Autophagy in Drosophila melanogaster.

PubMed

McPhee, Christina K; Baehrecke, Eric H

2009-09-01

Macroautophagy (autophagy) is a bulk cytoplasmic degradation process that is conserved from yeast to mammals. Autophagy is an important cellular response to starvation and stress, and plays critical roles in development, cell death, aging, immunity, and cancer. The fruit fly Drosophila melanogaster provides an excellent model system to study autophagy in vivo, in the context of a developing organism. Autophagy (atg) genes and their regulators are conserved in Drosophila, and autophagy is induced in response to nutrient starvation and hormones during development. In this review we provide an overview of how Drosophila research has contributed to our understanding of the role and regulation of autophagy in cell survival, growth, nutrient utilization, and cell death. Recent Drosophila research has also provided important mechanistic information about the role of autophagy in protein aggregation disorders, neurodegeneration, aging, and innate immunity. Differences in the role of autophagy in specific contexts and/or cell types suggest that there may be cell-context-specific regulators of autophagy, and studies in Drosophila are well-suited to yield discoveries about this specificity.

Ral GTPase and the exocyst regulate autophagy in a tissue-specific manner.

PubMed

Tracy, Kirsten; Velentzas, Panagiotis D; Baehrecke, Eric H

2016-01-01

Autophagy traffics cellular components to the lysosome for degradation. Ral GTPase and the exocyst have been implicated in the regulation of stress-induced autophagy, but it is unclear whether they are global regulators of this process. Here, we investigate Ral function in different cellular contexts in Drosophila and find that it is required for autophagy during developmentally regulated cell death in salivary glands, but does not affect starvation-induced autophagy in the fat body. Furthermore, knockdown of exocyst subunits has a similar effect, preventing autophagy in dying cells but not in cells of starved animals. Notch activity is elevated in dying salivary glands, this change in Notch signaling is influenced by Ral, and decreased Notch function influences autophagy. These data indicate that Ral and the exocyst regulate autophagy in a context-dependent manner, and that in dying salivary glands, Ral mediates autophagy, at least in part, by regulation of Notch. © 2015 The Authors.

Knowledge-guided fuzzy logic modeling to infer cellular signaling networks from proteomic data

PubMed Central

Liu, Hui; Zhang, Fan; Mishra, Shital Kumar; Zhou, Shuigeng; Zheng, Jie

2016-01-01

Modeling of signaling pathways is crucial for understanding and predicting cellular responses to drug treatments. However, canonical signaling pathways curated from literature are seldom context-specific and thus can hardly predict cell type-specific response to external perturbations; purely data-driven methods also have drawbacks such as limited biological interpretability. Therefore, hybrid methods that can integrate prior knowledge and real data for network inference are highly desirable. In this paper, we propose a knowledge-guided fuzzy logic network model to infer signaling pathways by exploiting both prior knowledge and time-series data. In particular, the dynamic time warping algorithm is employed to measure the goodness of fit between experimental and predicted data, so that our method can model temporally-ordered experimental observations. We evaluated the proposed method on a synthetic dataset and two real phosphoproteomic datasets. The experimental results demonstrate that our model can uncover drug-induced alterations in signaling pathways in cancer cells. Compared with existing hybrid models, our method can model feedback loops so that the dynamical mechanisms of signaling networks can be uncovered from time-series data. By calibrating generic models of signaling pathways against real data, our method supports precise predictions of context-specific anticancer drug effects, which is an important step towards precision medicine. PMID:27774993

Optogenetic Tools for Subcellular Applications in Neuroscience.

PubMed

Rost, Benjamin R; Schneider-Warme, Franziska; Schmitz, Dietmar; Hegemann, Peter

2017-11-01

The ability to study cellular physiology using photosensitive, genetically encoded molecules has profoundly transformed neuroscience. The modern optogenetic toolbox includes fluorescent sensors to visualize signaling events in living cells and optogenetic actuators enabling manipulation of numerous cellular activities. Most optogenetic tools are not targeted to specific subcellular compartments but are localized with limited discrimination throughout the cell. Therefore, optogenetic activation often does not reflect context-dependent effects of highly localized intracellular signaling events. Subcellular targeting is required to achieve more specific optogenetic readouts and photomanipulation. Here we first provide a detailed overview of the available optogenetic tools with a focus on optogenetic actuators. Second, we review established strategies for targeting these tools to specific subcellular compartments. Finally, we discuss useful tools and targeting strategies that are currently missing from the optogenetics repertoire and provide suggestions for novel subcellular optogenetic applications. Copyright © 2017 Elsevier Inc. All rights reserved.

Safe Genetic Modification of Cardiac Stem Cells Using a Site-Specific Integration Technique

PubMed Central

Lan, Feng; Liu, Junwei; Narsinh, Kazim H.; Hu, Shijun; Han, Leng; Lee, Andrew S.; Karow, Marisa; Nguyen, Patricia K.; Nag, Divya; Calos, Michele P.; Robbins, Robert C.; Wu, Joseph C.

2012-01-01

Background Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts. Methods and Results We employed the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells (hECs). Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared to unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging (BLI), and positron emission tomography (PET). Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function two weeks after cell delivery, as assessed by echocardiography (P = 0.002) and magnetic resonance imaging (P = 0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated hECs, which enhanced hindlimb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging. Conclusions The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types. PMID:22965984

Safe genetic modification of cardiac stem cells using a site-specific integration technique.

PubMed

Lan, Feng; Liu, Junwei; Narsinh, Kazim H; Hu, Shijun; Han, Leng; Lee, Andrew S; Karow, Marisa; Nguyen, Patricia K; Nag, Divya; Calos, Michele P; Robbins, Robert C; Wu, Joseph C

2012-09-11

Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts. We used the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells. Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared with unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging, and positron emission tomography. Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function 2 weeks after cell delivery, as assessed by echocardiography (P=0.002) and MRI (P=0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated human endothelial cells, which enhanced hind limb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging. The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types.

Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

PubMed Central

Kim, Doory; Deerinck, Thomas J.; Sigal, Yaron M.; Babcock, Hazen P.; Ellisman, Mark H.; Zhuang, Xiaowei

2015-01-01

Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets. PMID:25874453

Network analysis of transcriptomics expands regulatory landscapes in Synechococcus sp. PCC 7002

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

McClure, Ryan S.; Overall, Christopher C.; McDermott, Jason E.

Cyanobacterial regulation of gene expression must contend with a genome organization that lacks apparent functional context, as the majority of cellular processes and metabolic pathways are encoded by genes found at disparate locations across the genome. In addition, the fact that coordinated regulation of cyanobacterial cellular machinery takes place with significantly fewer transcription factors, compared to other Eubacteria, suggests the involvement of post-transcriptional mechanisms and regulatory adaptations which are not fully understood. Global transcript abundance from model cyanobacterium Synechococcus sp. PCC 7002 grown under 42 different conditions was analyzed using context-likelihood of relatedness. The resulting 903-gene network, which was organizedmore » into 11 modules, not only allowed classification of cyanobacterial responses to specific environmental variables but provided insight into the transcriptional network topology and led to the expansion of predicted regulons. When used in conjunction with genome sequence, the global transcript abundance allowed identification of putative post-transcriptional changes in expression as well as novel potential targets of both DNA binding proteins and asRNA regulators. The results offer a new perspective into the multi-level regulation that governs cellular adaptations of fast-growing physiologically robust cyanobacterium Synechococcus sp. PCC 7002 to changing environmental variables. It also extends a methodological knowledge-based framework for studying multi-scale regulatory mechanisms that operate in cyanobacteria. Finally, it provides valuable context for integrating systems-level data to enhance evidence-driven genomic annotation, especially in organisms where traditional context analyses cannot be implemented due to lack of operon-based functional organization.« less

NetDecoder: a network biology platform that decodes context-specific biological networks and gene activities.

PubMed

da Rocha, Edroaldo Lummertz; Ung, Choong Yong; McGehee, Cordelia D; Correia, Cristina; Li, Hu

2016-06-02

The sequential chain of interactions altering the binary state of a biomolecule represents the 'information flow' within a cellular network that determines phenotypic properties. Given the lack of computational tools to dissect context-dependent networks and gene activities, we developed NetDecoder, a network biology platform that models context-dependent information flows using pairwise phenotypic comparative analyses of protein-protein interactions. Using breast cancer, dyslipidemia and Alzheimer's disease as case studies, we demonstrate NetDecoder dissects subnetworks to identify key players significantly impacting cell behaviour specific to a given disease context. We further show genes residing in disease-specific subnetworks are enriched in disease-related signalling pathways and information flow profiles, which drive the resulting disease phenotypes. We also devise a novel scoring scheme to quantify key genes-network routers, which influence many genes, key targets, which are influenced by many genes, and high impact genes, which experience a significant change in regulation. We show the robustness of our results against parameter changes. Our network biology platform includes freely available source code (http://www.NetDecoder.org) for researchers to explore genome-wide context-dependent information flow profiles and key genes, given a set of genes of particular interest and transcriptome data. More importantly, NetDecoder will enable researchers to uncover context-dependent drug targets. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Protein structure in context: The molecular landscape of angiogenesis

PubMed Central

Span, Elise A.; Goodsell, David S.; Ramchandran, Ramani; Franzen, Margaret; Herman, Timothy; Sem, Daniel S.

2014-01-01

A team of students, educators, and researchers has developed new materials to teach cell signaling within its cellular context. Two non-traditional modalities are employed: physical models, to explore the atomic details of several of the proteins in the angiogenesis signaling cascade, and illustrations of the proteins in their cellular environment, to give an intuitive understanding of the cellular context of the pathway. The experiences of the team underscore the utility of these types of materials as an effective mode for fostering students’ understanding of the molecular world, and the scientific method used to define it. PMID:23868376

Twenty Years of Calcium Imaging: Cell Physiology to Dye For

PubMed Central

Knot, Harm J.; Laher, Ismail; Sobie, Eric A.; Guatimosim, Silvia; Gomez-Viquez, Leticia; Hartmann, Hali; Song, Long-Sheng; Lederer, W.J.; Graier, Wolfgang F.; Malli, Roland; Frieden, Maud; Petersen, Ole H.

2016-01-01

The use of fluorescent dyes over the past two decades has led to a revolution in our understanding of calcium signaling. Given the ubiquitous role of Ca2+ in signal transduction at the most fundamental levels of molecular, cellular, and organismal biology, it has been challenging to understand how the specificity and versatility of Ca2+ signaling is accomplished. In excitable cells, the coordination of changing Ca2+ concentrations at global (cellular) and well-defined subcellular spaces through the course of membrane depolarization can now be conceptualized in the context of disease processes such as cardiac arrhythmogenesis. The spatial and temporal dimensions of Ca2+ signaling are similarly important in non-excitable cells, such as endothelial and epithelial cells, to regulate multiple signaling pathways that participate in organ homeostasis as well as cellular organization and essential secretory processes. PMID:15821159

Amplifying Riboswitch Signal Output using Cellular Wiring

DTIC Science & Technology

2017-01-30

riboswitches are developed within a specific genetic context. This becomes challenging when using a riboswitch to control a reporter gene that it was...survive well outside of controlled environmental conditions. Biological circuits utilize molecules that connect different genetic ‘components’, so that the...engineering to construct genetic logic gates to form genetic programs within and between cells.8-10,12-14 We have applied biological circuitry to

Off and back-on again: a tumor suppressor's tale.

PubMed

Acosta, Jonuelle; Wang, Walter; Feldser, David M

2018-06-01

Tumor suppressor genes play critical roles orchestrating anti-cancer programs that are both context dependent and mechanistically diverse. Beyond canonical tumor suppressive programs that control cell division, cell death, and genome stability, unexpected tumor suppressor gene activities that regulate metabolism, immune surveillance, the epigenetic landscape, and others have recently emerged. This diversity underscores the important roles these genes play in maintaining cellular homeostasis to suppress cancer initiation and progression, but also highlights a tremendous challenge in discerning precise context-specific programs of tumor suppression controlled by a given tumor suppressor. Fortunately, the rapid sophistication of genetically engineered mouse models of cancer has begun to shed light on these context-dependent tumor suppressor activities. By using techniques that not only toggle "off" tumor suppressor genes in nascent tumors, but also facilitate the timely restoration of gene function "back-on again" in disease specific contexts, precise mechanisms of tumor suppression can be revealed in an unbiased manner. This review discusses the development and implementation of genetic systems designed to toggle tumor suppressor genes off and back-on again and their potential to uncover the tumor suppressor's tale.

Solubilization of human cells by the styrene-maleic acid copolymer: Insights from fluorescence microscopy.

PubMed

Dörr, Jonas M; van Coevorden-Hameete, Marleen H; Hoogenraad, Casper C; Killian, J Antoinette

2017-11-01

Extracting membrane proteins from biological membranes by styrene-maleic acid copolymers (SMAs) in the form of nanodiscs has developed into a powerful tool in membrane research. However, the mode of action of membrane (protein) solubilization in a cellular context is still poorly understood and potential specificity for cellular compartments has not been investigated. Here, we use fluorescence microscopy to visualize the process of SMA solubilization of human cells, exemplified by the immortalized human HeLa cell line. Using fluorescent protein fusion constructs that mark distinct subcellular compartments, we found that SMA solubilizes membranes in a concentration-dependent multi-stage process. While all major intracellular compartments were affected without a strong preference, plasma membrane solubilization was found to be generally slower than the solubilization of organelle membranes. Interestingly, some plasma membrane-localized proteins were more resistant against solubilization than others, which might be explained by their presence in specific membrane domains with differing properties. Our results support the general applicability of SMA for the isolation of membrane proteins from different types of (sub)cellular membranes. Copyright © 2017 Elsevier B.V. All rights reserved.

Xenophagic pathways and their bacterial subversion in cellular self-defense - παντα ρει - everything is in flux.

PubMed

Radomski, Nadine; Rebbig, Annica; Leonhardt, Ralf M; Knittler, Michael R

2017-11-02

Autophagy is an evolutionarily ancient and highly conserved eukaryotic mechanism that targets cytoplasmic material for degradation. Autophagic flux involves the formation of autophagosomes and their degradation by lysosomes. The process plays a crucial role in maintaining cellular homeostasis and responds to various environmental conditions. While autophagy had previously been thought to be a non-selective process, it is now clear that it can also selectively target cellular organelles, such as mitochondria (referred to as mitophagy) and/or invading pathogens (referred to as xenophagy). Selective autophagy is characterized by specific substrate recognition and requires distinct cellular adaptor proteins. Here we review xenophagic mechanisms involved in the recognition and autolysosomal or autophagolysosomal degradation of different intracellular bacteria. In this context, we also discuss a recently discovered cellular self-defense pathway, termed mito-xenophagy, which occurs during bacterial infection of dendritic cells and depends on a TNF-α-mediated metabolic switch from oxidative phosphorylation to glycolysis. Copyright © 2017 Elsevier GmbH. All rights reserved.

Acute neuroinflammation impairs context discrimination memory and disrupts pattern separation processes in hippocampus.

PubMed

Czerniawski, Jennifer; Guzowski, John F

2014-09-10

Although it is known that immune system activation can impair cognition, no study to date has linked cognitive deficits during acute neuroinflammation to dysregulation of task-relevant neuronal ensemble activity. Here, we assessed both neural circuit activity and context discrimination memory retrieval, in a within-subjects design, of male rats given systemic administration of saline or lipopolysaccharide (LPS). Rats were exposed over several days to two similar contexts: one of which was paired with weak foot shock and the other was not. After reaching criteria for discriminative freezing, rats were given systemic LPS or saline injection and tested for retrieval of context discrimination 6 h later. Importantly, LPS administration produced an acute neuroinflammatory response in dorsal hippocampus at this time (as assessed by elevation of proinflammatory cytokine mRNA levels) and abolished retrieval of the previously acquired discrimination. The impact of neuroinflammation on hippocampal CA3 and CA1 neural circuit activity was assessed using the Arc/Homer1a cellular analysis of temporal activity by fluorescence in situ hybridization imaging method. Whereas the saline-treated subjects discriminated and had low overlap of hippocampal ensembles activated in the two contexts, LPS-treated subjects did not discriminate and had greater ensemble overlap (i.e., reduced orthogonalization). Additionally, retrieval of standard contextual fear conditioning, which does not require context discrimination, was not affected by pretesting LPS administration. Together, the behavioral and circuit analyses data provide compelling evidence that LPS administration impairs context discrimination memory by disrupting cellular pattern separation processes within the hippocampus, thus linking acute neuroinflammation to disruption of specific neural circuit functions and cognitive impairment. Copyright © 2014 the authors 0270-6474/14/3412470-11$15.00/0.

Acute Neuroinflammation Impairs Context Discrimination Memory and Disrupts Pattern Separation Processes in Hippocampus

PubMed Central

Czerniawski, Jennifer

2014-01-01

Although it is known that immune system activation can impair cognition, no study to date has linked cognitive deficits during acute neuroinflammation to dysregulation of task-relevant neuronal ensemble activity. Here, we assessed both neural circuit activity and context discrimination memory retrieval, in a within-subjects design, of male rats given systemic administration of saline or lipopolysaccharide (LPS). Rats were exposed over several days to two similar contexts: one of which was paired with weak foot shock and the other was not. After reaching criteria for discriminative freezing, rats were given systemic LPS or saline injection and tested for retrieval of context discrimination 6 h later. Importantly, LPS administration produced an acute neuroinflammatory response in dorsal hippocampus at this time (as assessed by elevation of proinflammatory cytokine mRNA levels) and abolished retrieval of the previously acquired discrimination. The impact of neuroinflammation on hippocampal CA3 and CA1 neural circuit activity was assessed using the Arc/Homer1a cellular analysis of temporal activity by fluorescence in situ hybridization imaging method. Whereas the saline-treated subjects discriminated and had low overlap of hippocampal ensembles activated in the two contexts, LPS-treated subjects did not discriminate and had greater ensemble overlap (i.e., reduced orthogonalization). Additionally, retrieval of standard contextual fear conditioning, which does not require context discrimination, was not affected by pretesting LPS administration. Together, the behavioral and circuit analyses data provide compelling evidence that LPS administration impairs context discrimination memory by disrupting cellular pattern separation processes within the hippocampus, thus linking acute neuroinflammation to disruption of specific neural circuit functions and cognitive impairment. PMID:25209285

Specific Adoptive Cellular Immunotherapy in Allogeneic Stem Cell Transplantation.

PubMed

Audehm, Stefan; Krackhardt, Angela M

2017-01-01

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents a treatment option for a diversity of advanced hematopoietic malignancies providing hope for long-term responses especially due to immunogenic effects associated with the treatment modality. Despite respectable progress in the field, relapses and/or opportunistic infections are major reasons for the high treatment-related mortality. However, a number of novel immunotherapeutic approaches using defined cell populations have been developed to directly target residual malignant cells as well as defined infectious diseases. We here provide an overview of current adoptive cellular immunotherapies in the context of allo-HSCT and close with an outlook on new directions within the field. © 2017 S. Karger GmbH, Freiburg.

