellular signaling, and that some mitochondria chronically operate at a high ROS baseline. However, it is not completely understood how mitochondria adapt to persistently high ROS states and to environmental stressors that disturb the redox balance. Here we will review some of the current concepts regarding how mitochondria resist oxidative damage, how they are replaced when excessive oxidative damage compromises function, and the effect of environmental toxicants (i.e. heavy metals) on the regulation of mitochondrial ROS (mtROS) production and subsequent impact. Copyright © 2017 Elsevier B.V. All rights reserved.

Gene regulatory networks and the underlying biology of developmental toxicity

EPA Science Inventory

Embryonic cells are specified by large-scale networks of functionally linked regulatory genes. Knowledge of the relevant gene regulatory networks is essential for understanding phenotypic heterogeneity that emerges from disruption of molecular functions, cellular processes or sig...

Human HOIP and LUBAC deficiency underlies autoinflammation, immunodeficiency, amylopectinosis, and lymphangiectasia

PubMed Central

Boisson, Bertrand; Laplantine, Emmanuel; Dobbs, Kerry; Cobat, Aurélie; Tarantino, Nadine; Hazen, Melissa; Lidov, Hart G.W.; Hopkins, Gregory; Du, Likun; Belkadi, Aziz; Chrabieh, Maya; Itan, Yuval; Picard, Capucine; Fournet, Jean-Christophe; Eibel, Hermann; Tsitsikov, Erdyni; Pai, Sung-Yun; Abel, Laurent; Al-Herz, Waleed; Israel, Alain

2015-01-01

Inherited, complete deficiency of human HOIL-1, a component of the linear ubiquitination chain assembly complex (LUBAC), underlies autoinflammation, infections, and amylopectinosis. We report the clinical description and molecular analysis of a novel inherited disorder of the human LUBAC complex. A patient with multiorgan autoinflammation, combined immunodeficiency, subclinical amylopectinosis, and systemic lymphangiectasia, is homozygous for a mutation in HOIP, the gene encoding the catalytic component of LUBAC. The missense allele (L72P, in the PUB domain) is at least severely hypomorphic, as it impairs HOIP expression and destabilizes the whole LUBAC complex. Linear ubiquitination and NF-κB activation are impaired in the patient’s fibroblasts stimulated by IL-1β or TNF. In contrast, the patient’s monocytes respond to IL-1β more vigorously than control monocytes. However, the activation and differentiation of the patient’s B cells are impaired in response to CD40 engagement. These cellular and clinical phenotypes largely overlap those of HOIL-1-deficient patients. Clinical differences between HOIL-1- and HOIP-mutated patients may result from differences between the mutations, the loci, or other factors. Our findings show that human HOIP is essential for the assembly and function of LUBAC and for various processes governing inflammation and immunity in both hematopoietic and nonhematopoietic cells. PMID:26008899

Thiopeptide antibiotics stimulate biofilm formation in Bacillus subtilis.

PubMed

Bleich, Rachel; Watrous, Jeramie D; Dorrestein, Pieter C; Bowers, Albert A; Shank, Elizabeth A

2015-03-10

Bacteria have evolved the ability to produce a wide range of structurally complex natural products historically called "secondary" metabolites. Although some of these compounds have been identified as bacterial communication cues, more frequently natural products are scrutinized for antibiotic activities that are relevant to human health. However, there has been little regard for how these compounds might otherwise impact the physiology of neighboring microbes present in complex communities. Bacillus cereus secretes molecules that activate expression of biofilm genes in Bacillus subtilis. Here, we use imaging mass spectrometry to identify the thiocillins, a group of thiazolyl peptide antibiotics, as biofilm matrix-inducing compounds produced by B. cereus. We found that thiocillin increased the population of matrix-producing B. subtilis cells and that this activity could be abolished by multiple structural alterations. Importantly, a mutation that eliminated thiocillin's antibiotic activity did not affect its ability to induce biofilm gene expression in B. subtilis. We go on to show that biofilm induction appears to be a general phenomenon of multiple structurally diverse thiazolyl peptides and use this activity to confirm the presence of thiazolyl peptide gene clusters in other bacterial species. Our results indicate that the roles of secondary metabolites initially identified as antibiotics may have more complex effects--acting not only as killing agents, but also as specific modulators of microbial cellular phenotypes.

A Kinome RNAi Screen in Drosophila Identifies Novel Genes Interacting with Lgl, aPKC, and Crb Cell Polarity Genes in Epithelial Tissues.

PubMed

Parsons, Linda M; Grzeschik, Nicola A; Amaratunga, Kasun; Burke, Peter; Quinn, Leonie M; Richardson, Helena E

2017-08-07

In both Drosophila melanogaster and mammalian systems, epithelial structure and underlying cell polarity are essential for proper tissue morphogenesis and organ growth. Cell polarity interfaces with multiple cellular processes that are regulated by the phosphorylation status of large protein networks. To gain insight into the molecular mechanisms that coordinate cell polarity with tissue growth, we screened a boutique collection of RNAi stocks targeting the kinome for their capacity to modify Drosophila "cell polarity" eye and wing phenotypes. Initially, we identified kinase or phosphatase genes whose depletion modified adult eye phenotypes associated with the manipulation of cell polarity complexes (via overexpression of Crb or aPKC). We next conducted a secondary screen to test whether these cell polarity modifiers altered tissue overgrowth associated with depletion of Lgl in the wing. These screens identified Hippo, Jun kinase (JNK), and Notch signaling pathways, previously linked to cell polarity regulation of tissue growth. Furthermore, novel pathways not previously connected to cell polarity regulation of tissue growth were identified, including Wingless (Wg/Wnt), Ras, and lipid/Phospho-inositol-3-kinase (PI3K) signaling pathways. Additionally, we demonstrated that the "nutrient sensing" kinases Salt Inducible Kinase 2 and 3 ( SIK2 and 3 ) are potent modifiers of cell polarity phenotypes and regulators of tissue growth. Overall, our screen has revealed novel cell polarity-interacting kinases and phosphatases that affect tissue growth, providing a platform for investigating molecular mechanisms coordinating cell polarity and tissue growth during development. Copyright © 2017 Parsons et al.