Cellular context-dependent consequences of Apc mutations on gene regulation and cellular behavior.

PubMed

Hashimoto, Kyoichi; Yamada, Yosuke; Semi, Katsunori; Yagi, Masaki; Tanaka, Akito; Itakura, Fumiaki; Aoki, Hitomi; Kunisada, Takahiro; Woltjen, Knut; Haga, Hironori; Sakai, Yoshiharu; Yamamoto, Takuya; Yamada, Yasuhiro

2017-01-24

The spectrum of genetic mutations differs among cancers in different organs, implying a cellular context-dependent effect for genetic aberrations. However, the extent to which the cellular context affects the consequences of oncogenic mutations remains to be fully elucidated. We reprogrammed colon tumor cells in an Apc Min/+ (adenomatous polyposis coli) mouse model, in which the loss of the Apc gene plays a critical role in tumor development and subsequently, established reprogrammed tumor cells (RTCs) that exhibit pluripotent stem cell (PSC)-like signatures of gene expression. We show that the majority of the genes in RTCs that were affected by Apc mutations did not overlap with the genes affected in the intestine. RTCs lacked pluripotency but exhibited an increased expression of Cdx2 and a differentiation propensity that was biased toward the trophectoderm cell lineage. Genetic rescue of the mutated Apc allele conferred pluripotency on RTCs and enabled their differentiation into various cell types in vivo. The redisruption of Apc in RTC-derived differentiated cells resulted in neoplastic growth that was exclusive to the intestine, but the majority of the intestinal lesions remained as pretumoral microadenomas. These results highlight the significant influence of cellular context on gene regulation, cellular plasticity, and cellular behavior in response to the loss of the Apc function. Our results also imply that the transition from microadenomas to macroscopic tumors is reprogrammable, which underscores the importance of epigenetic regulation on tumor promotion.

Cellular context-dependent consequences of Apc mutations on gene regulation and cellular behavior

PubMed Central

Hashimoto, Kyoichi; Yamada, Yosuke; Semi, Katsunori; Yagi, Masaki; Tanaka, Akito; Itakura, Fumiaki; Aoki, Hitomi; Kunisada, Takahiro; Woltjen, Knut; Haga, Hironori; Sakai, Yoshiharu; Yamamoto, Takuya; Yamada, Yasuhiro

2017-01-01

The spectrum of genetic mutations differs among cancers in different organs, implying a cellular context-dependent effect for genetic aberrations. However, the extent to which the cellular context affects the consequences of oncogenic mutations remains to be fully elucidated. We reprogrammed colon tumor cells in an ApcMin/+ (adenomatous polyposis coli) mouse model, in which the loss of the Apc gene plays a critical role in tumor development and subsequently, established reprogrammed tumor cells (RTCs) that exhibit pluripotent stem cell (PSC)-like signatures of gene expression. We show that the majority of the genes in RTCs that were affected by Apc mutations did not overlap with the genes affected in the intestine. RTCs lacked pluripotency but exhibited an increased expression of Cdx2 and a differentiation propensity that was biased toward the trophectoderm cell lineage. Genetic rescue of the mutated Apc allele conferred pluripotency on RTCs and enabled their differentiation into various cell types in vivo. The redisruption of Apc in RTC-derived differentiated cells resulted in neoplastic growth that was exclusive to the intestine, but the majority of the intestinal lesions remained as pretumoral microadenomas. These results highlight the significant influence of cellular context on gene regulation, cellular plasticity, and cellular behavior in response to the loss of the Apc function. Our results also imply that the transition from microadenomas to macroscopic tumors is reprogrammable, which underscores the importance of epigenetic regulation on tumor promotion. PMID:28057861

The Protein Corona of Plant Virus Nanoparticles Influences their Dispersion Properties, Cellular Interactions, and In Vivo Fates.

PubMed

Pitek, Andrzej S; Wen, Amy M; Shukla, Sourabh; Steinmetz, Nicole F

2016-04-06

Biomolecules in bodily fluids such as plasma can adsorb to the surface of nanoparticles and influence their biological properties. This phenomenon, known as the protein corona, is well established in the field of synthetic nanotechnology but has not been described in the context of plant virus nanoparticles (VNPs). The interaction between VNPs derived from Tobacco mosaic virus (TMV) and plasma proteins is investigated, and it is found that the VNP protein corona is significantly less abundant compared to the corona of synthetic particles. The formed corona is dominated by complement proteins and immunoglobulins, the binding of which can be reduced by PEGylating the VNP surface. The impact of the VNP protein corona on molecular recognition and cell targeting in the context of cancer and thrombosis is investigated. A library of functionalized TMV rods with polyethylene glycol (PEG) and peptide ligands targeting integrins or fibrin(ogen) show different dispersion properties, cellular interactions, and in vivo fates depending on the properties of the protein corona, influencing target specificity, and non-specific scavenging by macrophages. Our results provide insight into the in vivo properties of VNPs and suggest that the protein corona effect should be considered during the development of efficacious, targeted VNP formulations. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Imaging Subcellular Structures in the Living Zebrafish Embryo.

PubMed

Engerer, Peter; Plucinska, Gabriela; Thong, Rachel; Trovò, Laura; Paquet, Dominik; Godinho, Leanne

2016-04-02

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Cytoskeletal self-organization in neuromorphogenesis.

PubMed

Dehmelt, Leif

2014-01-01

Self-organization of dynamic microtubules via interactions with associated motors plays a critical role in spindle formation. The microtubule-based mechanisms underlying other aspects of cellular morphogenesis, such as the formation and development of protrusions from neuronal cells is less well understood. In a recent study, we investigated the molecular mechanism that underlies the massive reorganization of microtubules induced in non-neuronal cells by expression of the neuronal microtubule stabilizer MAP2c. In that study we directly observed cortical dynein complexes and how they affect the dynamic behavior of motile microtubules in living cells. We found that stationary dynein complexes transiently associate with motile microtubules near the cell cortex and that their rapid turnover facilitates efficient microtubule transport. Here, we discuss our findings in the larger context of cellular morphogenesis with specific focus on self-organizing principles from which cellular shape patterns such as the thin protrusions of neurons can emerge.

Cytoskeletal self-organization in neuromorphogenesis

PubMed Central

Dehmelt, Leif

2014-01-01

Self-organization of dynamic microtubules via interactions with associated motors plays a critical role in spindle formation. The microtubule-based mechanisms underlying other aspects of cellular morphogenesis, such as the formation and development of protrusions from neuronal cells is less well understood. In a recent study, we investigated the molecular mechanism that underlies the massive reorganization of microtubules induced in non-neuronal cells by expression of the neuronal microtubule stabilizer MAP2c. In that study we directly observed cortical dynein complexes and how they affect the dynamic behavior of motile microtubules in living cells. We found that stationary dynein complexes transiently associate with motile microtubules near the cell cortex and that their rapid turnover facilitates efficient microtubule transport. Here, we discuss our findings in the larger context of cellular morphogenesis with specific focus on self-organizing principles from which cellular shape patterns such as the thin protrusions of neurons can emerge. PMID:24847718

Discrete Logic Modelling Optimization to Contextualize Prior Knowledge Networks Using PRUNET

PubMed Central

Androsova, Ganna; del Sol, Antonio

2015-01-01

High-throughput technologies have led to the generation of an increasing amount of data in different areas of biology. Datasets capturing the cell’s response to its intra- and extra-cellular microenvironment allows such data to be incorporated as signed and directed graphs or influence networks. These prior knowledge networks (PKNs) represent our current knowledge of the causality of cellular signal transduction. New signalling data is often examined and interpreted in conjunction with PKNs. However, different biological contexts, such as cell type or disease states, may have distinct variants of signalling pathways, resulting in the misinterpretation of new data. The identification of inconsistencies between measured data and signalling topologies, as well as the training of PKNs using context specific datasets (PKN contextualization), are necessary conditions to construct reliable, predictive models, which are current challenges in the systems biology of cell signalling. Here we present PRUNET, a user-friendly software tool designed to address the contextualization of a PKNs to specific experimental conditions. As the input, the algorithm takes a PKN and the expression profile of two given stable steady states or cellular phenotypes. The PKN is iteratively pruned using an evolutionary algorithm to perform an optimization process. This optimization rests in a match between predicted attractors in a discrete logic model (Boolean) and a Booleanized representation of the phenotypes, within a population of alternative subnetworks that evolves iteratively. We validated the algorithm applying PRUNET to four biological examples and using the resulting contextualized networks to predict missing expression values and to simulate well-characterized perturbations. PRUNET constitutes a tool for the automatic curation of a PKN to make it suitable for describing biological processes under particular experimental conditions. The general applicability of the implemented algorithm makes PRUNET suitable for a variety of biological processes, for instance cellular reprogramming or transitions between healthy and disease states. PMID:26058016

Ligand-independent TLR signals generated by ectopic overexpression of MyD88 generate local and systemic anti-tumor immunity

PubMed Central

Hartman, Zachary C.; Osada, Takuya; Glass, Oliver; Yang, Xiao Y.; Lei, Gang-jun; Lyerly, H. Kim; Clay, Timothy M.

2010-01-01

Although critical for initiating and regulating immune responses, the therapeutic use of individual cytokines as anti-cancer immunotherapeutic agents has achieved only modest clinical success. Consequently, many current strategies have focused on the use of specific immunotherapeutic agonists that engage individual receptors of innate immune networks, such as the Toll Like-Receptor (TLR) system, each resulting in specific patterns of gene expression, cytokine production and inflammatory outcome. However, these immunotherapeutics are constrained by variable cellular TLR expression and responsiveness to particular TLR agonists, as well as the specific cellular context of different tumors. We hypothesized that overexpression of MyD88, a pivotal regulator of multiple TLR signaling pathways, could circumvent these constraints and mimic coordinated TLR signaling across all cell types in a ligand independent fashion. To explore this hypothesis, we generated an adenoviral vector expressing MyD88 and demonstrate that Ad-MyD88 infection elicits extensive Th1-specific transcriptional and secreted cytokine signatures in all murine and human cell types tested in vitro and in vivo. Importantly, in vivo intratumoral injection of Ad-MyD88 into established tumor masses enhanced adaptive immune responses and inhibited local tumor immunosuppression, resulting in significantly inhibited local and systemic growth of multiple tumor types. Finally, Ad-MyD88 infection of primary human dendritic cells, tumor associated fibroblasts, and colorectal carcinoma cells elicited significant Th1-type cytokine responses, resulting in enhanced tumor cell lysis and expansion of human tumor antigen-specific T-cells. Thus, Ad-MyD88 initiated robust anti-tumor activity in established murine tumor microenvironments and in human contexts, suggesting its potential effectiveness as a clinical immunotherapeutic strategy. PMID:20823152

Context-specific metabolic networks are consistent with experiments.

PubMed

Becker, Scott A; Palsson, Bernhard O

2008-05-16

Reconstructions of cellular metabolism are publicly available for a variety of different microorganisms and some mammalian genomes. To date, these reconstructions are "genome-scale" and strive to include all reactions implied by the genome annotation, as well as those with direct experimental evidence. Clearly, many of the reactions in a genome-scale reconstruction will not be active under particular conditions or in a particular cell type. Methods to tailor these comprehensive genome-scale reconstructions into context-specific networks will aid predictive in silico modeling for a particular situation. We present a method called Gene Inactivity Moderated by Metabolism and Expression (GIMME) to achieve this goal. The GIMME algorithm uses quantitative gene expression data and one or more presupposed metabolic objectives to produce the context-specific reconstruction that is most consistent with the available data. Furthermore, the algorithm provides a quantitative inconsistency score indicating how consistent a set of gene expression data is with a particular metabolic objective. We show that this algorithm produces results consistent with biological experiments and intuition for adaptive evolution of bacteria, rational design of metabolic engineering strains, and human skeletal muscle cells. This work represents progress towards producing constraint-based models of metabolism that are specific to the conditions where the expression profiling data is available.

Protein Structure in Context: The Molecular Landscape of Angiogenesis

ERIC Educational Resources Information Center

Span, Elise A.; Goodsell, David S.; Ramchandran, Ramani; Franzen, Margaret A.; Herman, Tim; Sem, Daniel S.

2013-01-01

A team of students, educators, and researchers has developed new materials to teach cell signaling within its cellular context. Two nontraditional modalities are employed: physical models, to explore the atomic details of several of the proteins in the angiogenesis signaling cascade, and illustrations of the proteins in their cellular environment,…

30 years of NF-κB: a blossoming of relevance to human pathobiology

PubMed Central

Zhang, Qian; Lenardo, Michael J.; Baltimore, David

2016-01-01

NF-κB was discovered thirty years ago as a rapidly inducible transcription factor. Since that time it has been found to have a broad role in gene induction in diverse cellular responses, particularly throughout the immune system. Here we summarize elaborate regulatory pathways involving this transcription factor and use recent discoveries in human genetic diseases to place specific proteins within their relevant medical and biological contexts. PMID:28086098

Dissecting Cell-Fate Determination Through Integrated Mathematical Modeling of the ERK/MAPK Signaling Pathway.

PubMed

Shin, Sung-Young; Nguyen, Lan K

2017-01-01

The past three decades have witnessed an enormous progress in the elucidation of the ERK/MAPK signaling pathway and its involvement in various cellular processes. Because of its importance and complex wiring, the ERK pathway has been an intensive subject for mathematical modeling, which facilitates the unraveling of key dynamic properties and behaviors of the pathway. Recently, however, it became evident that the pathway does not act in isolation but closely interacts with many other pathways to coordinate various cellular outcomes under different pathophysiological contexts. This has led to an increasing number of integrated, large-scale models that link the ERK pathway to other functionally important pathways. In this chapter, we first discuss the essential steps in model development and notable models of the ERK pathway. We then use three examples of integrated, multipathway models to investigate how crosstalk of ERK signaling with other pathways regulates cell-fate decision-making in various physiological and disease contexts. Specifically, we focus on ERK interactions with the phosphoinositide-3 kinase (PI3K), c-Jun N-terminal kinase (JNK), and β-adrenergic receptor (β-AR) signaling pathways. We conclude that integrated modeling in combination with wet-lab experimentation have been and will be instrumental in gaining an in-depth understanding of ERK signaling in multiple biological contexts.

Physiological enzymology: The next frontier in understanding protein structure and function at the cellular level.

PubMed

Lee, Irene; Berdis, Anthony J

2016-01-01

Historically, the study of proteins has relied heavily on characterizing the activity of a single purified protein isolated from other cellular components. This classic approach allowed scientists to unambiguously define the intrinsic kinetic and chemical properties of that protein. The ultimate hope was to extrapolate this information toward understanding how the enzyme or receptor behaves within its native cellular context. These types of detailed in vitro analyses were necessary to reduce the innate complexities of measuring the singular activity and biochemical properties of a specific enzyme without interference from other enzymes and potential competing substrates. However, recent developments in fields encompassing cell biology, molecular imaging, and chemical biology now provide the unique chemical tools and instrumentation to study protein structure, function, and regulation in their native cellular environment. These advancements provide the foundation for a new field, coined physiological enzymology, which quantifies the function and regulation of enzymes and proteins at the cellular level. In this Special Edition, we explore the area of Physiological Enzymology and Protein Function through a series of review articles that focus on the tools and techniques used to measure the cellular activity of proteins inside living cells. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. Copyright © 2015 Elsevier B.V. All rights reserved.

A mechanistic pan-cancer pathway model informed by multi-omics data interprets stochastic cell fate responses to drugs and mitogens

PubMed Central

Bouhaddou, Mehdi; Koch, Rick J.; DiStefano, Matthew S.; Tan, Annie L.; Mertz, Alex E.

2018-01-01

Most cancer cells harbor multiple drivers whose epistasis and interactions with expression context clouds drug and drug combination sensitivity prediction. We constructed a mechanistic computational model that is context-tailored by omics data to capture regulation of stochastic proliferation and death by pan-cancer driver pathways. Simulations and experiments explore how the coordinated dynamics of RAF/MEK/ERK and PI-3K/AKT kinase activities in response to synergistic mitogen or drug combinations control cell fate in a specific cellular context. In this MCF10A cell context, simulations suggest that synergistic ERK and AKT inhibitor-induced death is likely mediated by BIM rather than BAD, which is supported by prior experimental studies. AKT dynamics explain S-phase entry synergy between EGF and insulin, but simulations suggest that stochastic ERK, and not AKT, dynamics seem to drive cell-to-cell proliferation variability, which in simulations is predictable from pre-stimulus fluctuations in C-Raf/B-Raf levels. Simulations suggest MEK alteration negligibly influences transformation, consistent with clinical data. Tailoring the model to an alternate cell expression and mutation context, a glioma cell line, allows prediction of increased sensitivity of cell death to AKT inhibition. Our model mechanistically interprets context-specific landscapes between driver pathways and cell fates, providing a framework for designing more rational cancer combination therapy. PMID:29579036

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

Endele, Max; Etzrodt, Martin; Schroeder, Timm, E-mail: timm.schroeder@bsse.ethz.ch

Hematopoiesis is the cumulative consequence of finely tuned signaling pathways activated through extrinsic factors, such as local niche signals and systemic hematopoietic cytokines. Whether extrinsic factors actively instruct the lineage choice of hematopoietic stem and progenitor cells or are only selectively allowing survival and proliferation of already intrinsically lineage-committed cells has been debated over decades. Recent results demonstrated that cytokines can instruct lineage choice. However, the precise function of individual cytokine-triggered signaling molecules in inducing cellular events like proliferation, lineage choice, and differentiation remains largely elusive. Signal transduction pathways activated by different cytokine receptors are highly overlapping, but support themore » production of distinct hematopoietic lineages. Cellular context, signaling dynamics, and the crosstalk of different signaling pathways determine the cellular response of a given extrinsic signal. New tools to manipulate and continuously quantify signaling events at the single cell level are therefore required to thoroughly interrogate how dynamic signaling networks yield a specific cellular response. - Highlights: • Recent studies provided definite proof for lineage-instructive action of cytokines. • Signaling pathways involved in hematopoietic lineage instruction remain elusive. • New tools are emerging to quantitatively study dynamic signaling networks over time.« less

Metabolic and Epigenetic Coordination of T Cell and Macrophage Immunity.

PubMed

Phan, Anthony T; Goldrath, Ananda W; Glass, Christopher K

2017-05-16

Recognition of pathogens by innate and adaptive immune cells instructs rapid alterations of cellular processes to promote effective resolution of infection. To accommodate increased bioenergetic and biosynthetic demands, metabolic pathways are harnessed to maximize proliferation and effector molecule production. In parallel, activation initiates context-specific gene-expression programs that drive effector functions and cell fates that correlate with changes in epigenetic landscapes. Many chromatin- and DNA-modifying enzymes make use of substrates and cofactors that are intermediates of metabolic pathways, providing potential cross talk between metabolism and epigenetic regulation of gene expression. In this review, we discuss recent studies of T cells and macrophages supporting a role for metabolic activity in integrating environmental signals with activation-induced gene-expression programs through modulation of the epigenome and speculate as to how this may influence context-specific macrophage and T cell responses to infection. Copyright © 2017 Elsevier Inc. All rights reserved.

Metabolic and epigenetic coordination of T cell and Macrophage immunity

PubMed Central

Phan, Anthony T.; Goldrath, Ananda W.; Glass, Christopher K.

2017-01-01

Recognition of pathogens by innate and adaptive immune cells instructs rapid alterations of cellular processes to promote effective resolution of infection. To accommodate increased bioenergetic and biosynthetic demands, metabolic pathways are harnessed to maximize proliferation and effector molecule production. In parallel, activation initiates context-specific gene-expression programs that drive effector functions and cell fates that correlate with changes in epigenetic landscapes. Many chromatin- and DNA-modifying enzymes make use of substrates and cofactors that are intermediates of metabolic pathways, providing potential cross talk between metabolism and epigenetic regulation of gene expression. In this review, we discuss recent studies of T cells and macrophages supporting a role for metabolic activity in integrating environmental signals with activation-induced gene-expression programs through modulation of the epigenome and speculate as to how this may influence context-specific macrophage and T cell responses to infection. PMID:28514673

Prospective Elementary Science Teachers' Understanding of Photosynthesis and Cellular Respiration in the Context of Multiple Biological Levels as Nested Systems

ERIC Educational Resources Information Center

Akçay, Süleyman

2017-01-01

In this study, Turkish prospective elementary science teachers' understanding of photosynthesis and cellular respiration has been analysed within the contexts of ecosystem knowledge, organism knowledge and interconnection knowledge (IK). In the analysis, concept maps developed by 74 prospective teachers were used. The study was carried out with…

Tissue-specific tumorigenesis – Context matters

PubMed Central

Schneider, Günter; Schmidt-Supprian, Marc; Rad, Roland; Saur, Dieter

2018-01-01

Preface How can we treat cancer more effectively? Traditionally, tumours from the same anatomical site are treated as one tumour entity. This concept has been challenged by recent breakthroughs in cancer genomics and translational research enabling molecular tumour profiling. The identification and validation of cancer drivers, which are shared between different tumour types, spurred the new paradigm to target driver pathways across anatomical sites by off-label drug use, or within so called “basket or umbrella trials”, which are designed to test whether molecular alterations in one tumour entity can be extrapolated to all others. However, recent clinical and preclinical studies suggest that there are tissue- and cell type-specific differences in tumourigenesis and the organization of oncogenic signalling pathways. In this Opinion article, we focus on the molecular, cellular, systemic and environmental determinants of organ-specific tumourigenesis and mechanisms of context-specific oncogenic signalling outputs. Investigation, recognition and in-depth biological understanding of these differences will be vital for the design of next-generation clinical trials and the implementation of molecularly-guided cancer therapies in the future. PMID:28256574

Airway epithelial repair in health and disease: Orchestrator or simply a player?

PubMed

Iosifidis, Thomas; Garratt, Luke W; Coombe, Deirdre R; Knight, Darryl A; Stick, Stephen M; Kicic, Anthony

2016-04-01

Epithelial cells represent the most important surface of contact in the body and form the first line of defence of the body to external environment. Consequently, epithelia have numerous roles in order to maintain a homeostatic defence barrier. Although the epithelium has been extensively studied over several decades, it remains the focus of new research, indicating a lack of understanding that continues to exist around these cells in specific disease settings. Importantly, evidence is emerging that airway epithelial cells in particular have varied complex functions rather than simple passive roles. One area of current interest is its role following injury. In particular, the epithelial-specific cellular mechanisms regulating their migration during wound repair remain poorly understood and remain an area that requires much needed investigation. A better understanding of the physiological, cellular and molecular wound repair mechanisms could assist in elucidating pathological processes that contribute to airway epithelial pathology. This review attempts to highlight migration-specific and cell-extracellular matrix (ECM) aspects of repair used by epithelial cells under normal and disease settings, in the context of human airways. © 2016 Asian Pacific Society of Respirology.

Manufacture of tumor- and virus-specific T lymphocytes for adoptive cell therapies

PubMed Central

Wang, X; Rivière, I

2015-01-01

Adoptive transfer of tumor-infiltrating lymphocytes (TILs) and genetically engineered T lymphocytes expressing chimeric antigen receptors (CARs) or conventional alpha/beta T-cell receptors (TCRs), collectively termed adoptive cell therapy (ACT), is an emerging novel strategy to treat cancer patients. Application of ACT has been constrained by the ability to isolate and expand functional tumor-reactive T cells. The transition of ACT from a promising experimental regimen to an established standard of care treatment relies largely on the establishment of safe, efficient, robust and cost-effective cell manufacturing protocols. The manufacture of cellular products under current good manufacturing practices (cGMPs) has a critical role in the process. Herein, we review current manufacturing methods for the large-scale production of clinical-grade TILs, virus-specific and genetically modified CAR or TCR transduced T cells in the context of phase I/II clinical trials as well as the regulatory pathway to get these complex personalized cellular products to the clinic. PMID:25721207

Manufacture of tumor- and virus-specific T lymphocytes for adoptive cell therapies.

PubMed

Wang, X; Rivière, I

2015-03-01

Adoptive transfer of tumor-infiltrating lymphocytes (TILs) and genetically engineered T lymphocytes expressing chimeric antigen receptors (CARs) or conventional alpha/beta T-cell receptors (TCRs), collectively termed adoptive cell therapy (ACT), is an emerging novel strategy to treat cancer patients. Application of ACT has been constrained by the ability to isolate and expand functional tumor-reactive T cells. The transition of ACT from a promising experimental regimen to an established standard of care treatment relies largely on the establishment of safe, efficient, robust and cost-effective cell manufacturing protocols. The manufacture of cellular products under current good manufacturing practices (cGMPs) has a critical role in the process. Herein, we review current manufacturing methods for the large-scale production of clinical-grade TILs, virus-specific and genetically modified CAR or TCR transduced T cells in the context of phase I/II clinical trials as well as the regulatory pathway to get these complex personalized cellular products to the clinic.

ADAM-17: The Enzyme That Does It All

PubMed Central

Gooz, Monika

2010-01-01

This review focuses on the role of ADAM-17 in disease. Since its debut as the tumor necrosis factor converting enzyme or TACE, ADAM-17 has been reported to be an indispensible regulator of almost every cellular event from proliferation to migration. The central role of ADAM-17 in cell regulation is rooted in its diverse array of substrates: cytokines, growth factors, and their receptors as well as adhesion molecules are activated or inactivated by their cleavage with ADAM-17. It is therefore not surprising that ADAM-17 is implicated in numerous human diseases including cancer, heart disease, diabetes, rheumatoid arthritis, kidney fibrosis, Alzheimer’s disease, and is a promising target for future treatments. The specific role of ADAM-17 in the pathophysiology of these diseases is very complex and depends on the cellular context. To exploit the therapeutic potential of ADAM-17, it is important to understand how its activity is regulated and how specific organs and cells can be targeted to inactivate or activate the enzyme. PMID:20184396

KDM1 Class Flavin-Dependent Protein Lysine Demethylases

PubMed Central

Burg, Jonathan M.; Link, Jennifer E.; Morgan, Brittany S.; Heller, Frederick J.; Hargrove, Amanda E.; McCafferty, Dewey G.

2015-01-01

Flavin-dependent, lysine-specific protein demethylases (KDM1s) are a subfamily of amine oxidases that catalyze the selective posttranslational oxidative demethylation of methyllysine side chains within protein and peptide substrates. KDM1s participate in the widespread epigenetic regulation of both normal and disease state transcriptional programs. Their activities are central to various cellular functions, such as hematopoietic and neuronal differentiation, cancer proliferation and metastasis, and viral lytic replication and establishment of latency. Interestingly, KDM1s function as catalytic subunits within complexes with coregulatory molecules that modulate enzymatic activity of the demethylases and coordinate their access to specific substrates at distinct sites within the cell and chromatin. Although several classes of KDM1 -selective small molecule inhibitors have been recently developed, these pan-active site inhibition strategies lack the ability to selectively discriminate between KDM1 activity in specific, and occasionally opposing, functional contexts within these complexes. Here we review the discovery of this class of demethylases, their structures, chemical mechanisms, and specificity. Additionally, we review inhibition of this class of enzymes as well as emerging interactions with coregulatory molecules that regulate demethylase activity in highly specific functional contexts of biological and potential therapeutic importance. PMID:25787087

A case of cellular alchemy: lineage reprogramming and its potential in regenerative medicine

PubMed Central

Asuelime, Grace E.; Shi, Yanhong

2012-01-01

The field of regenerative medicine is rapidly gaining momentum as an increasing number of reports emerge concerning the induced conversions observed in cellular fate reprogramming. While in recent years, much attention has been focused on the conversion of fate-committed somatic cells to an embryonic-like or pluripotent state, there are still many limitations associated with the applications of induced pluripotent stem cell reprogramming, including relatively low reprogramming efficiency, the times required for the reprogramming event to take place, the epigenetic instability, and the tumorigenicity associated with the pluripotent state. On the other hand, lineage reprogramming involves the conversion from one mature cell type to another without undergoing conversion to an unstable intermediate. It provides an alternative approach in regenerative medicine that has a relatively lower risk of tumorigenesis and increased efficiency within specific cellular contexts. While lineage reprogramming provides exciting potential, there is still much to be assessed before this technology is ready to be applied in a clinical setting. PMID:22371436

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.

Olfactory epithelium: Cells, clinical disorders, and insights from an adult stem cell niche

PubMed Central

Choi, Rhea

2018-01-01

Disorders causing a loss of the sense of smell remain a therapeutic challenge. Basic research has, however, greatly expanded our knowledge of the organization and function of the olfactory system. This review describes advances in our understanding of the cellular components of the peripheral olfactory system, specifically the olfactory epithelium in the nose. The article discusses recent findings regarding the mechanisms involved in regeneration and cellular renewal from basal stem cells in the adult olfactory epithelium, considering the strategies involved in embryonic olfactory development and insights from research on other stem cell niches. In the context of clinical conditions causing anosmia, the current view of adult olfactory neurogenesis, tissue homeostasis, and failures in these processes is considered, along with current and future treatment strategies. Level of Evidence NA PMID:29492466

Shaping Gene Expression by Landscaping Chromatin Architecture: Lessons from a Master.

PubMed

Sartorelli, Vittorio; Puri, Pier Lorenzo

2018-05-19

Since its discovery as a skeletal muscle-specific transcription factor able to reprogram somatic cells into differentiated myofibers, MyoD has provided an instructive model to understand how transcription factors regulate gene expression. Reciprocally, studies of other transcriptional regulators have provided testable hypotheses to further understand how MyoD activates transcription. Using MyoD as a reference, in this review, we discuss the similarities and differences in the regulatory mechanisms employed by tissue-specific transcription factors to access DNA and regulate gene expression by cooperatively shaping the chromatin landscape within the context of cellular differentiation. Copyright © 2018 Elsevier Inc. All rights reserved.

Optogenetic stimulation of a hippocampal engram activates fear memory recall.

PubMed

Liu, Xu; Ramirez, Steve; Pang, Petti T; Puryear, Corey B; Govindarajan, Arvind; Deisseroth, Karl; Tonegawa, Susumu

2012-03-22

A specific memory is thought to be encoded by a sparse population of neurons. These neurons can be tagged during learning for subsequent identification and manipulation. Moreover, their ablation or inactivation results in reduced memory expression, suggesting their necessity in mnemonic processes. However, the question of sufficiency remains: it is unclear whether it is possible to elicit the behavioural output of a specific memory by directly activating a population of neurons that was active during learning. Here we show in mice that optogenetic reactivation of hippocampal neurons activated during fear conditioning is sufficient to induce freezing behaviour. We labelled a population of hippocampal dentate gyrus neurons activated during fear learning with channelrhodopsin-2 (ChR2) and later optically reactivated these neurons in a different context. The mice showed increased freezing only upon light stimulation, indicating light-induced fear memory recall. This freezing was not detected in non-fear-conditioned mice expressing ChR2 in a similar proportion of cells, nor in fear-conditioned mice with cells labelled by enhanced yellow fluorescent protein instead of ChR2. Finally, activation of cells labelled in a context not associated with fear did not evoke freezing in mice that were previously fear conditioned in a different context, suggesting that light-induced fear memory recall is context specific. Together, our findings indicate that activating a sparse but specific ensemble of hippocampal neurons that contribute to a memory engram is sufficient for the recall of that memory. Moreover, our experimental approach offers a general method of mapping cellular populations bearing memory engrams.

Optogenetic stimulation of a hippocampal engram activates fear memory recall

PubMed Central

Liu, Xu; Ramirez, Steve; Pang, Petti T.; Puryear, Corey B.; Govindarajan, Arvind; Deisseroth, Karl; Tonegawa, Susumu

2012-01-01

A specific memory is thought to be encoded by a sparse population of neurons1,2. These neurons can be tagged during learning for subsequent identification3 and manipulation4,5,6. Moreover, their ablation or inactivation results in reduced memory expression, suggesting their necessity in mnemonic processes. However, a critical question of sufficiency remains: can one elicit the behavioral output of a specific memory by directly activating a population of neurons that was active during learning? Here we show that optogenetic reactivation of hippocampal neurons activated during fear conditioning is sufficient to induce freezing behavior. We labeled a population of hippocampal dentate gyrus neurons activated during fear learning with channelrhodopsin-2 (ChR2)7,8 and later optically reactivated these neurons in a different context. The mice showed increased freezing only upon light stimulation, indicating light-induced fear memory recall. This freezing was not detected in non-fear conditioned mice expressing ChR2 in a similar proportion of cells, nor in fear conditioned mice with cells labeled by EYFP instead of ChR2. Finally, activation of cells labeled in a context not associated with fear did not evoke freezing in mice that were previously fear conditioned in a different context, suggesting that light-induced fear memory recall is context-specific. Together, our findings indicate that activating a sparse but specific ensemble of hippocampal neurons that contribute to a memory engram is sufficient for the recall of that memory. Moreover, our experimental approach offers a general method of mapping cellular populations bearing memory engrams. PMID:22441246

In silico design of context-responsive mammalian promoters with user-defined functionality

PubMed Central

Gibson, Suzanne J.; Hatton, Diane

2017-01-01

Abstract Comprehensive de novo-design of complex mammalian promoters is restricted by unpredictable combinatorial interactions between constituent transcription factor regulatory elements (TFREs). In this study, we show that modular binding sites that do not function cooperatively can be identified by analyzing host cell transcription factor expression profiles, and subsequently testing cognate TFRE activities in varying homotypic and heterotypic promoter architectures. TFREs that displayed position-insensitive, additive function within a specific expression context could be rationally combined together in silico to create promoters with highly predictable activities. As TFRE order and spacing did not affect the performance of these TFRE-combinations, compositions could be specifically arranged to preclude the formation of undesirable sequence features. This facilitated simple in silico-design of promoters with context-required, user-defined functionalities. To demonstrate this, we de novo-created promoters for biopharmaceutical production in CHO cells that exhibited precisely designed activity dynamics and long-term expression-stability, without causing observable retroactive effects on cellular performance. The design process described can be utilized for applications requiring context-responsive, customizable promoter function, particularly where co-expression of synthetic TFs is not suitable. Although the synthetic promoter structure utilized does not closely resemble native mammalian architectures, our findings also provide additional support for a flexible billboard model of promoter regulation. PMID:28977454

Exploring the Spatial and Temporal Organization of a Cell’s Proteome

PubMed Central

Beck, Martin; Topf, Maya; Frazier, Zachary; Tjong, Harianto; Xu, Min; Zhang, Shihua; Alber, Frank

2013-01-01

To increase our current understanding of cellular processes, such as cell signaling and division, knowledge is needed about the spatial and temporal organization of the proteome at different organizational levels. These levels cover a wide range of length and time scales: from the atomic structures of macromolecules for inferring their molecular function, to the quantitative description of their abundance, and distribution in the cell. Emerging new experimental technologies are greatly increasing the availability of such spatial information on the molecular organization in living cells. This review addresses three fields that have significantly contributed to our understanding of the proteome’s spatial and temporal organization: first, methods for the structure determination of individual macromolecular assemblies, specifically the fitting of atomic structures into density maps generated from electron microscopy techniques; second, research that visualizes the spatial distributions of these complexes within the cellular context using cryo electron tomography techniques combined with computational image processing; and third, methods for the spatial modeling of the dynamic organization of the proteome, specifically those methods for simulating reaction and diffusion of proteins and complexes in crowded intracellular fluids. The long-term goal is to integrate the varied data about a proteome’s organization into a spatially explicit, predictive model of cellular processes. PMID:21094684

On the micro-indentation of plant cells in a tissue context

NASA Astrophysics Data System (ADS)

Mosca, Gabriella; Sapala, Aleksandra; Strauss, Soeren; Routier-Kierzkowska, Anne-Lise; Smith, Richard S.

2017-02-01

The effect of geometry on cell stiffness measured with micro-indentation techniques has been explored in single cells, however it is unclear if results on single cells can be readily transferred to indentation experiments performed on a tissue in vivo. Here we explored this question by using simulation models of osmotic treatments and micro-indentation experiments on 3D multicellular tissues with the finite element method. We found that the cellular context does affect measured cell stiffness, and that several cells of context in each direction are required for optimal results. We applied the model to micro-indentation data obtained with cellular force microscopy on the sepal of A. thaliana, and found that differences in measured stiffness could be explained by cellular geometry, and do not necessarily indicate differences in cell wall material properties or turgor pressure.

Self-organization versus Watchmaker: ambiguity of molecular recognition and design charts of cellular circuitry.