Genetic variants in Alzheimer disease – molecular and brain network approaches

PubMed Central

Gaiteri, Chris; Mostafavi, Sara; Honey, Christopher; De Jager, Philip L.; Bennett, David A.

2016-01-01

Genetic studies in late-onset Alzheimer disease (LOAD) are aimed at identifying core disease mechanisms and providing potential biomarkers and drug candidates to improve clinical care for AD. However, due to the complexity of LOAD, including pathological heterogeneity and disease polygenicity, extracting actionable guidance from LOAD genetics has been challenging. Past attempts to summarize the effects of LOAD-associated genetic variants have used pathway analysis and collections of small-scale experiments to hypothesize functional convergence across several variants. In this review, we discuss how the study of molecular, cellular and brain networks provides additional information on the effect of LOAD-associated genetic variants. We then discuss emerging combinations of omic data types in multiscale models, which provide a more comprehensive representation of the effect of LOAD-associated genetic variants at multiple biophysical scales. Further, we highlight the clinical potential of mechanistically coupling genetic variants and disease phenotypes with multiscale brain models. PMID:27282653

CHO microRNA engineering is growing up: Recent successes and future challenges☆

PubMed Central

Jadhav, Vaibhav; Hackl, Matthias; Druz, Aliaksandr; Shridhar, Smriti; Chung, Cheng-Yu; Heffner, Kelley M.; Kreil, David P.; Betenbaugh, Mike; Shiloach, Joseph; Barron, Niall; Grillari, Johannes; Borth, Nicole

2013-01-01

microRNAs with their ability to regulate complex pathways that control cellular behavior and phenotype have been proposed as potential targets for cell engineering in the context of optimization of biopharmaceutical production cell lines, specifically of Chinese Hamster Ovary cells. However, until recently, research was limited by a lack of genomic sequence information on this industrially important cell line. With the publication of the genomic sequence and other relevant data sets for CHO cells since 2011, the doors have been opened for an improved understanding of CHO cell physiology and for the development of the necessary tools for novel engineering strategies. In the present review we discuss both knowledge on the regulatory mechanisms of microRNAs obtained from other biological models and proof of concepts already performed on CHO cells, thus providing an outlook of potential applications of microRNA engineering in production cell lines. PMID:23916872

Of mice and men: molecular genetics of congenital heart disease.

PubMed

Andersen, Troels Askhøj; Troelsen, Karin de Linde Lind; Larsen, Lars Allan

2014-04-01

Congenital heart disease (CHD) affects nearly 1 % of the population. It is a complex disease, which may be caused by multiple genetic and environmental factors. Studies in human genetics have led to the identification of more than 50 human genes, involved in isolated CHD or genetic syndromes, where CHD is part of the phenotype. Furthermore, mapping of genomic copy number variants and exome sequencing of CHD patients have led to the identification of a large number of candidate disease genes. Experiments in animal models, particularly in mice, have been used to verify human disease genes and to gain further insight into the molecular pathology behind CHD. The picture emerging from these studies suggest that genetic lesions associated with CHD affect a broad range of cellular signaling components, from ligands and receptors, across down-stream effector molecules to transcription factors and co-factors, including chromatin modifiers.

Trash to treasure: production of biofuels and commodity chemicals via syngas fermenting microorganisms.

PubMed

Latif, Haythem; Zeidan, Ahmad A; Nielsen, Alex T; Zengler, Karsten

2014-06-01

Fermentation of syngas is a means through which unutilized organic waste streams can be converted biologically into biofuels and commodity chemicals. Despite recent advances, several issues remain which limit implementation of industrial-scale syngas fermentation processes. At the cellular level, the energy conservation mechanism of syngas fermenting microorganisms has not yet been entirely elucidated. Furthermore, there was a lack of genetic tools to study and ultimately enhance their metabolic capabilities. Recently, substantial progress has been made in understanding the intricate energy conservation mechanisms of these microorganisms. Given the complex relationship between energy conservation and metabolism, strain design greatly benefits from systems-level approaches. Numerous genetic manipulation tools have also been developed, paving the way for the use of metabolic engineering and systems biology approaches. Rational strain designs can now be deployed resulting in desirable phenotypic traits for large-scale production. Copyright © 2013 Elsevier Ltd. All rights reserved.

Comparison of the Virulence-Associated Phenotypes of Five Species of Acinetobacter baumannii Complex.

PubMed

Na, In Young; Chung, Eun Seon; Jung, Chang-Yun; Kim, Dae Hun; Shin, Juyoun; Kang, KyeongJin; Kim, Seong-Tae; Ko, Kwan Soo

2016-01-01

In this study, we compared the virulence-associated factors of Acinetobacter baumannii complex species. Sixty-three isolates of five A. baumannii complex species, including 19 A. baumannii, 15 A. nosocomialis, 13 A. seifertii, 13 A. pittii, and 3 A. calcoaceticus isolates, were included in this study. For all isolates, biofilm formation, A549 cell adherence, resistance to normal human serum, and motility were evaluated. A. baumannii complex isolates showed diversity in biofilm formation, A549 cell adherence, and serum resistance, and no strong positive relationships among these virulence characteristics. However, A. seifertii showed relatively consistent virulence-associated phenotypes. In addition, A. baumannii clone ST110 exhibited consistently high virulence-associated phenotypes. Motility was observed in seven isolates, and all four A. baumannii ST110 isolates showed twitching motility. Although some inconsistencies in virulence-associated phenotypes were seen, high virulence characteristics were observed in A. seifertii, which has been mainly reported in Korea and shows high rates of colistin resistance.

Differential cellular responses by oncogenic levels of c-Myc expression in long-term confluent retinal pigment epithelial cells.

PubMed

Wang, Yiping; Cheng, Xiangdong; Samma, Muhammad Kaleem; Kung, Sam K P; Lee, Clement M; Chiu, Sung Kay

2018-06-01

c-Myc is a highly pleiotropic transcription factor known to control cell cycle progression, apoptosis, and cellular transformation. Normally, ectopic expression of c-Myc is associated with promoting cell proliferation or triggering cell death via activating p53. However, it is not clear how the levels of c-Myc lead to different cellular responses. Here, we generated a series of stable RPE cell clones expressing c-Myc at different levels, and found that consistent low level of c-Myc induced cellular senescence by activating AP4 in post-confluent RPE cells, while the cells underwent cell death at high level of c-Myc. In addition, high level of c-Myc could override the effect of AP4 on cellular senescence. Further knockdown of AP4 abrogated senescence-like phenotype in cells expressing low level of c-Myc, and accelerated cell death in cells with medium level of c-Myc, indicating that AP4 was required for cellular senescence induced by low level of c-Myc.

Chimeric Protein Complexes in Hybrid Species Generate Novel Phenotypes

PubMed Central

Piatkowska, Elzbieta M.; Naseeb, Samina; Knight, David; Delneri, Daniela

2013-01-01

Hybridization between species is an important mechanism for the origin of novel lineages and adaptation to new environments. Increased allelic variation and modification of the transcriptional network are the two recognized forces currently deemed to be responsible for the phenotypic properties seen in hybrids. However, since the majority of the biological functions in a cell are carried out by protein complexes, inter-specific protein assemblies therefore represent another important source of natural variation upon which evolutionary forces can act. Here we studied the composition of six protein complexes in two different Saccharomyces “sensu stricto” hybrids, to understand whether chimeric interactions can be freely formed in the cell in spite of species-specific co-evolutionary forces, and whether the different types of complexes cause a change in hybrid fitness. The protein assemblies were isolated from the hybrids via affinity chromatography and identified via mass spectrometry. We found evidence of spontaneous chimericity for four of the six protein assemblies tested and we showed that different types of complexes can cause a variety of phenotypes in selected environments. In the case of TRP2/TRP3 complex, the effect of such chimeric formation resulted in the fitness advantage of the hybrid in an environment lacking tryptophan, while only one type of parental combination of the MBF complex allowed the hybrid to grow under respiratory conditions. These phenotypes were dependent on both genetic and environmental backgrounds. This study provides empirical evidence that chimeric protein complexes can freely assemble in cells and reveals a new mechanism to generate phenotypic novelty and plasticity in hybrids to complement the genomic innovation resulting from gene duplication. The ability to exchange orthologous members has also important implications for the adaptation and subsequent genome evolution of the hybrids in terms of pattern of gene loss. PMID:24137105

Assessing the value of phenotypic information from non-genotyped animals for QTL mapping of complex traits in real and simulated populations.

PubMed

Melo, Thaise P; Takada, Luciana; Baldi, Fernando; Oliveira, Henrique N; Dias, Marina M; Neves, Haroldo H R; Schenkel, Flavio S; Albuquerque, Lucia G; Carvalheiro, Roberto

2016-06-21

QTL mapping through genome-wide association studies (GWAS) is challenging, especially in the case of low heritability complex traits and when few animals possess genotypic and phenotypic information. When most of the phenotypic information is from non-genotyped animals, GWAS can be performed using the weighted single-step GBLUP (WssGBLUP) method, which permits to combine all available information, even that of non-genotyped animals. However, it is not clear to what extent phenotypic information from non-genotyped animals increases the power of QTL detection, and whether factors such as the extent of linkage disequilibrium (LD) in the population and weighting SNPs in WssGBLUP affect the importance of using information from non-genotyped animals in GWAS. These questions were investigated in this study using real and simulated data. Analysis of real data showed that the use of phenotypes of non-genotyped animals affected SNP effect estimates and, consequently, QTL mapping. Despite some coincidence, the most important genomic regions identified by the analyses, either using or ignoring phenotypes of non-genotyped animals, were not the same. The simulation results indicated that the inclusion of all available phenotypic information, even that of non-genotyped animals, tends to improve QTL detection for low heritability complex traits. For populations with low levels of LD, this trend of improvement was less pronounced. Stronger shrinkage on SNPs explaining lower variance was not necessarily associated with better QTL mapping. The use of phenotypic information from non-genotyped animals in GWAS may improve the ability to detect QTL for low heritability complex traits, especially in populations in which the level of LD is high.