PubMed

Kurakin, Alexei

2007-01-01

A large body of experimental evidence indicates that the specific molecular interactions and/or chemical conversions depicted as links in the conventional diagrams of cellular signal transduction and metabolic pathways are inherently probabilistic, ambiguous and context-dependent. Being the inevitable consequence of the dynamic nature of protein structure in solution, the ambiguity of protein-mediated interactions and conversions challenges the conceptual adequacy and practical usefulness of the mechanistic assumptions and inferences embodied in the design charts of cellular circuitry. It is argued that the reconceptualization of molecular recognition and cellular organization within the emerging interpretational framework of self-organization, which is expanded here to include such concepts as bounded stochasticity, evolutionary memory, and adaptive plasticity offers a significantly more adequate representation of experimental reality than conventional mechanistic conceptions do. Importantly, the expanded framework of self-organization appears to be universal and scale-invariant, providing conceptual continuity across multiple scales of biological organization, from molecules to societies. This new conceptualization of biological phenomena suggests that such attributes of intelligence as adaptive plasticity, decision-making, and memory are enforced by evolution at different scales of biological organization and may represent inherent properties of living matter. (c) 2007 John Wiley & Sons, Ltd.

Drosophila as a genetic and cellular model for studies on axonal growth

PubMed Central

Sánchez-Soriano, Natalia; Tear, Guy; Whitington, Paul; Prokop, Andreas

2007-01-01

One of the most fascinating processes during nervous system development is the establishment of stereotypic neuronal networks. An essential step in this process is the outgrowth and precise navigation (pathfinding) of axons and dendrites towards their synaptic partner cells. This phenomenon was first described more than a century ago and, over the past decades, increasing insights have been gained into the cellular and molecular mechanisms regulating neuronal growth and navigation. Progress in this area has been greatly assisted by the use of simple and genetically tractable invertebrate model systems, such as the fruit fly Drosophila melanogaster. This review is dedicated to Drosophila as a genetic and cellular model to study axonal growth and demonstrates how it can and has been used for this research. We describe the various cellular systems of Drosophila used for such studies, insights into axonal growth cones and their cytoskeletal dynamics, and summarise identified molecular signalling pathways required for growth cone navigation, with particular focus on pathfinding decisions in the ventral nerve cord of Drosophila embryos. These Drosophila-specific aspects are viewed in the general context of our current knowledge about neuronal growth. PMID:17475018

Interaction of the Spo20 membrane-sensor motif with phosphatidic acid and other anionic lipids, and influence of the membrane environment.

PubMed

Horchani, Habib; de Saint-Jean, Maud; Barelli, Hélène; Antonny, Bruno

2014-01-01

The yeast protein Spo20 contains a regulatory amphipathic motif that has been suggested to recognize phosphatidic acid, a lipid involved in signal transduction, lipid metabolism and membrane fusion. We have investigated the interaction of the Spo20 amphipathic motif with lipid membranes using a bioprobe strategy that consists in appending this motif to the end of a long coiled-coil, which can be coupled to a GFP reporter for visualization in cells. The resulting construct is amenable to in vitro and in vivo experiments and allows unbiased comparison between amphipathic helices of different chemistry. In vitro, the Spo20 bioprobe responded to small variations in the amount of phosphatidic acid. However, this response was not specific. The membrane binding of the probe depended on the presence of phosphatidylethanolamine and also integrated the contribution of other anionic lipids, including phosphatidylserine and phosphatidyl-inositol-(4,5)bisphosphate. Inverting the sequence of the Spo20 motif neither affected the ability of the probe to interact with anionic liposomes nor did it modify its cellular localization, making a stereo-specific mode of phosphatidic acid recognition unlikely. Nevertheless, the lipid binding properties and the cellular localization of the Spo20 alpha-helix differed markedly from that of another amphipathic motif, Amphipathic Lipid Packing Sensor (ALPS), suggesting that even in the absence of stereo specific interactions, amphipathic helices can act as subcellular membrane targeting determinants in a cellular context.

Mechanisms of phagocytosis and host clearance of Pseudomonas aeruginosa

PubMed Central

Lovewell, Rustin R.; Patankar, Yash R.

2014-01-01

Pseudomonas aeruginosa is an opportunistic bacterial pathogen responsible for a high incidence of acute and chronic pulmonary infection. These infections are particularly prevalent in patients with chronic obstructive pulmonary disease and cystic fibrosis: much of the morbidity and pathophysiology associated with these diseases is due to a hypersusceptibility to bacterial infection. Innate immunity, primarily through inflammatory cytokine production, cellular recruitment, and phagocytic clearance by neutrophils and macrophages, is the key to endogenous control of P. aeruginosa infection. In this review, we highlight recent advances toward understanding the innate immune response to P. aeruginosa, with a focus on the role of phagocytes in control of P. aeruginosa infection. Specifically, we summarize the cellular and molecular mechanisms of phagocytic recognition and uptake of P. aeruginosa, and how current animal models of P. aeruginosa infection reflect clinical observations in the context of phagocytic clearance of the bacteria. Several notable phenotypic changes to the bacteria are consistently observed during chronic pulmonary infections, including changes to mucoidy and flagellar motility, that likely enable or reflect their ability to persist. These traits are likewise examined in the context of how the bacteria avoid phagocytic clearance, inflammation, and sterilizing immunity. PMID:24464809

Mechanisms of phagocytosis and host clearance of Pseudomonas aeruginosa.

PubMed

Lovewell, Rustin R; Patankar, Yash R; Berwin, Brent

2014-04-01

Pseudomonas aeruginosa is an opportunistic bacterial pathogen responsible for a high incidence of acute and chronic pulmonary infection. These infections are particularly prevalent in patients with chronic obstructive pulmonary disease and cystic fibrosis: much of the morbidity and pathophysiology associated with these diseases is due to a hypersusceptibility to bacterial infection. Innate immunity, primarily through inflammatory cytokine production, cellular recruitment, and phagocytic clearance by neutrophils and macrophages, is the key to endogenous control of P. aeruginosa infection. In this review, we highlight recent advances toward understanding the innate immune response to P. aeruginosa, with a focus on the role of phagocytes in control of P. aeruginosa infection. Specifically, we summarize the cellular and molecular mechanisms of phagocytic recognition and uptake of P. aeruginosa, and how current animal models of P. aeruginosa infection reflect clinical observations in the context of phagocytic clearance of the bacteria. Several notable phenotypic changes to the bacteria are consistently observed during chronic pulmonary infections, including changes to mucoidy and flagellar motility, that likely enable or reflect their ability to persist. These traits are likewise examined in the context of how the bacteria avoid phagocytic clearance, inflammation, and sterilizing immunity.

Modeling oscillatory dynamics in brain microcircuits as a way to help uncover neurological disease mechanisms: A proposal

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

Skinner, F. K.; Department of Medicine; Department of Physiology, University of Toronto Medical Sciences Building, 3rd Floor, 1 King's College Circle, Toronto, Ontario M5S 1A8

There is an undisputed need and requirement for theoretical and computational studies in Neuroscience today. Furthermore, it is clear that oscillatory dynamical output from brain networks is representative of various behavioural states, and it is becoming clear that one could consider these outputs as measures of normal and pathological brain states. Although mathematical modeling of oscillatory dynamics in the context of neurological disease exists, it is a highly challenging endeavour because of the many levels of organization in the nervous system. This challenge is coupled with the increasing knowledge of cellular specificity and network dysfunction that is associated with disease.more » Recently, whole hippocampus in vitro preparations from control animals have been shown to spontaneously express oscillatory activities. In addition, when using preparations derived from animal models of disease, these activities show particular alterations. These preparations present an opportunity to address challenges involved with using models to gain insight because of easier access to simultaneous cellular and network measurements, and pharmacological modulations. We propose that by developing and using models with direct links to experiment at multiple levels, which at least include cellular and microcircuit, a cycling can be set up and used to help us determine critical mechanisms underlying neurological disease. We illustrate our proposal using our previously developed inhibitory network models in the context of these whole hippocampus preparations and show the importance of having direct links at multiple levels.« less

Smooth Muscle Cell Genome Browser: Enabling the Identification of Novel Serum Response Factor Target Genes

PubMed Central

Lee, Moon Young; Park, Chanjae; Berent, Robyn M.; Park, Paul J.; Fuchs, Robert; Syn, Hannah; Chin, Albert; Townsend, Jared; Benson, Craig C.; Redelman, Doug; Shen, Tsai-wei; Park, Jong Kun; Miano, Joseph M.; Sanders, Kenton M.; Ro, Seungil

2015-01-01

Genome-scale expression data on the absolute numbers of gene isoforms offers essential clues in cellular functions and biological processes. Smooth muscle cells (SMCs) perform a unique contractile function through expression of specific genes controlled by serum response factor (SRF), a transcription factor that binds to DNA sites known as the CArG boxes. To identify SRF-regulated genes specifically expressed in SMCs, we isolated SMC populations from mouse small intestine and colon, obtained their transcriptomes, and constructed an interactive SMC genome and CArGome browser. To our knowledge, this is the first online resource that provides a comprehensive library of all genetic transcripts expressed in primary SMCs. The browser also serves as the first genome-wide map of SRF binding sites. The browser analysis revealed novel SMC-specific transcriptional variants and SRF target genes, which provided new and unique insights into the cellular and biological functions of the cells in gastrointestinal (GI) physiology. The SRF target genes in SMCs, which were discovered in silico, were confirmed by proteomic analysis of SMC-specific Srf knockout mice. Our genome browser offers a new perspective into the alternative expression of genes in the context of SRF binding sites in SMCs and provides a valuable reference for future functional studies. PMID:26241044

Context Processing and the Neurobiology of Post-Traumatic Stress Disorder

PubMed Central

Liberzon, Israel; Abelson, James L.

2016-01-01

Summary Progress in clinical and affective neuroscience is redefining psychiatric illness as symptomatic expression of cellular/molecular dysfunctions in specific brain circuits. Post-traumatic stress disorder (PTSD) has been an exemplar of this progress, with improved understanding of neurobiological systems subserving fear learning, salience detection, and emotion regulation explaining much of its phenomenology and neurobiology. However, many features remain unexplained and a parsimonious model that more fully accounts for symptoms and the core neurobiology remains elusive. Contextual processing is a key modulatory function of hippocampal-prefrontal-thalamic circuitry, allowing organisms to disambiguate cues and derive situation-specific meaning from the world. We propose that dysregulation within this context-processing circuit is at the core of PTSD pathophysiology, accounting for much of its phenomenology and most of its biological findings. Understanding core mechanisms like this, and their underlying neural circuits, will sharpen diagnostic precision and understanding of risk factors, enhancing our ability to develop preventive and “personalized” interventions. PMID:27710783

Hippocampal mechanisms for the context-dependent retrieval of episodes

PubMed Central

Hasselmo, Michael E.; Eichenbaum, Howard B.

2008-01-01

Behaviors ranging from delivering newspapers to waiting tables depend on remembering previous episodes to avoid incorrect repetition. Physiologically, this requires mechanisms for long-term storage and selective retrieval of episodes based on time of occurrence, despite variable intervals and similarity of events in a familiar environment. Here, this process has been modeled based on physiological properties of the hippocampal formation, including mechanisms for sustained activity in entorhinal cortex and theta rhythm oscillations in hippocampal subregions. The model simulates the context-sensitive firing properties of hippocampal neurons including trial specific firing during spatial alternation and trial by trial changes in theta phase precession on a linear track. This activity is used to guide behavior, and lesions of the hippocampal network impair memory-guided behavior. The model links data at the cellular level to behavior at the systems level, describing a physiologically plausible mechanism for the brain to recall a given episode which occurred at a specific place and time. PMID:16263240

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

THE RGM/DRAGON FAMILY OF BMP CO-RECEPTORS

PubMed Central

Corradini, Elena; Babitt, Jodie L.; Lin, Herbert Y.

2013-01-01

The BMP signaling pathway controls a number of cell processes during development and in adult tissues. At the cellular level, ligands of the BMP family act by binding a hetero-tetrameric signaling complex, composed of two type I and two type II receptors. BMP ligands make use of a limited number of receptors, which in turn activate a common signal transduction cascade at the intracellular level. A complex regulatory network is required in order to activate the signaling cascade at proper times and locations, and to generate specific downstream effects in the appropriate cellular context. One such regulatory mechanism is the repulsive guidance molecule (RGM) family of BMP co-receptors. This article reviews the current knowledge regarding the structure, regulation, and function of RGMs, focusing on known and potential roles of RGMs in physiology and pathophysiology. PMID:19897400

Role of microRNAs in herpesvirus latency and persistence.

PubMed

Grey, Finn

2015-04-01

The identification of virally encoded microRNAs (miRNAs) has had a major impact on the field of herpes virology. Given their ability to target cellular and viral transcripts, and the lack of immune response to small RNAs, miRNAs represent an ideal mechanism of gene regulation during viral latency and persistence. In this review, we discuss the role of miRNAs in virus latency and persistence, specifically focusing on herpesviruses. We cover the current knowledge on miRNAs in establishing and maintaining virus latency and promoting survival of infected cells through targeting of both viral and cellular transcripts, highlighting key publications in the field. We also discuss potential areas of future research and how novel technologies may aid in determining how miRNAs shape virus latency in the context of herpesvirus infections. © 2015 The Author.

Regulation of mammalian cell differentiation by long non-coding RNAs

PubMed Central

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

2012-01-01

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

Cancer prevention and therapy through the modulation of the tumor microenvironment

PubMed Central

Casey, Stephanie C.; Amedei, Amedeo; Aquilano, Katia; Benencia, Fabian; Bhakta, Dipita; Boosani, Chandra S.; Chen, Sophie; Ciriolo, Maria Rosa; Crawford, Sarah; Fujii, Hiromasa; Georgakilas, Alexandros G.; Guha, Gunjan; Halicka, Dorota; Helferich, William G.; Heneberg, Petr; Honoki, Kanya; Kerkar, Sid P.; Mohammed, Sulma I.; Niccolai, Elena; Nowsheen, Somaira; Rupasinghe, H. P. Vasantha; Samadi, Abbas; Singh, Neetu; Talib, Wamidh H.; Venkateswaran, Vasundara; Whelan, Richard; Yang, Xujuan; Felsher, Dean W.

2015-01-01

Cancer arises in the context of an in vivo tumor microenvironment. This microenvironment is both a cause and consequence of tumorigenesis. Tumor and host cells co-evolve dynamically through indirect and direct cellular interactions, eliciting multiscale effects on many biological programs, including cellular proliferation, growth, and metabolism, as well as angiogenesis and hypoxia and innate and adapative immunity. Here we highlight specific biological processes that could be exploited as targets for the prevention and therapy of cancer. Specifically, we describe how inhibition of targets such as cholesterol synthesis and metabolites, reactive oxygen species and hypoxia, macrophage activation and conversion, indoleamine 2, 3-dioxygenase regulation of dendritic cells, vascular endothelial growth factor regulation of angiogenesis, fibrosis inhibition, endoglin, and Janus kinase signaling emerge as examples of important potential nexuses in the regulation of tumorigenesis and the tumor microenvironment that can be targeted. We have also identified therapeutic agents as approaches, in particular natural products such as berberine, resveratrol, onionin A, epigallocatechin gallate, genistein, curcumin, naringenin, desoxyrhapontigenin, piperine, and zerumbone, that may warrant further investigation to target the tumor microenvironment for the treatment and/or prevention of cancer. PMID:25865775

Mutations in NONO lead to syndromic intellectual disability and inhibitory synaptic defects.

PubMed

Mircsof, Dennis; Langouët, Maéva; Rio, Marlène; Moutton, Sébastien; Siquier-Pernet, Karine; Bole-Feysot, Christine; Cagnard, Nicolas; Nitschke, Patrick; Gaspar, Ludmila; Žnidarič, Matej; Alibeu, Olivier; Fritz, Ann-Kristina; Wolfer, David P; Schröter, Aileen; Bosshard, Giovanna; Rudin, Markus; Koester, Christina; Crestani, Florence; Seebeck, Petra; Boddaert, Nathalie; Prescott, Katrina; Hines, Rochelle; Moss, Steven J; Fritschy, Jean-Marc; Munnich, Arnold; Amiel, Jeanne; Brown, Steven A; Tyagarajan, Shiva K; Colleaux, Laurence

2015-12-01

The NONO protein has been characterized as an important transcriptional regulator in diverse cellular contexts. Here we show that loss of NONO function is a likely cause of human intellectual disability and that NONO-deficient mice have cognitive and affective deficits. Correspondingly, we find specific defects at inhibitory synapses, where NONO regulates synaptic transcription and gephyrin scaffold structure. Our data identify NONO as a possible neurodevelopmental disease gene and highlight the key role of the DBHS protein family in functional organization of GABAergic synapses.

Genetically targeted 3D visualisation of Drosophila neurons under Electron Microscopy and X-Ray Microscopy using miniSOG

PubMed Central

Ng, Julian; Browning, Alyssa; Lechner, Lorenz; Terada, Masako; Howard, Gillian; Jefferis, Gregory S. X. E.

2016-01-01

Large dimension, high-resolution imaging is important for neural circuit visualisation as neurons have both long- and short-range patterns: from axons and dendrites to the numerous synapses at terminal endings. Electron Microscopy (EM) is the favoured approach for synaptic resolution imaging but how such structures can be segmented from high-density images within large volume datasets remains challenging. Fluorescent probes are widely used to localise synapses, identify cell-types and in tracing studies. The equivalent EM approach would benefit visualising such labelled structures from within sub-cellular, cellular, tissue and neuroanatomical contexts. Here we developed genetically-encoded, electron-dense markers using miniSOG. We demonstrate their ability in 1) labelling cellular sub-compartments of genetically-targeted neurons, 2) generating contrast under different EM modalities, and 3) segmenting labelled structures from EM volumes using computer-assisted strategies. We also tested non-destructive X-ray imaging on whole Drosophila brains to evaluate contrast staining. This enabled us to target specific regions for EM volume acquisition. PMID:27958322

Anticancer activity of metal complexes: involvement of redox processes.

PubMed

Jungwirth, Ute; Kowol, Christian R; Keppler, Bernhard K; Hartinger, Christian G; Berger, Walter; Heffeter, Petra

2011-08-15

Cells require tight regulation of the intracellular redox balance and consequently of reactive oxygen species for proper redox signaling and maintenance of metal (e.g., of iron and copper) homeostasis. In several diseases, including cancer, this balance is disturbed. Therefore, anticancer drugs targeting the redox systems, for example, glutathione and thioredoxin, have entered focus of interest. Anticancer metal complexes (platinum, gold, arsenic, ruthenium, rhodium, copper, vanadium, cobalt, manganese, gadolinium, and molybdenum) have been shown to strongly interact with or even disturb cellular redox homeostasis. In this context, especially the hypothesis of "activation by reduction" as well as the "hard and soft acids and bases" theory with respect to coordination of metal ions to cellular ligands represent important concepts to understand the molecular modes of action of anticancer metal drugs. The aim of this review is to highlight specific interactions of metal-based anticancer drugs with the cellular redox homeostasis and to explain this behavior by considering chemical properties of the respective anticancer metal complexes currently either in (pre)clinical development or in daily clinical routine in oncology.