Phenotypic Diagnosis of Lineage and Differentiation During Sake Yeast Breeding

PubMed Central

Ohnuki, Shinsuke; Okada, Hiroki; Friedrich, Anne; Kanno, Yoichiro; Goshima, Tetsuya; Hasuda, Hirokazu; Inahashi, Masaaki; Okazaki, Naoto; Tamura, Hiroyasu; Nakamura, Ryo; Hirata, Dai; Fukuda, Hisashi; Shimoi, Hitoshi; Kitamoto, Katsuhiko; Watanabe, Daisuke; Schacherer, Joseph; Akao, Takeshi; Ohya, Yoshikazu

2017-01-01

Sake yeast was developed exclusively in Japan. Its diversification during breeding remains largely uncharacterized. To evaluate the breeding processes of the sake lineage, we thoroughly investigated the phenotypes and differentiation of 27 sake yeast strains using high-dimensional, single-cell, morphological phenotyping. Although the genetic diversity of the sake yeast lineage is relatively low, its morphological diversity has expanded substantially compared to that of the Saccharomyces cerevisiae species as a whole. Evaluation of the different types of breeding processes showed that the generation of hybrids (crossbreeding) has more profound effects on cell morphology than the isolation of mutants (mutation breeding). Analysis of phenotypic robustness revealed that some sake yeast strains are more morphologically heterogeneous, possibly due to impairment of cellular network hubs. This study provides a new perspective for studying yeast breeding genetics and micro-organism breeding strategies. PMID:28642365

Structure and composition of the courtship phenotype in the bird of paradise Parotia lawesii (Aves: Paradisaeidae).

PubMed

Scholes, Edwin

2008-01-01

Ethology is rooted in the idea that behavior is composed of discrete units and sub-units that can be compared among taxa in a phylogenetic framework. This means that behavior, like morphology and genes, is inherently modular. Yet, the concept of modularity is not well integrated into how we envision the behavioral components of phenotype. Understanding ethological modularity, and its implications for animal phenotype organization and evolution, requires that we construct interpretive schemes that permit us to examine it. In this study, I describe the structure and composition of a complex part of the behavioral phenotype of Parotia lawesii Ramsay, 1885--a bird of paradise (Aves: Paradisaeidae) from the forests of eastern New Guinea. I use archived voucher video clips, photographic ethograms, and phenotype ontology diagrams to describe the modular units comprising courtship at various levels of integration. Results show P. lawesii to have 15 courtship and mating behaviors (11 males, 4 females) hierarchically arranged within a complex seven-level structure. At the finest level examined, male displays are comprised of 49 modular sub-units (elements) differentially employed to form more complex modular units (phases and versions) at higher-levels of integration. With its emphasis on hierarchical modularity, this study provides an important conceptual framework for understanding courtship-related phenotypic complexity and provides a solid basis for comparative study of the genus Parotia.

β-Catenin Serves as a Clutch between Low and High Intercellular E-Cadherin Bond Strengths

PubMed Central

Bajpai, Saumendra; Feng, Yunfeng; Wirtz, Denis; Longmore, Gregory D.

2013-01-01

A wide range of invasive pathological outcomes originate from the loss of epithelial phenotype and involve either loss of function or downregulation of transmembrane adhesive receptor complexes, including Ecadherin (Ecad) and binding partners β-catenin and α-catenin at adherens junctions. Cellular pathways regulating wild-type β-catenin level, or direct mutations in β-catenin that affect the turnover of the protein have been shown to contribute to cancer development, through induction of uncontrolled proliferation of transformed tumor cells, particularly in colon cancer. Using single-molecule force spectroscopy, we show that depletion of β-catenin or the prominent cancer-related S45 deletion mutation in β-catenin present in human colon cancers both weaken tumor intercellular Ecad/Ecad bond strength and diminishes the capacity of specific extracellular matrix proteins—including collagen I, collagen IV, and laminin V—to modulate intercellular Ecad/Ecad bond strength through α-catenin and the kinase activity of glycogen synthase kinase 3 (GSK-3β). Thus, in addition to regulating tumor cell proliferation, cancer-related mutations in β-catenin can influence tumor progression by weakening the adhesion of tumor cells to one another through reduced individual Ecad/Ecad bond strength and cellular adhesion to specific components of the extracellular matrix and the basement membrane. PMID:24268141

Three-dimensional scaffolding to investigate neuronal derivatives of human embryonic stem cells.

PubMed

Soman, Pranav; Tobe, Brian T D; Lee, Jin Woo; Winquist, Alicia M; Singec, Ilyas; Vecchio, Kenneth S; Snyder, Evan Y; Chen, Shaochen

2012-10-01

Access to unlimited numbers of live human neurons derived from stem cells offers unique opportunities for in vitro modeling of neural development, disease-related cellular phenotypes, and drug testing and discovery. However, to develop informative cellular in vitro assays, it is important to consider the relevant in vivo environment of neural tissues. Biomimetic 3D scaffolds are tools to culture human neurons under defined mechanical and physico-chemical properties providing an interconnected porous structure that may potentially enable a higher or more complex organization than traditional two-dimensional monolayer conditions. It is known that even minor variations in the internal geometry and mechanical properties of 3D scaffolds can impact cell behavior including survival, growth, and cell fate choice. In this report, we describe the design and engineering of 3D synthetic polyethylene glycol (PEG)-based and biodegradable gelatin-based scaffolds generated by a free form fabrication technique with precise internal geometry and elastic stiffnesses. We show that human neurons, derived from human embryonic stem (hESC) cells, are able to adhere to these scaffolds and form organoid structures that extend in three dimensions as demonstrated by confocal and electron microscopy. Future refinements of scaffold structure, size and surface chemistries may facilitate long term experiments and designing clinically applicable bioassays.

A damaged DNA binding protein 2 mutation disrupting interaction with proliferating-cell nuclear antigen affects DNA repair and confers proliferation advantage.

PubMed

Perucca, Paola; Mocchi, Roberto; Guardamagna, Isabella; Bassi, Elisabetta; Sommatis, Sabrina; Nardo, Tiziana; Prosperi, Ennio; Stivala, Lucia Anna; Cazzalini, Ornella

2018-06-01

In mammalian cells, Nucleotide Excision Repair (NER) plays a role in removing DNA damage induced by UV radiation. In Global Genome-NER subpathway, DDB2 protein forms a complex with DDB1 (UV-DDB), recognizing photolesions. During DNA repair, DDB2 interacts directly with PCNA through a conserved region in N-terminal tail and this interaction is important for DDB2 degradation. In this work, we sought to investigate the role of DDB2-PCNA association in DNA repair and cell proliferation after UV-induced DNA damage. To this end, stable clones expressing DDB2 Wt and DDB2 PCNA- were used. We have found that cells expressing a mutant DDB2 show inefficient photolesions removal, and a concomitant lack of binding to damaged DNA in vitro. Unexpected cellular behaviour after DNA damage, such as UV-resistance, increased cell growth and motility were found in DDB2 PCNA- stable cell clones, in which the most significant defects in cell cycle checkpoint were observed, suggesting a role in the new cellular phenotype. Based on these findings, we propose that DDB2-PCNA interaction may contribute to a correct DNA damage response for maintaining genome integrity. Copyright © 2018 Elsevier B.V. All rights reserved.

Disease models for the development of therapies for lysosomal storage diseases.

PubMed

Xu, Miao; Motabar, Omid; Ferrer, Marc; Marugan, Juan J; Zheng, Wei; Ottinger, Elizabeth A

2016-05-01

Lysosomal storage diseases (LSDs) are a group of rare diseases in which the function of the lysosome is disrupted by the accumulation of macromolecules. The complexity underlying the pathogenesis of LSDs and the small, often pediatric, population of patients make the development of therapies for these diseases challenging. Current treatments are only available for a small subset of LSDs and have not been effective at treating neurological symptoms. Disease-relevant cellular and animal models with high clinical predictability are critical for the discovery and development of new treatments for LSDs. In this paper, we review how LSD patient primary cells and induced pluripotent stem cell-derived cellular models are providing novel assay systems in which phenotypes are more similar to those of the human LSD physiology. Furthermore, larger animal disease models are providing additional tools for evaluation of the efficacy of drug candidates. Early predictors of efficacy and better understanding of disease biology can significantly affect the translational process by focusing efforts on those therapies with the higher probability of success, thus decreasing overall time and cost spent in clinical development and increasing the overall positive outcomes in clinical trials. © 2016 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals Inc. on behalf of The New York Academy of Sciences.

Mutations in the murine homologue of TUBB5 cause microcephaly by perturbing cell cycle progression and inducing p53-associated apoptosis.

PubMed

Breuss, Martin; Fritz, Tanja; Gstrein, Thomas; Chan, Kelvin; Ushakova, Lyubov; Yu, Nuo; Vonberg, Frederick W; Werner, Barbara; Elling, Ulrich; Keays, David A

2016-04-01

Microtubules play a crucial role in the generation, migration and differentiation of nascent neurons in the developing vertebrate brain. Mutations in the constituents of microtubules, the tubulins, are known to cause an array of neurological disorders, including lissencephaly, polymicrogyria and microcephaly. In this study we explore the genetic and cellular mechanisms that cause TUBB5-associated microcephaly by exploiting two new mouse models: a conditional E401K knock-in, and a conditional knockout animal. These mice present with profound microcephaly due to a loss of upper-layer neurons that correlates with massive apoptosis and upregulation of p53. This phenotype is associated with a delay in cell cycle progression and ectopic DNA elements in progenitors, which is dependent on the dosage of functional Tubb5. Strikingly, we report ectopic Sox2-positive progenitors and defects in spindle orientation in our knock-in mouse line, which are absent in knockout animals. This work sheds light on the functional repertoire of Tubb5, reveals that the E401K mutation acts by a complex mechanism, and demonstrates that the cellular pathology driving TUBB5-associated microcephaly is cell death. © 2016. Published by The Company of Biologists Ltd.

Analytical approaches to the diagnosis and treatment of aging and aging-related disease: redox status and proteomics.

PubMed

Calabrese, V; Dattilo, S; Petralia, A; Parenti, R; Pennisi, M; Koverech, G; Calabrese, V; Graziano, A; Monte, I; Maiolino, L; Ferreri, T; Calabrese, E J

2015-05-01

Basal levels of oxidants are indispensible for redox signaling to produce adaptive cellular responses such as vitagenes linked to cell survival; however, at higher levels, they are detrimental to cells, contributing to aging and to the pathogenesis of numerous age-related diseases. Aging is a complex systemic process and the major gap in aging research reminds the insufficient knowledge about pathways shifting from normal "healthy" aging to disease-associated pathological aging. The major complication of normal "healthy" aging is in fact the increasing risk of age-related diseases such as cardiovascular diseases, diabetes mellitus, and neurodegenerative pathologies that can adversely affect the quality of life in general, with enhanced incidences of comorbidities and mortality. In this context, global "omics" approaches may help to dissect and fully study the cellular and molecular mechanisms of aging and age-associated processes. The proteome, being more close to the phenotype than the transcriptome and more stable than the metabolome, represents the most promising "omics" field in aging research. In the present study, we exploit recent advances in the redox biology of aging and discuss the potential of proteomics approaches as innovative tools for monitoring at the proteome level the extent of protein oxidative insult and related modifications with the identification of targeted proteins.