Anticancer Activity of Metal Complexes: Involvement of Redox Processes

PubMed Central

Jungwirth, Ute; Kowol, Christian R.; Keppler, Bernhard K.; Hartinger, Christian G.; Berger, Walter; Heffeter, Petra

2012-01-01

Cells require tight regulation of the intracellular redox balance and consequently of reactive oxygen species for proper redox signaling and maintenance of metal (e.g., of iron and copper) homeostasis. In several diseases, including cancer, this balance is disturbed. Therefore, anticancer drugs targeting the redox systems, for example, glutathione and thioredoxin, have entered focus of interest. Anticancer metal complexes (platinum, gold, arsenic, ruthenium, rhodium, copper, vanadium, cobalt, manganese, gadolinium, and molybdenum) have been shown to strongly interact with or even disturb cellular redox homeostasis. In this context, especially the hypothesis of “activation by reduction” as well as the “hard and soft acids and bases” theory with respect to coordination of metal ions to cellular ligands represent important concepts to understand the molecular modes of action of anticancer metal drugs. The aim of this review is to highlight specific interactions of metal-based anticancer drugs with the cellular redox homeostasis and to explain this behavior by considering chemical properties of the respective anticancer metal complexes currently either in (pre)clinical development or in daily clinical routine in oncology. PMID:21275772

Identification of shared and unique susceptibility pathways among cancers of the lung, breast, and prostate from genome-wide association studies and tissue-specific protein interactions

PubMed Central

Qian, David C.; Byun, Jinyoung; Han, Younghun; Greene, Casey S.; Field, John K.; Hung, Rayjean J.; Brhane, Yonathan; Mclaughlin, John R.; Fehringer, Gordon; Landi, Maria Teresa; Rosenberger, Albert; Bickeböller, Heike; Malhotra, Jyoti; Risch, Angela; Heinrich, Joachim; Hunter, David J.; Henderson, Brian E.; Haiman, Christopher A.; Schumacher, Fredrick R.; Eeles, Rosalind A.; Easton, Douglas F.; Seminara, Daniela; Amos, Christopher I.

2015-01-01

Results from genome-wide association studies (GWAS) have indicated that strong single-gene effects are the exception, not the rule, for most diseases. We assessed the joint effects of germline genetic variations through a pathway-based approach that considers the tissue-specific contexts of GWAS findings. From GWAS meta-analyses of lung cancer (12 160 cases/16 838 controls), breast cancer (15 748 cases/18 084 controls) and prostate cancer (14 160 cases/12 724 controls) in individuals of European ancestry, we determined the tissue-specific interaction networks of proteins expressed from genes that are likely to be affected by disease-associated variants. Reactome pathways exhibiting enrichment of proteins from each network were compared across the cancers. Our results show that pathways associated with all three cancers tend to be broad cellular processes required for growth and survival. Significant examples include the nerve growth factor (P = 7.86 × 10−33), epidermal growth factor (P = 1.18 × 10−31) and fibroblast growth factor (P = 2.47 × 10−31) signaling pathways. However, within these shared pathways, the genes that influence risk largely differ by cancer. Pathways found to be unique for a single cancer focus on more specific cellular functions, such as interleukin signaling in lung cancer (P = 1.69 × 10−15), apoptosis initiation by Bad in breast cancer (P = 3.14 × 10−9) and cellular responses to hypoxia in prostate cancer (P = 2.14 × 10−9). We present the largest comparative cross-cancer pathway analysis of GWAS to date. Our approach can also be applied to the study of inherited mechanisms underlying risk across multiple diseases in general. PMID:26483192

Transcriptome-Wide Analysis of Hepatitis B Virus-Mediated Changes to Normal Hepatocyte Gene Expression.

PubMed

Lamontagne, Jason; Mell, Joshua C; Bouchard, Michael J

2016-02-01

Globally, a chronic hepatitis B virus (HBV) infection remains the leading cause of primary liver cancer. The mechanisms leading to the development of HBV-associated liver cancer remain incompletely understood. In part, this is because studies have been limited by the lack of effective model systems that are both readily available and mimic the cellular environment of a normal hepatocyte. Additionally, many studies have focused on single, specific factors or pathways that may be affected by HBV, without addressing cell physiology as a whole. Here, we apply RNA-seq technology to investigate transcriptome-wide, HBV-mediated changes in gene expression to identify single factors and pathways as well as networks of genes and pathways that are affected in the context of HBV replication. Importantly, these studies were conducted in an ex vivo model of cultured primary hepatocytes, allowing for the transcriptomic characterization of this model system and an investigation of early HBV-mediated effects in a biologically relevant context. We analyzed differential gene expression within the context of time-mediated gene-expression changes and show that in the context of HBV replication a number of genes and cellular pathways are altered, including those associated with metabolism, cell cycle regulation, and lipid biosynthesis. Multiple analysis pipelines, as well as qRT-PCR and an independent, replicate RNA-seq analysis, were used to identify and confirm differentially expressed genes. HBV-mediated alterations to the transcriptome that we identified likely represent early changes to hepatocytes following an HBV infection, suggesting potential targets for early therapeutic intervention. Overall, these studies have produced a valuable resource that can be used to expand our understanding of the complex network of host-virus interactions and the impact of HBV-mediated changes to normal hepatocyte physiology on viral replication.

Dissecting the Calcium-Induced Differentiation of Human Primary Keratinocytes Stem Cells by Integrative and Structural Network Analyses

PubMed Central

Toufighi, Kiana; Yang, Jae-Seong; Luis, Nuno Miguel; Aznar Benitah, Salvador; Lehner, Ben; Serrano, Luis; Kiel, Christina

2015-01-01

The molecular details underlying the time-dependent assembly of protein complexes in cellular networks, such as those that occur during differentiation, are largely unexplored. Focusing on the calcium-induced differentiation of primary human keratinocytes as a model system for a major cellular reorganization process, we look at the expression of genes whose products are involved in manually-annotated protein complexes. Clustering analyses revealed only moderate co-expression of functionally related proteins during differentiation. However, when we looked at protein complexes, we found that the majority (55%) are composed of non-dynamic and dynamic gene products (‘di-chromatic’), 19% are non-dynamic, and 26% only dynamic. Considering three-dimensional protein structures to predict steric interactions, we found that proteins encoded by dynamic genes frequently interact with a common non-dynamic protein in a mutually exclusive fashion. This suggests that during differentiation, complex assemblies may also change through variation in the abundance of proteins that compete for binding to common proteins as found in some cases for paralogous proteins. Considering the example of the TNF-α/NFκB signaling complex, we suggest that the same core complex can guide signals into diverse context-specific outputs by addition of time specific expressed subunits, while keeping other cellular functions constant. Thus, our analysis provides evidence that complex assembly with stable core components and competition could contribute to cell differentiation. PMID:25946651

Intravital FRET: Probing Cellular and Tissue Function in Vivo

PubMed Central

Radbruch, Helena; Bremer, Daniel; Mothes, Ronja; Günther, Robert; Rinnenthal, Jan Leo; Pohlan, Julian; Ulbricht, Carolin; Hauser, Anja E.; Niesner, Raluca

2015-01-01

The development of intravital Förster Resonance Energy Transfer (FRET) is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss the advantages and pitfalls of two strategies to quantify FRET in vivo—ratiometrically and time-resolved by fluorescence lifetime imaging—and show their concrete application in the context of neuroinflammation in adult mice. PMID:26006244

Reference materials for cellular therapeutics.

PubMed

Bravery, Christopher A; French, Anna

2014-09-01

The development of cellular therapeutics (CTP) takes place over many years, and, where successful, the developer will anticipate the product to be in clinical use for decades. Successful demonstration of manufacturing and quality consistency is dependent on the use of complex analytical methods; thus, the risk of process and method drift over time is high. The use of reference materials (RM) is an established scientific principle and as such also a regulatory requirement. The various uses of RM in the context of CTP manufacturing and quality are discussed, along with why they are needed for living cell products and the analytical methods applied to them. Relatively few consensus RM exist that are suitable for even common methods used by CTP developers, such as flow cytometry. Others have also identified this need and made proposals; however, great care will be needed to ensure any consensus RM that result are fit for purpose. Such consensus RM probably will need to be applied to specific standardized methods, and the idea that a single RM can have wide applicability is challenged. Written standards, including standardized methods, together with appropriate measurement RM are probably the most appropriate way to define specific starting cell types. The characteristics of a specific CTP will to some degree deviate from those of the starting cells; consequently, a product RM remains the best solution where feasible. Each CTP developer must consider how and what types of RM should be used to ensure the reliability of their own analytical measurements. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Engineering peptide ligase specificity by proteomic identification of ligation sites.

PubMed

Weeks, Amy M; Wells, James A

2018-01-01

Enzyme-catalyzed peptide ligation is a powerful tool for site-specific protein bioconjugation, but stringent enzyme-substrate specificity limits its utility. We developed an approach for comprehensively characterizing peptide ligase specificity for N termini using proteome-derived peptide libraries. We used this strategy to characterize the ligation efficiency for >25,000 enzyme-substrate pairs in the context of the engineered peptide ligase subtiligase and identified a family of 72 mutant subtiligases with activity toward N-terminal sequences that were previously recalcitrant to modification. We applied these mutants individually for site-specific bioconjugation of purified proteins, including antibodies, and in algorithmically selected combinations for sequencing of the cellular N terminome with reduced sequence bias. We also developed a web application to enable algorithmic selection of the most efficient subtiligase variant(s) for bioconjugation to user-defined sequences. Our methods provide a new toolbox of enzymes for site-specific protein modification and a general approach for rapidly defining and engineering peptide ligase specificity.

Drosophila as a model system to study autophagy.

PubMed

Zirin, Jonathan; Perrimon, Norbert

2010-12-01

Originally identified as a response to starvation in yeast, autophagy is now understood to fulfill a variety of roles in higher eukaryotes, from the maintenance of cellular homeostasis to the cellular response to stress, starvation, and infection. Although genetics and biochemical studies in yeast have identified many components involved in autophagy, the findings that some of the essential components of the yeast pathway are missing in higher organisms underscore the need to study autophagy in more complex systems. This review focuses on the use of the fruitfly, Drosophila melanogaster as a model system for analysis of autophagy. Drosophila is an organism well-suited for genetic analysis and represents an intermediate between yeast and mammals with respect to conservation of the autophagy machinery. Furthermore, the complex biology and physiology of Drosophila presents an opportunity to model human diseases in a tissue specific and analogous context.

New Insights into Thyroid Hormone Action

PubMed Central

Mendoza, Arturo; Hollenberg, Anthony N.

2017-01-01

Thyroid hormones (TH) are endocrine messengers essential for normal development and function of virtually every vertebrate. The hypothalamic-pituitary-thyroid axis is exquisitely modulated to maintain nearly constant TH (T4 and T3) concentrations in circulation. However peripheral tissues and the CNS control the intracellular availability of TH, suggesting that circulating concentrations of TH are not fully representative of what each cell type sees. Indeed, recent work in the field has identified that TH transporters, deiodinases and thyroid hormone receptor coregulators can strongly control tissue-specific sensitivity to a set amount of TH. Furthermore, the mechanism by which the thyroid hormone receptors regulate target gene expression can vary by gene, tissue and cellular context. This review will highlight novel insights into the machinery that controls the cellular response to TH, which include unique signaling cascades. These findings shed new light into the pathophysiology of human diseases caused by abnormal TH signaling. PMID:28174093

Expanding the metabolic engineering toolbox with directed evolution.

PubMed

Abatemarco, Joseph; Hill, Andrew; Alper, Hal S

2013-12-01

Cellular systems can be engineered into factories that produce high-value chemicals from renewable feedstock. Such an approach requires an expanded toolbox for metabolic engineering. Recently, protein engineering and directed evolution strategies have started to play a growing and critical role within metabolic engineering. This review focuses on the various ways in which directed evolution can be applied in conjunction with metabolic engineering to improve product yields. Specifically, we discuss the application of directed evolution on both catalytic and non-catalytic traits of enzymes, on regulatory elements, and on whole genomes in a metabolic engineering context. We demonstrate how the goals of metabolic pathway engineering can be achieved in part through evolving cellular parts as opposed to traditional approaches that rely on gene overexpression and deletion. Finally, we discuss the current limitations in screening technology that hinder the full implementation of a metabolic pathway-directed evolution approach. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Regulatory T cells as suppressors of anti-tumor immunity: Role of metabolism.

PubMed

De Rosa, Veronica; Di Rella, Francesca; Di Giacomo, Antonio; Matarese, Giuseppe

2017-06-01

Novel concepts in immunometabolism support the hypothesis that glucose consumption is also used to modulate anti-tumor immune responses, favoring growth and expansion of specific cellular subsets defined in the past as suppressor T cells and currently reborn as regulatory T (Treg) cells. During the 1920s, Otto Warburg and colleagues observed that tumors consumed high amounts of glucose compared to normal tissues, even in the presence of oxygen and completely functioning mitochondria. However, the role of the Warburg Effect is still not completely understood, particularly in the context of an ongoing anti-tumor immune response. Current experimental evidence suggests that tumor-derived metabolic restrictions can drive T cell hyporesponsiveness and immune tolerance. For example, several glycolytic enzymes, deregulated in cancer, contribute to tumor progression independently from their canonical metabolic activity. Indeed, they can control apoptosis, gene expression and activation of specific intracellular pathways, thus suggesting a direct link between metabolic switches and pro-tumorigenic transcriptional programs. Focus of this review is to define the specific metabolic pathways controlling Treg cell immunobiology in the context of anti-tumor immunity and tumor progression. Copyright © 2017 Elsevier Ltd. All rights reserved.

Technology Learning Activities: Columbus Sailed the Ocean Blue, the Cellular Connection, Emergency Shelter.

ERIC Educational Resources Information Center

Etchison, Cindy; Deal, Walter F., III

1992-01-01

Presents learning activities such as planning and building a sailboat, manufacturing cellular phone cases, and designing and building emergency shelters. Includes the context, the challenge, resources used, objectives, materials needed, and an evaluation. (JOW)

A C-terminally truncated form of β-catenin acts as a novel regulator of Wnt/β-catenin signaling in planarians

PubMed Central

Rabaneda-Lombarte, Neus; Gelabert, Maria; Xie, Jianlei; Wu, Wei

2017-01-01

β-Catenin, the core element of the Wnt/β-catenin pathway, is a multifunctional and evolutionarily conserved protein which performs essential roles in a variety of developmental and homeostatic processes. Despite its crucial roles, the mechanisms that control its context-specific functions in time and space remain largely unknown. The Wnt/β-catenin pathway has been extensively studied in planarians, flatworms with the ability to regenerate and remodel the whole body, providing a ‘whole animal’ developmental framework to approach this question. Here we identify a C-terminally truncated β-catenin (β-catenin4), generated by gene duplication, that is required for planarian photoreceptor cell specification. Our results indicate that the role of β-catenin4 is to modulate the activity of β-catenin1, the planarian β-catenin involved in Wnt signal transduction in the nucleus, mediated by the transcription factor TCF-2. This inhibitory form of β-catenin, expressed in specific cell types, would provide a novel mechanism to modulate nuclear β-catenin signaling levels. Genomic searches and in vitro analysis suggest that the existence of a C-terminally truncated form of β-catenin could be an evolutionarily conserved mechanism to achieve a fine-tuned regulation of Wnt/β-catenin signaling in specific cellular contexts. PMID:28976975

A C-terminally truncated form of β-catenin acts as a novel regulator of Wnt/β-catenin signaling in planarians.

PubMed

Su, Hanxia; Sureda-Gomez, Miquel; Rabaneda-Lombarte, Neus; Gelabert, Maria; Xie, Jianlei; Wu, Wei; Adell, Teresa

2017-10-01

β-Catenin, the core element of the Wnt/β-catenin pathway, is a multifunctional and evolutionarily conserved protein which performs essential roles in a variety of developmental and homeostatic processes. Despite its crucial roles, the mechanisms that control its context-specific functions in time and space remain largely unknown. The Wnt/β-catenin pathway has been extensively studied in planarians, flatworms with the ability to regenerate and remodel the whole body, providing a 'whole animal' developmental framework to approach this question. Here we identify a C-terminally truncated β-catenin (β-catenin4), generated by gene duplication, that is required for planarian photoreceptor cell specification. Our results indicate that the role of β-catenin4 is to modulate the activity of β-catenin1, the planarian β-catenin involved in Wnt signal transduction in the nucleus, mediated by the transcription factor TCF-2. This inhibitory form of β-catenin, expressed in specific cell types, would provide a novel mechanism to modulate nuclear β-catenin signaling levels. Genomic searches and in vitro analysis suggest that the existence of a C-terminally truncated form of β-catenin could be an evolutionarily conserved mechanism to achieve a fine-tuned regulation of Wnt/β-catenin signaling in specific cellular contexts.

Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche.

PubMed

Van Doorslaer, Koenraad; DeSalle, Rob; Einstein, Mark H; Burk, Robert D

2015-06-01

In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution.

Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche

PubMed Central

Van Doorslaer, Koenraad; DeSalle, Rob; Einstein, Mark H.; Burk, Robert D.

2015-01-01

In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution. PMID:26086730

Comprehensive structural and substrate specificity classification of the Saccharomyces cerevisiae methyltransferome.

PubMed

Wlodarski, Tomasz; Kutner, Jan; Towpik, Joanna; Knizewski, Lukasz; Rychlewski, Leszek; Kudlicki, Andrzej; Rowicka, Maga; Dziembowski, Andrzej; Ginalski, Krzysztof

2011-01-01

Methylation is one of the most common chemical modifications of biologically active molecules and it occurs in all life forms. Its functional role is very diverse and involves many essential cellular processes, such as signal transduction, transcriptional control, biosynthesis, and metabolism. Here, we provide further insight into the enzymatic methylation in S. cerevisiae by conducting a comprehensive structural and functional survey of all the methyltransferases encoded in its genome. Using distant homology detection and fold recognition, we found that the S. cerevisiae methyltransferome comprises 86 MTases (53 well-known and 33 putative with unknown substrate specificity). Structural classification of their catalytic domains shows that these enzymes may adopt nine different folds, the most common being the Rossmann-like. We also analyzed the domain architecture of these proteins and identified several new domain contexts. Interestingly, we found that the majority of MTase genes are periodically expressed during yeast metabolic cycle. This finding, together with calculated isoelectric point, fold assignment and cellular localization, was used to develop a novel approach for predicting substrate specificity. Using this approach, we predicted the general substrates for 24 of 33 putative MTases and confirmed these predictions experimentally in both cases tested. Finally, we show that, in S. cerevisiae, methylation is carried out by 34 RNA MTases, 32 protein MTases, eight small molecule MTases, three lipid MTases, and nine MTases with still unknown substrate specificity.