Induction of appropriate Th-cell phenotypes: cellular decision-making in heterogeneous environments.

PubMed

van den Ham, H-J; Andeweg, A C; de Boer, R J

2013-11-01

Helper T (Th)-cell differentiation is a key event in the development of the adaptive immune response. By the production of a range of cytokines, Th cells determine the type of immune response that is raised against an invading pathogen. Th cells can adopt many different phenotypes, and Th-cell phenotype decision-making is crucial in mounting effective host responses. This review discusses the different Th-cell phenotypes that have been identified and how Th cells adopt a particular phenotype. The regulation of Th-cell phenotypes has been studied extensively using mathematical models, which have explored the role of regulatory mechanisms such as autocrine cytokine signalling and cross-inhibition between self-activating transcription factors. At the single cell level, Th responses tend to be heterogeneous, but corrections can be made soon after T-cell activation. Although pathogens and the innate immune system provide signals that direct the induction of Th-cell phenotypes, these instructive mechanisms could be easily subverted by pathogens. We discuss that a model of success-driven feedback would select the most appropriate phenotype for clearing a pathogen. Given the heterogeneity in the induction phase of the Th response, such a success-driven feedback loop would allow the selection of effective Th-cell phenotypes while terminating incorrect responses. © 2013 John Wiley & Sons Ltd.

Co-delivery of doxorubicin and siRNA using octreotide-conjugated gold nanorods for targeted neuroendocrine cancer therapy

NASA Astrophysics Data System (ADS)

Xiao, Yuling; Jaskula-Sztul, Renata; Javadi, Alireza; Xu, Wenjin; Eide, Jacob; Dammalapati, Ajitha; Kunnimalaiyaan, Muthusamy; Chen, Herbert; Gong, Shaoqin

2012-10-01

A multifunctional gold (Au) nanorod (NR)-based nanocarrier capable of co-delivering small interfering RNA (siRNA) against achaete-scute complex-like 1 (ASCL1) and an anticancer drug (doxorubicin (DOX)) specifically to neuroendocrine (NE) cancer cells was developed and characterized for combined chemotherapy and siRNA-mediated gene silencing. The Au NR was conjugated with (1) DOX, an anticancer drug, via a pH-labile hydrazone linkage to enable pH-controlled drug release, (2) polyarginine, a cationic polymer for complexing siRNA, and (3) octreotide (OCT), a tumor-targeting ligand, to specifically target NE cancer cells with overexpressed somatostatin receptors. The Au NR-based nanocarriers exhibited a uniform size distribution as well as pH-sensitive drug release. The OCT-conjugated Au NR-based nanocarriers (Au-DOX-OCT, targeted) exhibited a much higher cellular uptake in a human carcinoid cell line (BON cells) than non-targeted Au NR-based nanocarriers (Au-DOX) as measured by both flow cytometry and confocal laser scanning microscopy (CLSM). Moreover, Au-DOX-OCT-ASCL1 siRNA (Au-DOX-OCT complexed with ASCL1 siRNA) resulted in significantly higher gene silencing in NE cancer cells than Au-DOX-ASCL1 siRNA (non-targeted Au-DOX complexed with ASCL1 siRNA) as measured by an immunoblot analysis. Additionally, Au-DOX-OCT-ASCL1 siRNA was the most efficient nanocarrier at altering the NE phenotype of NE cancer cells and showed the strongest anti-proliferative effect. Thus, combined chemotherapy and RNA silencing using NE tumor-targeting Au NR-based nanocarriers could potentially enhance the therapeutic outcomes in treating NE cancers.A multifunctional gold (Au) nanorod (NR)-based nanocarrier capable of co-delivering small interfering RNA (siRNA) against achaete-scute complex-like 1 (ASCL1) and an anticancer drug (doxorubicin (DOX)) specifically to neuroendocrine (NE) cancer cells was developed and characterized for combined chemotherapy and siRNA-mediated gene silencing. The Au NR was conjugated with (1) DOX, an anticancer drug, via a pH-labile hydrazone linkage to enable pH-controlled drug release, (2) polyarginine, a cationic polymer for complexing siRNA, and (3) octreotide (OCT), a tumor-targeting ligand, to specifically target NE cancer cells with overexpressed somatostatin receptors. The Au NR-based nanocarriers exhibited a uniform size distribution as well as pH-sensitive drug release. The OCT-conjugated Au NR-based nanocarriers (Au-DOX-OCT, targeted) exhibited a much higher cellular uptake in a human carcinoid cell line (BON cells) than non-targeted Au NR-based nanocarriers (Au-DOX) as measured by both flow cytometry and confocal laser scanning microscopy (CLSM). Moreover, Au-DOX-OCT-ASCL1 siRNA (Au-DOX-OCT complexed with ASCL1 siRNA) resulted in significantly higher gene silencing in NE cancer cells than Au-DOX-ASCL1 siRNA (non-targeted Au-DOX complexed with ASCL1 siRNA) as measured by an immunoblot analysis. Additionally, Au-DOX-OCT-ASCL1 siRNA was the most efficient nanocarrier at altering the NE phenotype of NE cancer cells and showed the strongest anti-proliferative effect. Thus, combined chemotherapy and RNA silencing using NE tumor-targeting Au NR-based nanocarriers could potentially enhance the therapeutic outcomes in treating NE cancers. Electronic supplementary information (ESI) available: Additional flow cytometry histogram profiles of DOX fluorescence and ASCL1 knockdown results. See DOI: 10.1039/c2nr31853a

Defects in lymphocyte telomere homeostasis contribute to cellular immune phenotype in patients with cartilage-hair hypoplasia.