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

PubMed Central

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

2018-01-01

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

Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming

PubMed Central

McClellan, Michael J.; Wood, C. David; Ojeniyi, Opeoluwa; Cooper, Tim J.; Kanhere, Aditi; Arvey, Aaron; Webb, Helen M.; Palermo, Richard D.; Harth-Hertle, Marie L.; Kempkes, Bettina; Jenner, Richard G.; West, Michelle J.

2013-01-01

Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors. PMID:24068937

The similia principle: results obtained in a cellular model system.

PubMed

Wiegant, Fred; Van Wijk, Roeland

2010-01-01

This paper describes the results of a research program focused on the beneficial effect of low dose stress conditions that were applied according to the similia principle to cells previously disturbed by more severe stress conditions. In first instance, we discuss criteria for research on the similia principle at the cellular level. Then, the homologous ('isopathic') approach is reviewed, in which the initial (high dose) stress used to disturb cellular physiology and the subsequent (low dose) stress are identical. Beneficial effects of low dose stress are described in terms of increased cellular survival capacity and at the molecular level as an increase in the synthesis of heat shock proteins (hsps). Both phenomena reflect a stimulation of the endogenous cellular self-recovery capacity. Low dose stress conditions applied in a homologous approach stimulate the synthesis of hsps and enhance survival in comparison with stressed cells that were incubated in the absence of low dose stress conditions. Thirdly, the specificity of the low dose stress condition is described where the initial (high dose) stress is different in nature from the subsequently applied (low dose) stress; the heterologous or 'heteropathic' approach. The results support the similia principle at the cellular level and add to understanding of how low dose stress conditions influence the regulatory processes underlying self-recovery. In addition, the phenomenon of 'symptom aggravation' which is also observed at the cellular level, is discussed in the context of self-recovery. Finally, the difference in efficiency between the homologous and the heterologous approach is discussed; a perspective is indicated for further research; and the relationship between studies on the similia principle and the recently introduced concept of 'postconditioning hormesis' is emphasized. Copyright 2009 The Faculty of Homeopathy. Published by Elsevier Ltd. All rights reserved.

The agglomeration state of nanoparticles can influence the mechanism of their cellular internalisation.

PubMed

Halamoda-Kenzaoui, Blanka; Ceridono, Mara; Urbán, Patricia; Bogni, Alessia; Ponti, Jessica; Gioria, Sabrina; Kinsner-Ovaskainen, Agnieszka

2017-06-26

Significant progress of nanotechnology, including in particular biomedical and pharmaceutical applications, has resulted in a high number of studies describing the biological effects of nanomaterials. Moreover, a determination of so-called "critical quality attributes", that is specific physicochemical properties of nanomaterials triggering the observed biological response, has been recognised as crucial for the evaluation and design of novel safe and efficacious therapeutics. In the context of in vitro studies, a thorough physicochemical characterisation of nanoparticles (NPs), also in the biological medium, is necessary to allow a correlation with a cellular response. Following this concept, we examined whether the main and frequently reported characteristics of NPs such as size and the agglomeration state can influence the level and the mechanism of NP cellular internalization. We employed fluorescently-labelled 30 and 80 nm silicon dioxide NPs, both in agglomerated and non-agglomerated form. Using flow cytometry, transmission electron microscopy, the inhibitors of endocytosis and gene silencing we determined the most probable routes of cellular uptake for each form of tested silica NPs. We observed differences in cellular uptake depending on the size and the agglomeration state of NPs. Caveolae-mediated endocytosis was implicated particularly in the internalisation of well dispersed silica NPs but with an increase of the agglomeration state of NPs a combination of endocytic pathways with a predominant role of macropinocytosis was noted. We demonstrated that the agglomeration state of NPs is an important factor influencing the level of cell uptake and the mechanism of endocytosis of silica NPs.

Studying the Brain in a Dish: 3D Cell Culture Models of Human Brain Development and Disease.

PubMed

Brown, Juliana; Quadrato, Giorgia; Arlotta, Paola

2018-01-01

The study of the cellular and molecular processes of the developing human brain has been hindered by access to suitable models of living human brain tissue. Recently developed 3D cell culture models offer the promise of studying fundamental brain processes in the context of human genetic background and species-specific developmental mechanisms. Here, we review the current state of 3D human brain organoid models and consider their potential to enable investigation of complex aspects of human brain development and the underpinning of human neurological disease. © 2018 Elsevier Inc. All rights reserved.

Biomaterials at the interface of nano- and micro-scale vector-cellular interactions in genetic vaccine design.

PubMed

Jones, Charles H; Hakansson, Anders P; Pfeifer, Blaine A

2014-01-01

The development of safe and effective vaccines for the prevention of elusive infectious diseases remains a public health priority. Immunization, characterized by adaptive immune responses to specific antigens, can be raised by an array of delivery vectors. However, current commercial vaccination strategies are predicated on the retooling of archaic technology. This review will discuss current and emerging strategies designed to elicit immune responses in the context of genetic vaccination. Selected strategies at the biomaterial-biological interface will be emphasized to illustrate the potential of coupling both fields towards a common goal.

Activation Loop Dynamics Determine the Different Catalytic Efficiencies of B Cell- and T Cell-Specific Tec Kinases

PubMed Central

Joseph, Raji E.; Kleino, Iivari; Wales, Thomas E.; Xie, Qian; Fulton, D. Bruce; Engen, John R.; Berg, Leslie J.; Andreotti, Amy H.

2014-01-01

Itk and Btk are nonreceptor tyrosine kinases of the Tec family that signal downstream of the T cell receptor (TCR) and B cell receptor (BCR), respectively. Despite their high sequence similarity and related signaling roles, Btk is a substantially more active kinase than Itk. We showed that substitution of six of the 619 amino acid residues of Itk with those of Btk was sufficient to completely switch the activities of Itk and Btk. The substitutions responsible for the swap in activity are all localized to the activation segment of the kinase domain. Nuclear magnetic resonance and hydrogen-deuterium exchange mass spectrometry analyses revealed that Itk and Btk had distinct protein dynamics in this region, which could explain the observed differences in catalytic efficiency between these kinases. Introducing Itk with enhanced activity into T cells led to enhanced and prolonged TCR signaling compared to that in cells with wild-type Itk. These findings imply that evolutionary pressures have led to Tec kinases having distinct enzymatic properties depending on the cellular context. We suggest that the weaker catalytic activities observed for T cell–specific kinases is one mechanism to regulate cellular activation and prevent aberrant immune responses. PMID:23982207

Autophagy Driven by a Master Regulator of Hematopoiesis

PubMed Central

Kang, Yoon-A; Sanalkumar, Rajendran; O'Geen, Henriette; Linnemann, Amelia K.; Chang, Chan-Jung; Bouhassira, Eric E.; Farnham, Peggy J.; Keles, Sunduz

2012-01-01

Developmental and homeostatic remodeling of cellular organelles is mediated by a complex process termed autophagy. The cohort of proteins that constitute the autophagy machinery functions in a multistep biochemical pathway. Though components of the autophagy machinery are broadly expressed, autophagy can occur in specialized cellular contexts, and mechanisms underlying cell-type-specific autophagy are poorly understood. We demonstrate that the master regulator of hematopoiesis, GATA-1, directly activates transcription of genes encoding the essential autophagy component microtubule-associated protein 1 light chain 3B (LC3B) and its homologs (MAP1LC3A, GABARAP, GABARAPL1, and GATE-16). In addition, GATA-1 directly activates genes involved in the biogenesis/function of lysosomes, which mediate autophagic protein turnover. We demonstrate that GATA-1 utilizes the forkhead protein FoxO3 to activate select autophagy genes. GATA-1-dependent LC3B induction is tightly coupled to accumulation of the active form of LC3B and autophagosomes, which mediate mitochondrial clearance as a critical step in erythropoiesis. These results illustrate a novel mechanism by which a master regulator of development establishes a genetic network to instigate cell-type-specific autophagy. PMID:22025678

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.

Generative mechanistic explanation building in undergraduate molecular and cellular biology

NASA Astrophysics Data System (ADS)

Southard, Katelyn M.; Espindola, Melissa R.; Zaepfel, Samantha D.; Bolger, Molly S.

2017-09-01

When conducting scientific research, experts in molecular and cellular biology (MCB) use specific reasoning strategies to construct mechanistic explanations for the underlying causal features of molecular phenomena. We explored how undergraduate students applied this scientific practice in MCB. Drawing from studies of explanation building among scientists, we created and applied a theoretical framework to explore the strategies students use to construct explanations for 'novel' biological phenomena. Specifically, we explored how students navigated the multi-level nature of complex biological systems using generative mechanistic reasoning. Interviews were conducted with introductory and upper-division biology students at a large public university in the United States. Results of qualitative coding revealed key features of students' explanation building. Students used modular thinking to consider the functional subdivisions of the system, which they 'filled in' to varying degrees with mechanistic elements. They also hypothesised the involvement of mechanistic entities and instantiated abstract schema to adapt their explanations to unfamiliar biological contexts. Finally, we explored the flexible thinking that students used to hypothesise the impact of mutations on multi-leveled biological systems. Results revealed a number of ways that students drew mechanistic connections between molecules, functional modules (sets of molecules with an emergent function), cells, tissues, organisms and populations.

B-1 Cell Immunoglobulin Directed Against Oxidation-Specific Epitopes

PubMed Central

Tsiantoulas, Dimitrios; Gruber, Sabrina; Binder, Christoph J.

2013-01-01

Natural antibodies (NAbs) are pre-existing antibodies with germline origin that arise in the absence of previous exposure to foreign antigens. NAbs are produced by B-1 lymphocytes and are primarily of the IgM isotype. There is accumulating evidence that – in addition to their role in antimicrobial host defense – NAbs exhibit important housekeeping functions by facilitating the non-immunogenic clearance of apoptotic cells as well as the removal of (neo-)self antigens. These properties are largely mediated by the ability of NAbs to recognize highly conserved and endogenously generated structures, which are exemplified by so-called oxidation-specific epitopes (OSEs) that are products of lipid peroxidation. The generation of OSEs as well as their interaction with the immune system have been studied extensively in the context of atherosclerosis, a chronic inflammatory disease of the vascular wall that is characterized by the accumulation of cellular debris and oxidized low-density lipoproteins (OxLDL). Both apoptotic cells as well as OxLDL carry OSEs that are targeted by NAbs. Therefore, OSEs represent stress-induced neo self-structures that mediate recognition of metabolic waste (e.g., cellular debris) by NAbs, allowing its safe disposal, which has fundamental implications in health and disease. PMID:23316200

In vivo dynamical behavior of yeast chromatin modeled as an entangled polymer network with constraint release

NASA Astrophysics Data System (ADS)

Wang, Chenxi; Kilfoil, Maria L.

2013-03-01

The high fidelity segregation of chromatin is the central problem in cell mitosis. The role of mechanics underlying this, however, is undetermined. Work in this area has largely focused on cytoskeletal elements of the process. Preliminary work in our lab suggests the mechanical properties of chromatin are fundamental in this process. Nevertheless, the mechanical properties of chromatin in the cellular context are not well-characterized. For better understanding of the role of mechanics in this cellular process, and of the chromatin mechanics in vivo generally, a systematic dynamical description of chromatin in vivo is required. Accordingly, we label specific sites on chromatin with fluorescent proteins of different wave lengths, enabling us to detect multiple spots separately in 3D and track their displacements in time inside living yeast cells. We analyze the pairwise cross-correlated motion between spots as a function of relative distance along the DNA contour. Comparison between the reptation model and our data serves to test our conjecture that chromatin in the cell is basically an entangled polymer network under constraints to thermal motion, and removal of constraints by non-thermal cellular processes is expected to affect its dynamic behavior.

A Model of How Different Biology Experts Explain Molecular and Cellular Mechanisms

PubMed Central

Trujillo, Caleb M.; Anderson, Trevor R.; Pelaez, Nancy J.

2015-01-01

Constructing explanations is an essential skill for all science learners. The goal of this project was to model the key components of expert explanation of molecular and cellular mechanisms. As such, we asked: What is an appropriate model of the components of explanation used by biology experts to explain molecular and cellular mechanisms? Do explanations made by experts from different biology subdisciplines at a university support the validity of this model? Guided by the modeling framework of R. S. Justi and J. K. Gilbert, the validity of an initial model was tested by asking seven biologists to explain a molecular mechanism of their choice. Data were collected from interviews, artifacts, and drawings, and then subjected to thematic analysis. We found that biologists explained the specific activities and organization of entities of the mechanism. In addition, they contextualized explanations according to their biological and social significance; integrated explanations with methods, instruments, and measurements; and used analogies and narrated stories. The derived methods, analogies, context, and how themes informed the development of our final MACH model of mechanistic explanations. Future research will test the potential of the MACH model as a guiding framework for instruction to enhance the quality of student explanations. PMID:25999313

Protein S-Nitrosylation: Determinants of Specificity and Enzymatic Regulation of S-Nitrosothiol-Based Signaling.

PubMed

Stomberski, Colin T; Hess, Douglas T; Stamler, Jonathan S

2018-01-10

Protein S-nitrosylation, the oxidative modification of cysteine by nitric oxide (NO) to form protein S-nitrosothiols (SNOs), mediates redox-based signaling that conveys, in large part, the ubiquitous influence of NO on cellular function. S-nitrosylation regulates protein activity, stability, localization, and protein-protein interactions across myriad physiological processes, and aberrant S-nitrosylation is associated with diverse pathophysiologies. Recent Advances: It is recently recognized that S-nitrosylation endows S-nitroso-protein (SNO-proteins) with S-nitrosylase activity, that is, the potential to trans-S-nitrosylate additional proteins, thereby propagating SNO-based signals, analogous to kinase-mediated signaling cascades. In addition, it is increasingly appreciated that cellular S-nitrosylation is governed by dynamically coupled equilibria between SNO-proteins and low-molecular-weight SNOs, which are controlled by a growing set of enzymatic denitrosylases comprising two main classes (high and low molecular weight). S-nitrosylases and denitrosylases, which together control steady-state SNO levels, may be identified with distinct physiology and pathophysiology ranging from cardiovascular and respiratory disorders to neurodegeneration and cancer. The target specificity of protein S-nitrosylation and the stability and reactivity of protein SNOs are determined substantially by enzymatic machinery comprising highly conserved transnitrosylases and denitrosylases. Understanding the differential functionality of SNO-regulatory enzymes is essential, and is amenable to genetic and pharmacological analyses, read out as perturbation of specific equilibria within the SNO circuitry. The emerging picture of NO biology entails equilibria among potentially thousands of different SNOs, governed by denitrosylases and nitrosylases. Thus, to elucidate the operation and consequences of S-nitrosylation in cellular contexts, studies should consider the roles of SNO-proteins as both targets and transducers of S-nitrosylation, functioning according to enzymatically governed equilibria. Antioxid. Redox Signal. 00, 000-000.

Rewiring of the inferred protein interactome during blood development studied with the tool PPICompare.

PubMed

Will, Thorsten; Helms, Volkhard

2017-04-04

Differential analysis of cellular conditions is a key approach towards understanding the consequences and driving causes behind biological processes such as developmental transitions or diseases. The progress of whole-genome expression profiling enabled to conveniently capture the state of a cell's transcriptome and to detect the characteristic features that distinguish cells in specific conditions. In contrast, mapping the physical protein interactome for many samples is experimentally infeasible at the moment. For the understanding of the whole system, however, it is equally important how the interactions of proteins are rewired between cellular states. To overcome this deficiency, we recently showed how condition-specific protein interaction networks that even consider alternative splicing can be inferred from transcript expression data. Here, we present the differential network analysis tool PPICompare that was specifically designed for isoform-sensitive protein interaction networks. Besides detecting significant rewiring events between the interactomes of grouped samples, PPICompare infers which alterations to the transcriptome caused each rewiring event and what is the minimal set of alterations necessary to explain all between-group changes. When applied to the development of blood cells, we verified that a reasonable amount of rewiring events were reported by the tool and found that differential gene expression was the major determinant of cellular adjustments to the interactome. Alternative splicing events were consistently necessary in each developmental step to explain all significant alterations and were especially important for rewiring in the context of transcriptional control. Applying PPICompare enabled us to investigate the dynamics of the human protein interactome during developmental transitions. A platform-independent implementation of the tool PPICompare is available at https://sourceforge.net/projects/ppicompare/ .

Transcriptional Regulation and Transport of Terpenoid Indole Alkaloid in Catharanthus roseus: Exploration of New Research Directions

PubMed Central

Liu, Jiaqi; Cai, Junjun; Wang, Rui; Yang, Shihai

2016-01-01

As one of the model medicinal plants for exploration of biochemical pathways and molecular biological questions on complex metabolic pathways, Catharanthus roseus synthesizes more than 100 terpenoid indole alkaloids (TIAs) used for clinical treatment of various diseases and for new drug discovery. Given that extensive studies have revealed the major metabolic pathways and the spatial-temporal biosynthesis of TIA in C. roseus plant, little is known about subcellular and inter-cellular trafficking or long-distance transport of TIA end products or intermediates, as well as their regulation. While these transport processes are indispensable for multi-organelle, -tissue and -cell biosynthesis, storage and their functions, great efforts have been made to explore these dynamic cellular processes. Progress has been made in past decades on transcriptional regulation of TIA biosynthesis by transcription factors as either activators or repressors; recent studies also revealed several transporters involved in subcellular and inter-cellular TIA trafficking. However, many details and the regulatory network for controlling the tissue-or cell-specific biosynthesis, transport and storage of serpentine and ajmalicine in root, catharanthine in leaf and root, vindoline specifically in leaf and vinblastine and vincristine only in green leaf and their biosynthetic intermediates remain to be determined. This review is to summarize the progress made in biosynthesis, transcriptional regulation and transport of TIAs. Based on analysis of organelle, tissue and cell-type specific biosynthesis and progresses in transport and trafficking of similar natural products, the transporters that might be involved in transport of TIAs and their synthetic intermediates are discussed; according to transcriptome analysis and bioinformatic approaches, the transcription factors that might be involved in TIA biosynthesis are analyzed. Further discussion is made on a broad context of transcriptional and transport regulation in order to guide our future research. PMID:28036025

COMP-1 promotes competitive advantage of nematode sperm.

PubMed

Hansen, Jody M; Chavez, Daniela R; Stanfield, Gillian M

2015-03-19

Competition among sperm to fertilize oocytes is a ubiquitous feature of sexual reproduction as well as a profoundly important aspect of sexual selection. However, little is known about the cellular mechanisms sperm use to gain competitive advantage or how these mechanisms are regulated genetically. In this study, we utilize a forward genetic screen in Caenorhabditis elegans to identify a gene, comp-1, whose function is specifically required in competitive contexts. We show that comp-1 functions in sperm to modulate their migration through and localization within the reproductive tract, thereby promoting their access to oocytes. Contrary to previously described models, comp-1 mutant sperm show no defects in size or velocity, thereby defining a novel pathway for preferential usage. Our results indicate not only that sperm functional traits can influence the outcome of sperm competition, but also that these traits can be modulated in a context-dependent manner depending on the presence of competing sperm.