PubMed

Aubert, Geraldine; Strauss, Kevin A; Lansdorp, Peter M; Rider, Nicholas L

2017-10-01

Mutations in the long noncoding RNA RNase component of the mitochondrial RNA processing endoribonuclease (RMRP) give rise to the autosomal recessive condition cartilage-hair hypoplasia (CHH). The CHH disease phenotype has some overlap with dyskeratosis congenita, a well-known "telomere disorder." RMRP binds the telomerase reverse transcriptase (catalytic subunit) in some cell lines, raising the possibility that RMRP might play a role in telomere biology. We sought to determine whether a telomere phenotype is present in immune cells from patients with CHH and explore mechanisms underlying these observations. We assessed proliferative capacity and telomere length using flow-fluorescence in situ hybridization (in situ hybridization and flow cytometry) of primary lymphocytes from patients with CHH, carrier relatives, and control subjects. The role of telomerase holoenzyme components in gene expression and activity were assessed by using quantitative PCR and the telomere repeat amplification protocol from PBMCs and enriched lymphocyte cultures. Lymphocyte cultures from patients with CHH display growth defects in vitro, which is consistent with an immune deficiency cellular phenotype. Here we show that telomere length and telomerase activity are impaired in primary lymphocyte subsets from patients with CHH. Notably, telomerase activity is affected in a gene dose-dependent manner when comparing heterozygote RMRP carriers with patients with CHH. Telomerase deficiency in patients with CHH is not mediated by abnormal telomerase gene transcript levels relative to those of endogenous genes. These findings suggest that telomere deficiency is implicated in the CHH disease phenotype through an as yet unidentified mechanism. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Superoxide dismutating molecules rescue the toxic effects of PINK1 and parkin loss.

PubMed

Biosa, Alice; Sanchez-Martinez, Alvaro; Filograna, Roberta; Terriente-Felix, Ana; Alam, Sarah M; Beltramini, Mariano; Bubacco, Luigi; Bisaglia, Marco; Whitworth, Alexander J

2018-05-01

Reactive oxygen species exert important functions in regulating several cellular signalling pathways. However, an excessive accumulation of reactive oxygen species can perturb the redox homeostasis leading to oxidative stress, a condition which has been associated to many neurodegenerative disorders. Accordingly, alterations in the redox state of cells and mitochondrial homeostasis are established hallmarks in both familial and sporadic Parkinson's disease cases. PINK1 and Parkin are two genes which account for a large fraction of autosomal recessive early-onset forms of Parkinson's disease and are now firmly associated to both mitochondria and redox homeostasis. In this study we explored the hypothesis that superoxide anions participate in the generation of the Parkin and PINK1 associated phenotypic effect by testing the capacity of endogenous and exogenous superoxide dismutating molecules to rescue the toxic effects induced by loss of PINK1 or Parkin, in both cellular and fly models. Our results demonstrate the positive effect of an increased level of superoxide dismutase proteins on the pathological phenotypes, both in vitro and in vivo. A more pronounced effectiveness for mitochondrial SOD2 activity points to the superoxide radicals generated in the mitochondrial matrix as the prime suspect in the definition of the observed phenotypes. Moreover, we also demonstrate the efficacy of a SOD-mimetic compound, M40403, to partially ameliorate PINK1/Parkin phenotypes in vitro and in vivo. These results support the further exploration of SOD-mimetic compounds as a therapeutic strategy against Parkinson's disease.

Point Mutations in the Stem Region and the Fourth AAA Domain of Cytoplasmic Dynein Heavy Chain Partially Suppress the Phenotype of NUDF/LIS1 Loss in Aspergillus nidulans

PubMed Central

Zhuang, Lei; Zhang, Jun; Xiang, Xin

2007-01-01

Cytoplasmic dynein performs multiple cellular tasks but its regulation remains unclear. The dynein heavy chain has a N-terminal stem that binds to other subunits and a C-terminal motor unit that contains six AAA (ATPase associated with cellular activities) domains and a microtubule-binding site located between AAA4 and AAA5. In Aspergillus nidulans, NUDF (a LIS1 homolog) functions in the dynein pathway, and two nudF6 partial suppressors were mapped to the nudA dynein heavy chain locus. Here we identified these two mutations. The nudAL1098F mutation resides in the stem region, and nudAR3086C is in the end of AAA4. These mutations partially suppress the phenotype of nudF deletion but do not suppress the phenotype exhibited by mutants of dynein intermediate chain and Arp1. Surprisingly, the stronger ΔnudF suppressor, nudAR3086C, causes an obvious decrease in the basal level of dynein's ATPase activity and an increase in dynein's distribution along microtubules. Thus, suppression of the ΔnudF phenotype may result from mechanisms other than simply the enhancement of dynein's ATPase activity. The fact that a mutation in the end of AAA4 negatively regulates dynein's ATPase activity but partially compensates for NUDF loss indicates the importance of the AAA4 domain in dynein regulation in vivo. PMID:17237507

Noise-Driven Phenotypic Heterogeneity with Finite Correlation Time in Clonal Populations.

PubMed

Lee, UnJin; Skinner, John J; Reinitz, John; Rosner, Marsha Rich; Kim, Eun-Jin

2015-01-01

There has been increasing awareness in the wider biological community of the role of clonal phenotypic heterogeneity in playing key roles in phenomena such as cellular bet-hedging and decision making, as in the case of the phage-λ lysis/lysogeny and B. Subtilis competence/vegetative pathways. Here, we report on the effect of stochasticity in growth rate, cellular memory/intermittency, and its relation to phenotypic heterogeneity. We first present a linear stochastic differential model with finite auto-correlation time, where a randomly fluctuating growth rate with a negative average is shown to result in exponential growth for sufficiently large fluctuations in growth rate. We then present a non-linear stochastic self-regulation model where the loss of coherent self-regulation and an increase in noise can induce a shift from bounded to unbounded growth. An important consequence of these models is that while the average change in phenotype may not differ for various parameter sets, the variance of the resulting distributions may considerably change. This demonstrates the necessity of understanding the influence of variance and heterogeneity within seemingly identical clonal populations, while providing a mechanism for varying functional consequences of such heterogeneity. Our results highlight the importance of a paradigm shift from a deterministic to a probabilistic view of clonality in understanding selection as an optimization problem on noise-driven processes, resulting in a wide range of biological implications, from robustness to environmental stress to the development of drug resistance.

A full computation-relevant topological dynamics classification of elementary cellular automata.

PubMed

Schüle, Martin; Stoop, Ruedi

2012-12-01

Cellular automata are both computational and dynamical systems. We give a complete classification of the dynamic behaviour of elementary cellular automata (ECA) in terms of fundamental dynamic system notions such as sensitivity and chaoticity. The "complex" ECA emerge to be sensitive, but not chaotic and not eventually weakly periodic. Based on this classification, we conjecture that elementary cellular automata capable of carrying out complex computations, such as needed for Turing-universality, are at the "edge of chaos."

Epigenetic mechanisms of nutrient-induced modulation of gene expression and cellular functions

USDA-ARS?s Scientific Manuscript database

Utilizing next-generation sequencing technology in combination with chromatin immunoprecipitation (ChIP) technology, our study provides systematic and novel insights into the relationships between nutrition and epigenetics. One paradigmatic example of nutrient-epigenetic-phenotype relationship is th...

Advances in molecular labeling, high throughput imaging and machine intelligence portend powerful functional cellular biochemistry tools.

PubMed

Price, Jeffrey H; Goodacre, Angela; Hahn, Klaus; Hodgson, Louis; Hunter, Edward A; Krajewski, Stanislaw; Murphy, Robert F; Rabinovich, Andrew; Reed, John C; Heynen, Susanne

2002-01-01

Cellular behavior is complex. Successfully understanding systems at ever-increasing complexity is fundamental to advances in modern science and unraveling the functional details of cellular behavior is no exception. We present a collection of prospectives to provide a glimpse of the techniques that will aid in collecting, managing and utilizing information on complex cellular processes via molecular imaging tools. These include: 1) visualizing intracellular protein activity with fluorescent markers, 2) high throughput (and automated) imaging of multilabeled cells in statistically significant numbers, and 3) machine intelligence to analyze subcellular image localization and pattern. Although not addressed here, the importance of combining cell-image-based information with detailed molecular structure and ligand-receptor binding models cannot be overlooked. Advanced molecular imaging techniques have the potential to impact cellular diagnostics for cancer screening, clinical correlations of tissue molecular patterns for cancer biology, and cellular molecular interactions for accelerating drug discovery. The goal of finally understanding all cellular components and behaviors will be achieved by advances in both instrumentation engineering (software and hardware) and molecular biochemistry. Copyright 2002 Wiley-Liss, Inc.

Deregulation of obesity-relevant genes is associated with progression in BMI and the amount of adipose tissue in pigs.

PubMed

Mentzel, Caroline M Junker; Cardoso, Tainã Figueiredo; Pipper, Christian Bressen; Jacobsen, Mette Juul; Jørgensen, Claus Bøttcher; Cirera, Susanna; Fredholm, Merete

2018-02-01

The aim of this study was to elucidate the relative impact of three phenotypes often used to characterize obesity on perturbation of molecular pathways involved in obesity. The three obesity-related phenotypes are (1) body mass index (BMI), (2) amount of subcutaneous adipose tissue (SATa), and (3) amount of retroperitoneal adipose tissue (RPATa). Although it is generally accepted that increasing amount of RPATa is 'unhealthy', a direct comparison of the relative impact of the three obesity-related phenotypes on gene expression has, to our knowledge, not been performed previously. We have used multiple linear models to analyze altered gene expression of selected obesity-related genes in tissues collected from 19 female pigs phenotypically characterized with respect to the obesity-related phenotypes. Gene expression was assessed by high-throughput qPCR in RNA from liver, skeletal muscle and abdominal adipose tissue. The stringent statistical approach used in the study has increased the power of the analysis compared to the classical approach of analysis in divergent groups of individuals. Our approach led to the identification of key components of cellular pathways that are modulated in the three tissues in association with changes in the three obesity-relevant phenotypes (BMI, SATa and RPATa). The deregulated pathways are involved in biosynthesis and transcript regulation in adipocytes, in lipid transport, lipolysis and metabolism, and in inflammatory responses. Deregulation seemed more comprehensive in liver (23 genes) compared to abdominal adipose tissue (10 genes) and muscle (3 genes). Notably, the study supports the notion that excess amount of intra-abdominal adipose tissue is associated with a greater metabolic disease risk. Our results provide molecular support for this notion by demonstrating that increasing amount of RPATa has a higher impact on perturbation of cellular pathways influencing obesity and obesity-related metabolic traits compared to increase in BMI and amount of SATa.