The emergence of ECM mechanics and cytoskeletal tension as important regulators of cell function.

PubMed

Peyton, Shelly R; Ghajar, Cyrus M; Khatiwala, Chirag B; Putnam, Andrew J

2007-01-01

The ability to harvest and maintain viable cells from mammalian tissues represented a critical advance in biomedical research, enabling individual cells to be cultured and studied in molecular detail. However, in these traditional cultures, cells are grown on rigid glass or polystyrene substrates, the mechanical properties of which often do not match those of the in vivo tissue from which the cells were originally derived. This mechanical mismatch likely contributes to abrupt changes in cellular phenotype. In fact, it has been proposed that mechanical changes in the cellular microenvironment may alone be responsible for driving specific cellular behaviors. Recent multidisciplinary efforts from basic scientists and engineers have begun to address this hypothesis more explicitly by probing the effects of ECM mechanics on cell and tissue function. Understanding the consequences of such mechanical changes is physiologically relevant in the context of a number of tissues in which altered mechanics may either correlate with or play an important role in the onset of pathology. Examples include changes in the compliance of blood vessels associated with atherosclerosis and intimal hyperplasia, as well as changes in the mechanical properties of developing tumors. Compelling evidence from 2-D in vitro model systems has shown that substrate mechanical properties induce changes in cell shape, migration, proliferation, and differentiation, but it remains to be seen whether or not these same effects translate to 3-D systems or in vivo. Furthermore, the molecular "mechanotransduction" mechanisms by which cells respond to changes in ECM mechanics remain unclear. Here, we provide some historical context for this emerging area of research, and discuss recent evidence that regulation of cytoskeletal tension by changes in ECM mechanics (either directly or indirectly) may provide a critical switch that controls cell function.

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

RNA imaging: tracking in real-time RNA transport in neurons using molecular beacons and confocal microscopy.

PubMed

Zepeda, Angélica; Arias, Clorinda; Flores-Jasso, Fabian; Vaca, Luis

2013-01-01

RNAs are present within eukaryotic cells and are involved in several biological processes. RNA transport within cell compartments is important for proper cell function. To understand in depth the cellular processes in which RNA is involved requires a method that reveals RNA localization in real time in a sub-cellular context in living cells. In this protocol we describe a method for imaging RNA in living cells and in particular in neuronal cultures based on cell microinjection of molecular beacons in conjunction with confocal microscopy. This methodology overcomes some of the main obstacles for imaging RNA in live cells since microinjection allows the delivery of the probe to a desired cellular compartment and MBs bind with high specificity to its target RNA without inhibiting its function. The proper design of the MBs is essential to obtain RNA-MB association at the temperature of the cell cytosol. MBs design with other purposes in mind (such as PCR experiments) have a design that facilitates association to its target at high temperatures, rendering them unsuitable for live cell imaging. Using the methodology described in this chapter allows the study of RNA transport to different regions of neurons and may be combined with the tagging of proteins of interest to measure co-transport of the protein and the RNA to different cellular regions. Copyright © 2013 Elsevier Inc. All rights reserved.

Hydrodynamics in Cell Studies

PubMed Central

2018-01-01

Hydrodynamic phenomena are ubiquitous in living organisms and can be used to manipulate cells or emulate physiological microenvironments experienced in vivo. Hydrodynamic effects influence multiple cellular properties and processes, including cell morphology, intracellular processes, cell–cell signaling cascades and reaction kinetics, and play an important role at the single-cell, multicellular, and organ level. Selected hydrodynamic effects can also be leveraged to control mechanical stresses, analyte transport, as well as local temperature within cellular microenvironments. With a better understanding of fluid mechanics at the micrometer-length scale and the advent of microfluidic technologies, a new generation of experimental tools that provide control over cellular microenvironments and emulate physiological conditions with exquisite accuracy is now emerging. Accordingly, we believe that it is timely to assess the concepts underlying hydrodynamic control of cellular microenvironments and their applications and provide some perspective on the future of such tools in in vitro cell-culture models. Generally, we describe the interplay between living cells, hydrodynamic stressors, and fluid flow-induced effects imposed on the cells. This interplay results in a broad range of chemical, biological, and physical phenomena in and around cells. More specifically, we describe and formulate the underlying physics of hydrodynamic phenomena affecting both adhered and suspended cells. Moreover, we provide an overview of representative studies that leverage hydrodynamic effects in the context of single-cell studies within microfluidic systems. PMID:29420889

Adapter reagents for protein site specific dye labeling.

PubMed

Thompson, Darren A; Evans, Eric G B; Kasza, Tomas; Millhauser, Glenn L; Dawson, Philip E

2014-05-01

Chemoselective protein labeling remains a significant challenge in chemical biology. Although many selective labeling chemistries have been reported, the practicalities of matching the reaction with appropriately functionalized proteins and labeling reagents is often a challenge. For example, we encountered the challenge of site specifically labeling the cellular form of the murine Prion protein with a fluorescent dye. To facilitate this labeling, a protein was expressed with site specific p-acetylphenylalanine. However, the utility of this acetophenone reactive group is hampered by the severe lack of commercially available aminooxy fluorophores. Here we outline a general strategy for the efficient solid phase synthesis of adapter reagents capable of converting maleimido-labels into aminooxy or azide functional groups that can be further tuned for desired length or solubility properties. The utility of the adapter strategy is demonstrated in the context of fluorescent labeling of the murine Prion protein through an adapted aminooxy-Alexa dye. © 2014 Wiley Periodicals, Inc.

Adapter Reagents for Protein Site Specific Dye Labeling

PubMed Central

Thompson, Darren A.; Evans, Eric G. B.; Kasza, Tomas; Millhauser, Glenn L.; Dawson, Philip E.

2016-01-01

Chemoselective protein labeling remains a significant challenge in chemical biology. Although many selective labeling chemistries have been reported, the practicalities of matching the reaction with appropriately functionalized proteins and labeling reagents is often a challenge. For example, we encountered the challenge of site specifically labeling the cellular form of the murine Prion protein with a fluorescent dye. To facilitate this labeling, a protein was expressed with site specific p-acetylphenylalanine. However, the utility of this aceto-phenone reactive group is hampered by the severe lack of commercially available aminooxy fluorophores. Here we outline a general strategy for the efficient solid phase synthesis of adapter reagents capable of converting maleimido-labels into aminooxy or azide functional groups that can be further tuned for desired length or solubility properties. The utility of the adapter strategy is demonstrated in the context of fluorescent labeling of the murine Prion protein through an adapted aminooxy-Alexa dye. PMID:24599728

RNA helicase, DDX27 regulates skeletal muscle growth and regeneration by modulation of translational processes

PubMed Central

Gundry, Stacey R.; Chan, Aye T.; Widrick, Jeffrey; Draper, Isabelle; Chakraborty, Anirban; Zhou, Yi; Zon, Leonard I.; Gleizes, Pierre-Emmanuel

2018-01-01

Gene expression in a tissue-specific context depends on the combined efforts of epigenetic, transcriptional and post-transcriptional processes that lead to the production of specific proteins that are important determinants of cellular identity. Ribosomes are a central component of the protein biosynthesis machinery in cells; however, their regulatory roles in the translational control of gene expression in skeletal muscle remain to be defined. In a genetic screen to identify critical regulators of myogenesis, we identified a DEAD-Box RNA helicase, DDX27, that is required for skeletal muscle growth and regeneration. We demonstrate that DDX27 regulates ribosomal RNA (rRNA) maturation, and thereby the ribosome biogenesis and the translation of specific transcripts during myogenesis. These findings provide insight into the translational regulation of gene expression in myogenesis and suggest novel functions for ribosomes in regulating gene expression in skeletal muscles. PMID:29518074

Ahr function in lymphocytes: emerging concepts

PubMed Central

Zhou, Liang

2015-01-01

The aryl hydrocarbon receptor (Ahr) is an important regulator of the development and function of both innate and adaptive immune cells through roles associated with Ahr's ability to respond to cellular and dietary ligands. Recent findings have revealed tissue and context-specific functions for Ahr in both homeostasis and in during an immune response. I review these findings here, and integrate them into the current understanding of the mechanisms that regulate Ahr transcription and function. I propose a conceptual framework in which Ahr function is determined by three factors: the amount of Ahr in any given cell, the abundance and potency of Ahr ligands within certain tissues, and the tissue microenvironment wherein Ahr+ cells reside. This complexity emphasizes the necessity cell-type specific genetic approaches towards the study of Ahr function. PMID:26700314

Prevalence and multiplicity of cutaneous beta papilloma viruses in plucked hairs depend on cellular DNA input.

PubMed

Weissenborn, S J; Neale, R; de Koning, M N C; Waterboer, T; Abeni, D; Bouwes Bavinck, J N; Wieland, U; Pfister, H J

2009-11-01

In view of the low loads of beta human papillomaviruses in skin samples, amounts of cellular DNA used in qualitative PCR may become limiting for virus detection and introduce variations in prevalence and multiplicity. This issue was explored within the context of a multicentre study and increasing prevalence and multiplicity was found with increasing input amounts of cellular DNA extracted from hair bulbs. To improve the quality and comparability between different epidemiologic studies ideally equal amounts of cellular DNA should be employed. When cellular DNA input varies this should be clearly taken into account in assessing viral prevalence and multiplicity.

Landscape of Conditional eQTL in Dorsolateral Prefrontal Cortex and Co-localization with Schizophrenia GWAS.

PubMed

Dobbyn, Amanda; Huckins, Laura M; Boocock, James; Sloofman, Laura G; Glicksberg, Benjamin S; Giambartolomei, Claudia; Hoffman, Gabriel E; Perumal, Thanneer M; Girdhar, Kiran; Jiang, Yan; Raj, Towfique; Ruderfer, Douglas M; Kramer, Robin S; Pinto, Dalila; Akbarian, Schahram; Roussos, Panos; Domenici, Enrico; Devlin, Bernie; Sklar, Pamela; Stahl, Eli A; Sieberts, Solveig K

2018-06-07

Causal genes and variants within genome-wide association study (GWAS) loci can be identified by integrating GWAS statistics with expression quantitative trait loci (eQTL) and determining which variants underlie both GWAS and eQTL signals. Most analyses, however, consider only the marginal eQTL signal, rather than dissect this signal into multiple conditionally independent signals for each gene. Here we show that analyzing conditional eQTL signatures, which could be important under specific cellular or temporal contexts, leads to improved fine mapping of GWAS associations. Using genotypes and gene expression levels from post-mortem human brain samples (n = 467) reported by the CommonMind Consortium (CMC), we find that conditional eQTL are widespread; 63% of genes with primary eQTL also have conditional eQTL. In addition, genomic features associated with conditional eQTL are consistent with context-specific (e.g., tissue-, cell type-, or developmental time point-specific) regulation of gene expression. Integrating the 2014 Psychiatric Genomics Consortium schizophrenia (SCZ) GWAS and CMC primary and conditional eQTL data reveals 40 loci with strong evidence for co-localization (posterior probability > 0.8), including six loci with co-localization of conditional eQTL. Our co-localization analyses support previously reported genes, identify novel genes associated with schizophrenia risk, and provide specific hypotheses for their functional follow-up. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Induction of the neural crest state: Control of stem cell attributes by gene regulatory, post-transcriptional and epigenetic interactions

PubMed Central

Prasad, Maneeshi S.; Sauka-Spengler, Tatjana; LaBonne, Carole

2012-01-01

Neural crest cells are a population of multipotent stem cell-like progenitors that arise at the neural plate border in vertebrates, migrate extensively, and give rise to diverse derivatives such as melanocytes, craniofacial cartilage and bone, smooth muscle, peripheral and enteric neurons and glia. The neural crest gene regulatory network (NC-GRN) includes a number of key factors that are used reiteratively to control multiple steps in the development of neural crest cells, including the acquisition of stem cell attributes. It is therefore essential to understand the mechanisms that control the distinct functions of such reiteratively used factors in different cellular contexts. The context-dependent control of neural crest specification is achieved through combinatorial interaction with other factors, post-transcriptional and post-translational modifications, and the epigenetic status and chromatin state of target genes. Here we review the current understanding of the NC-GRN, including the role of the neural crest specifiers, their links to the control of “stemness,” and their dynamic context-dependent regulation during the formation of neural crest progenitors. PMID:22583479

Imaging Cell Shape Change in Living Drosophila Embryos

PubMed Central

Figard, Lauren; Sokac, Anna Marie

2011-01-01

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells. The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle. Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope. PMID:21490577

Formin homology 2 domains occur in multiple contexts in angiosperms

PubMed Central

Cvrčková, Fatima; Novotný, Marian; Pícková, Denisa; Žárský, Viktor

2004-01-01

Background Involvement of conservative molecular modules and cellular mechanisms in the widely diversified processes of eukaryotic cell morphogenesis leads to the intriguing question: how do similar proteins contribute to dissimilar morphogenetic outputs. Formins (FH2 proteins) play a central part in the control of actin organization and dynamics, providing a good example of evolutionarily versatile use of a conserved protein domain in the context of a variety of lineage-specific structural and signalling interactions. Results In order to identify possible plant-specific sequence features within the FH2 protein family, we performed a detailed analysis of angiosperm formin-related sequences available in public databases, with particular focus on the complete Arabidopsis genome and the nearly finished rice genome sequence. This has led to revision of the current annotation of half of the 22 Arabidopsis formin-related genes. Comparative analysis of the two plant genomes revealed a good conservation of the previously described two subfamilies of plant formins (Class I and Class II), as well as several subfamilies within them that appear to predate the separation of monocot and dicot plants. Moreover, a number of plant Class II formins share an additional conserved domain, related to the protein phosphatase/tensin/auxilin fold. However, considerable inter-species variability sets limits to generalization of any functional conclusions reached on a single species such as Arabidopsis. Conclusions The plant-specific domain context of the conserved FH2 domain, as well as plant-specific features of the domain itself, may reflect distinct functional requirements in plant cells. The variability of formin structures found in plants far exceeds that known from both fungi and metazoans, suggesting a possible contribution of FH2 proteins in the evolution of the plant type of multicellularity. PMID:15256004

The neurite growth inhibitory effects of soluble TNFα on developing sympathetic neurons are dependent on developmental age.

PubMed

Nolan, Aoife M; Collins, Louise M; Wyatt, Sean L; Gutierrez, Humberto; O'Keeffe, Gerard W

2014-01-01

During development, the growth of neural processes is regulated by an array of cellular and molecular mechanisms which influence growth rate, direction and branching. Recently, many members of the TNF superfamily have been shown to be key regulators of neurite growth during development. The founder member of this family, TNFα can both promote and inhibit neurite growth depending on the cellular context. Specifically, transmembrane TNFα promotes neurite growth, while soluble TNFα inhibits it. While the growth promoting effects of TNFα are restricted to a defined developmental window of early postnatal development, whether the growth inhibitory effects of soluble TNFα occur throughout development is unknown. In this study we used the extensively studied, well characterised neurons of the superior cervical ganglion to show that the growth inhibitory effects of soluble TNFα are restricted to a specific period of late embryonic and early postnatal development. Furthermore, we show that this growth inhibitory effect of soluble TNFα requires NF-κB signalling at all developmental stages at which soluble TNFα inhibits neurite growth. These findings raise the possibility that increases in the amount of soluble TNFα in vivo, for example as a result of maternal inflammation, could negatively affect neurite growth in developing neurons at specific stages of development. Copyright © 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Inhibition and Regulation of the Ergothioneine Biosynthetic Methyltransferase EgtD.

PubMed

Misson, Laëtitia; Burn, Reto; Vit, Allegra; Hildesheim, Julia; Beliaeva, Mariia A; Blankenfeldt, Wulf; Seebeck, Florian P

2018-05-18

Ergothioneine is an emerging factor in cellular redox homeostasis in bacteria, fungi, plants, and animals. Reports that ergothioneine biosynthesis may be important for the pathogenicity of bacteria and fungi raise the question as to how this pathway is regulated and whether the corresponding enzymes may be therapeutic targets. The first step in ergothioneine biosynthesis is catalyzed by the methyltransferase EgtD that converts histidine into N-α-trimethylhistidine. This report examines the kinetic, thermodynamic and structural basis for substrate, product, and inhibitor binding by EgtD from Mycobacterium smegmatis. This study reveals an unprecedented substrate binding mechanism and a fine-tuned affinity landscape as determinants for product specificity and product inhibition. Both properties are evolved features that optimize the function of EgtD in the context of cellular ergothioneine production. On the basis of these findings, we developed a series of simple histidine derivatives that inhibit methyltransferase activity at low micromolar concentrations. Crystal structures of inhibited complexes validate this structure- and mechanism-based design strategy.

Oncogenomic disruptions in arsenic-induced carcinogenesis

PubMed Central

Ng, Kevin W.; Stewart, Greg L.; Dummer, Trevor J.B.; Lam, Wan L.; Martinez, Victor D

2017-01-01

Chronic exposure to arsenic affects more than 200 million people worldwide, and has been associated with many adverse health effects, including cancer in several organs. There is accumulating evidence that arsenic biotransformation, a step in the elimination of arsenic from the human body, can induce changes at a genetic and epigenetic level, leading to carcinogenesis. At the genetic level, arsenic interferes with key cellular processes such as DNA damage-repair and chromosomal structure, leading to genomic instability. At the epigenetic level, arsenic places a high demand on the cellular methyl pool, leading to global hypomethylation and hypermethylation of specific gene promoters. These arsenic-associated DNA alterations result in the deregulation of both oncogenic and tumour-suppressive genes. Furthermore, recent reports have implicated aberrant expression of non-coding RNAs and the consequential disruption of signaling pathways in the context of arsenic-induced carcinogenesis. This article provides an overview of the oncogenomic anomalies associated with arsenic exposure and conveys the importance of non-coding RNAs in the arsenic-induced carcinogenic process. PMID:28179585

Dietary Glucosinolates Sulforaphane, Phenethyl Isothiocyanate, Indole-3-Carbinol/3,3'-Diindolylmethane: Anti-Oxidative Stress/Inflammation, Nrf2, Epigenetics/Epigenomics and In Vivo Cancer Chemopreventive Efficacy.

PubMed

Fuentes, Francisco; Paredes-Gonzalez, Ximena; Kong, Ah-Ng Tony

2015-05-01

Glucosinolates are a group of sulfur-containing glycosides found in many plant species, including cruciferous vegetables such as broccoli, cabbage, brussels sprouts, and cauliflower. Accumulating evidence increasingly supports the beneficial effects of dietary glucosinolates on overall health, including as potential anti-cancer agents, because of their role in the prevention of the initiation of carcinogenesis via the induction of cellular defense detoxifying/antioxidant enzymes and their epigenetic mechanisms, including modification of the CpG methylation of cancer-related genes, histone modification regulation and changes in the expression of miRNAs. In this context, the defense mechanism mediated by Nrf2-antioxidative stress and anti-inflammatory signaling pathways can contribute to cellular protection against oxidative stress and reactive metabolites of carcinogens. In this review, we summarize the cancer chemopreventive role of naturally occurring glucosinolate derivatives as inhibitors of carcinogenesis, with particular emphasis on specific molecular targets and epigenetic alterations in in vitro and in vivo human cancer animal models.

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy.

PubMed

Peckys, Diana B; de Jonge, Niels

2015-09-11

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e., one emitting at 655 nm and at 800 revealed similar characteristic results.

The Yin and Yang of YY1 in the nervous system

PubMed Central

He, Ye; Casaccia-Bonnefil, Patrizia

2008-01-01

The transcription factor Yin Yang 1 (YY1) is a multifunctional protein that can activate or repress gene expression depending on the cellular context. YY1 is ubiquitously expressed and highly conserved between species. However its role varies in diverse cell types and includes proliferation, differentiation and apoptosis. This review will focus on the function of YY1 in the nervous system including its role in neural development, neuronal function, developmental myelination and neurological disease. The multiple functions of YY1 in distinct cell types are reviewed and the possible mechanisms underlying the cell specificity for these functions are discussed. PMID:18485096

Formin' actin in the nucleus.

PubMed

Baarlink, Christian; Grosse, Robert

2014-01-01

Many if not most proteins can, under certain conditions, change cellular compartments, such as, for example, shuttling from the cytoplasm to the nucleus. Thus, many proteins may exert functions in various and very different subcellular locations, depending on the signaling context. A large amount of actin regulatory proteins has been detected in the mammalian cell nucleus, although their potential roles are much debated and are just beginning to emerge. Recently, members of the formin family of actin nucleators were also reported to dynamically localize to the nuclear environment. Here we discuss our findings that specific diaphanous-related formins can promote nuclear actin assembly in a signal-dependent manner.

Cellular therapies supplement: strategies for improving transplant efficiency in the context of cellular therapeutics.

PubMed

Jimenez, Antonio; Fung, Henry C; Christopherson, Kent W

2011-11-01

The field of hematopoietic stem cell transplantation (HSCT) has overcome many obstacles that have led to our current clinical ability to utilize cells collected from marrow, mobilized peripheral blood, or umbilical cord blood for the treatment of malignant and nonmalignant hematologic diseases. It is in this context that it becomes evident that future progress will lie in our development of an understanding of the biology by which the process of HSCT is regulated. By understanding the cellular components and the mechanisms by which HSCT is either enhanced or suppressed it will then be possible to design therapeutic strategies to improve rates of engraftment that will have a positive impact on immune reconstitution post-HSCT. In this review we focus primarily on allogeneic hematopoietic stem cell transplantation (allo-HSCT), the current challenges associated with allo-HSCT, and some developing strategies to improve engraftment in this setting. © 2011 American Association of Blood Banks.

Role of host cell factors in flavivirus infection: Implications for pathogenesis and development of antiviral drugs.

PubMed

Pastorino, Boris; Nougairède, Antoine; Wurtz, Nathalie; Gould, Ernest; de Lamballerie, Xavier

2010-09-01

The genus Flavivirus contains approximately 70 arthropod-borne enveloped RNA viruses many of which cause severe human and in some cases, animal disease. They include dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and tick-borne encephalitis virus. Hundreds of thousands of deaths due to flavivirus infections occur each year, many of which are unpreventable due to lack of availability of appropriate vaccines and/or antiviral drugs. Flaviviruses exploit the cytoplasmic cellular machinery to facilitate propagation of infectious progeny virions. They engage in dynamic and antagonistic interactions with host cell membranes and biochemical processes. Following infection, the cells initiate various antiviral strategies to counteract viral invasion. In its defense, the virus has alternative strategies to suppress these host responses to infection. The fine balance between these interactions determines the outcome of the viral infection and disease progression. Published studies have revealed specific effects of flaviviruses on cellular processes, but the underlying mechanisms that determine the specific cytopathogenetic changes induced by different flaviviruses have not, as yet, been elucidated. Independently of the suppression of the type I IFN response which has been described in detail elsewhere, this review focuses on recent discoveries relating to alterations of host metabolism following viral infection. Such studies may contribute to new approaches to antiviral drug development. The role of host cellular factors will be examined in the context of protection and/or pathogenesis resulting from flavivirus infection, with particular emphasis on West Nile virus and dengue virus. 2010 Elsevier B.V. All rights reserved.

Creating Context for the Use of DNA Adduct Data in Risk Assessment

EPA Science Inventory

Assessments of human cancer risk require the integration of diverse types of data. Advancing technologies for quantitative measurements at the sub-cellular domain raise the critical issue of interpretation and use of DNA adduct data in context with current understanding of cancer...

Modeling Cell and Tumor-Metastasis Dosimetry with the Particle and Heavy Ion Transport Code System (PHITS) Software for Targeted Alpha-Particle Radionuclide Therapy.

PubMed

Lee, Dongyoul; Li, Mengshi; Bednarz, Bryan; Schultz, Michael K

2018-06-26

The use of targeted radionuclide therapy for cancer is on the rise. While beta-particle-emitting radionuclides have been extensively explored for targeted radionuclide therapy, alpha-particle-emitting radionuclides are emerging as effective alternatives. In this context, fundamental understanding of the interactions and dosimetry of these emitted particles with cells in the tumor microenvironment is critical to ascertaining the potential of alpha-particle-emitting radionuclides. One important parameter that can be used to assess these metrics is the S-value. In this study, we characterized several alpha-particle-emitting radionuclides (and their associated radionuclide progeny) regarding S-values in the cellular and tumor-metastasis environments. The Particle and Heavy Ion Transport code System (PHITS) was used to obtain S-values via Monte Carlo simulation for cell and tumor metastasis resulting from interactions with the alpha-particle-emitting radionuclides, lead-212 ( 212 Pb), actinium-225 ( 225 Ac) and bismuth-213 ( 213 Bi); these values were compared to the beta-particle-emitting radionuclides yttrium-90 ( 90 Y) and lutetium-177 ( 177 Lu) and an Auger-electron-emitting radionuclide indium-111 ( 111 In). The effect of cellular internalization on S-value was explored at increasing degree of internalization for each radionuclide. This aspect of S-value determination was further explored in a cell line-specific fashion for six different cancer cell lines based on the cell dimensions obtained by confocal microscopy. S-values from PHITS were in good agreement with MIRDcell S-values (cellular S-values) and the values found by Hindié et al. (tumor S-values). In the cellular model, 212 Pb and 213 Bi decay series produced S-values that were 50- to 120-fold higher than 177 Lu, while 225 Ac decay series analysis suggested S-values that were 240- to 520-fold higher than 177 Lu. S-values arising with 100% cellular internalization were two- to sixfold higher for the nucleus when compared to 0% internalization. The tumor dosimetry model defines the relative merit of radionuclides and suggests alpha particles may be effective for large tumors as well as small tumor metastases. These results from PHITS modeling substantiate emerging evidence that alpha-particle-emitting radionuclides may be an effective alternative to beta-particle-emitting radionuclides for targeted radionuclide therapy due to preferred dose-deposition profiles in the cellular and tumor metastasis context. These results further suggest that internalization of alpha-particle-emitting radionuclides via radiolabeled ligands may increase the relative biological effectiveness of radiotherapeutics.

Unique aspects of the perinatal immune system.

PubMed

Zhang, Xiaoming; Zhivaki, Dania; Lo-Man, Richard

2017-08-01

The early stages of life are associated with increased susceptibility to infection, which is in part due to an ineffective immune system. In the context of infection, the immune system must be stimulated to provide efficient protection while avoiding insufficient or excessive activation. Yet, in early life, age-dependent immune regulation at molecular and cellular levels contributes to a reduced immunological fitness in terms of pathogen clearance and response to vaccines. To enable microbial colonization to be tolerated at birth, epigenetic immune cell programming and early life-specific immune regulatory and effector mechanisms ensure that vital functions and organ development are supported and that tissue damage is avoided. Advancement in our understanding of age-related remodelling of immune networks and the consequent tuning of immune responsiveness will open up new possibilities for immune intervention and vaccine strategies that are designed specifically for early life.

Role of biological membranes in slow-wave sleep.

PubMed

Karnovsky, M L

1991-02-01

Two involvements of cellular membranes in slow-wave sleep (SWS) are discussed. In the first the endoplasmic reticulum (ER) is focussed upon, and in the second, the plasmalemma, where specific binding sites (receptors?) for promoters of slow-wave sleep are believed to be located. The study concerning the ER focuses on an enzyme in the brain, glucose-6-phosphatase, which, although present at low levels, manifests greatly increased activity during SWS compared to the waking state. The work on the plasmalemma has to do with the specific binding of muramyl peptides, inducers of slow-wave sleep, to various cells, and membrane preparations of various sorts, including those from brain tissue. Such cells as macrophages from mice, B-lymphocytes from human blood, and cells from a cell line (C-6 glioma) have been examined in this context.

In silico prediction of protein-protein interactions in human macrophages

PubMed Central

2014-01-01

Background Protein-protein interaction (PPI) network analyses are highly valuable in deciphering and understanding the intricate organisation of cellular functions. Nevertheless, the majority of available protein-protein interaction networks are context-less, i.e. without any reference to the spatial, temporal or physiological conditions in which the interactions may occur. In this work, we are proposing a protocol to infer the most likely protein-protein interaction (PPI) network in human macrophages. Results We integrated the PPI dataset from the Agile Protein Interaction DataAnalyzer (APID) with different meta-data to infer a contextualized macrophage-specific interactome using a combination of statistical methods. The obtained interactome is enriched in experimentally verified interactions and in proteins involved in macrophage-related biological processes (i.e. immune response activation, regulation of apoptosis). As a case study, we used the contextualized interactome to highlight the cellular processes induced upon Mycobacterium tuberculosis infection. Conclusion Our work confirms that contextualizing interactomes improves the biological significance of bioinformatic analyses. More specifically, studying such inferred network rather than focusing at the gene expression level only, is informative on the processes involved in the host response. Indeed, important immune features such as apoptosis are solely highlighted when the spotlight is on the protein interaction level. PMID:24636261

Protein Corona Influences Cell-Biomaterial Interactions in Nanostructured Tissue Engineering Scaffolds.

PubMed

Serpooshan, Vahid; Mahmoudi, Morteza; Zhao, Mingming; Wei, Ke; Sivanesan, Senthilkumar; Motamedchaboki, Khatereh; Malkovskiy, Andrey V; Gladstone, Andrew B; Cohen, Jeffrey E; Yang, Phillip C; Rajadas, Jayakumar; Bernstein, Daniel; Woo, Y Joseph; Ruiz-Lozano, Pilar

2015-07-22

Biomaterials are extensively used to restore damaged tissues, in the forms of implants (e.g. tissue engineered scaffolds) or biomedical devices (e.g. pacemakers). Once in contact with the physiological environment, nanostructured biomaterials undergo modifications as a result of endogenous proteins binding to their surface. The formation of this macromolecular coating complex, known as 'protein corona', onto the surface of nanoparticles and its effect on cell-particle interactions are currently under intense investigation. In striking contrast, protein corona constructs within nanostructured porous tissue engineering scaffolds remain poorly characterized. As organismal systems are highly dynamic, it is conceivable that the formation of distinct protein corona on implanted scaffolds might itself modulate cell-extracellular matrix interactions. Here, we report that corona complexes formed onto the fibrils of engineered collagen scaffolds display specific, distinct, and reproducible compositions that are a signature of the tissue microenvironment as well as being indicative of the subject's health condition. Protein corona formed on collagen matrices modulated cellular secretome in a context-specific manner ex-vivo , demonstrating their role in regulating scaffold-cellular interactions. Together, these findings underscore the importance of custom-designing personalized nanostructured biomaterials, according to the biological milieu and disease state. We propose the use of protein corona as in situ biosensor of temporal and local biomarkers.

A systems approach to integrative biology: an overview of statistical methods to elucidate association and architecture.

PubMed

Ciaccio, Mark F; Finkle, Justin D; Xue, Albert Y; Bagheri, Neda

2014-07-01

An organism's ability to maintain a desired physiological response relies extensively on how cellular and molecular signaling networks interpret and react to environmental cues. The capacity to quantitatively predict how networks respond to a changing environment by modifying signaling regulation and phenotypic responses will help inform and predict the impact of a changing global enivronment on organisms and ecosystems. Many computational strategies have been developed to resolve cue-signal-response networks. However, selecting a strategy that answers a specific biological question requires knowledge both of the type of data being collected, and of the strengths and weaknesses of different computational regimes. We broadly explore several computational approaches, and we evaluate their accuracy in predicting a given response. Specifically, we describe how statistical algorithms can be used in the context of integrative and comparative biology to elucidate the genomic, proteomic, and/or cellular networks responsible for robust physiological response. As a case study, we apply this strategy to a dataset of quantitative levels of protein abundance from the mussel, Mytilus galloprovincialis, to uncover the temperature-dependent signaling network. © The Author 2014. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

Electrophysiological experiments in microgravity: lessons learned and future challenges.

PubMed

Wuest, Simon L; Gantenbein, Benjamin; Ille, Fabian; Egli, Marcel

2018-01-01

Advances in electrophysiological experiments have led to the discovery of mechanosensitive ion channels (MSCs) and the identification of the physiological function of specific MSCs. They are believed to play important roles in mechanosensitive pathways by allowing for cells to sense their mechanical environment. However, the physiological function of many MSCs has not been conclusively identified. Therefore, experiments have been developed that expose cells to various mechanical loads, such as shear flow, membrane indentation, osmotic challenges and hydrostatic pressure. In line with these experiments, mechanical unloading, as experienced in microgravity, represents an interesting alternative condition, since exposure to microgravity leads to a series of physiological adaption processes. As outlined in this review, electrophysiological experiments performed in microgravity have shown an influence of gravity on biological functions depending on ion channels at all hierarchical levels, from the cellular level to organs. In this context, calcium signaling represents an interesting cellular pathway, as it involves the direct action of calcium-permeable ion channels, and specific gravitatic cells have linked graviperception to this pathway. Multiple key proteins in the graviperception pathways have been identified. However, measurements on vertebrae cells have revealed controversial results. In conclusion, electrophysiological experiments in microgravity have shown that ion-channel-dependent physiological processes are altered in mechanically unloaded conditions. Future experiments may provide a better understanding of the underlying mechanisms.

Asynchronous adaptive time step in quantitative cellular automata modeling

PubMed Central

Zhu, Hao; Pang, Peter YH; Sun, Yan; Dhar, Pawan

2004-01-01

Background The behaviors of cells in metazoans are context dependent, thus large-scale multi-cellular modeling is often necessary, for which cellular automata are natural candidates. Two related issues are involved in cellular automata based multi-cellular modeling: how to introduce differential equation based quantitative computing to precisely describe cellular activity, and upon it, how to solve the heavy time consumption issue in simulation. Results Based on a modified, language based cellular automata system we extended that allows ordinary differential equations in models, we introduce a method implementing asynchronous adaptive time step in simulation that can considerably improve efficiency yet without a significant sacrifice of accuracy. An average speedup rate of 4–5 is achieved in the given example. Conclusions Strategies for reducing time consumption in simulation are indispensable for large-scale, quantitative multi-cellular models, because even a small 100 × 100 × 100 tissue slab contains one million cells. Distributed and adaptive time step is a practical solution in cellular automata environment. PMID:15222901

Anakoinosis: Communicative Reprogramming of Tumor Systems - for Rescuing from Chemorefractory Neoplasia.

PubMed

Hart, Christina; Vogelhuber, Martin; Wolff, Daniel; Klobuch, Sebastian; Ghibelli, Lina; Foell, Jürgen; Corbacioglu, Selim; Rehe, Klaus; Haegeman, Guy; Thomas, Simone; Herr, Wolfgang; Reichle, Albrecht

2015-08-01

Disruptive technologies, such as communicative reprogramming (anakoinosis) with cellular therapies in situ for treating refractory metastatic cancer allow patient care to accelerate along a totally new trajectory and highlight what may well become the next sea change in the care of patients with many types of advanced neoplasia. Cellular therapy in situ consisted of repurposed drugs, pioglitazone plus all-trans retinoic acid or dexamethasone or interferon-alpha (dual transcriptional modulation) combined with metronomic low-dose chemotherapy or low-dose 5-azacytidine, plus/minus classic targeted therapy. The novel therapeutic tools for specifically designing communication processes within tumor diseases focus on redirecting (1) rationalizations of cancer hallmarks (constitution of single cancer hallmarks), (2) modular events, (3) the 'metabolism' of evolutionary processes (the sum of therapeutically and intrinsically inducible evolutionary processes) and (4) the holistic communicative context, which determines validity and denotation of tumor promoting communication lines. Published data on cellular therapies in situ (6 histologic tumor types, 144 patients, age 0.9-83 years) in castration-resistant prostate cancer, pretreated renal clear cell carcinoma, chemorefractory acute myelocytic leukemia, multiple myeloma > second-line, chemorefractory Hodgkin lymphoma or multivisceral Langerhans cell histiocytosis, outline the possibility for treating refractory metastatic cancer with the hope that this type of reprogrammed communication will be scalable with minimal toxicity. Accessibility to anakoinosis is a tumor inherent feature, and cellular therapy in situ addresses extrinsic and intrinsic drug resistance, by redirecting convergent organized communication tools, while been supported by quite different pattern of (molecular-)genetic aberrations.

Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay.

PubMed

De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M

2011-10-01

Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin.

Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay

PubMed Central

De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M.

2011-01-01

Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin. PMID:22006542

Phosphatidic acid - a simple phospholipid with multiple faces.

PubMed

Zegarlińska, Jolanta; Piaścik, Magda; Sikorski, Aleksander F; Czogalla, Aleksander

2018-01-01

Phosphatidic acid (PA) is the simplest glycerophospholipid naturally occurring in living organisms, and even though its content among other cellular lipids is minor, it is drawing more and more attention due to its multiple biological functions. PA is a precursor for other phospholipids, acts as a lipid second messenger and, due to its structural properties, is also a modulator of membrane shape. Although much is known about interaction of PA with its effectors, the molecular mechanisms remain unresolved to a large degree. Throughout many of the well-characterized PA cellular sensors, no conserved binding domain can be recognized. Moreover, not much is known about the cellular dynamics of PA and how it is distributed among subcellular compartments. Remarkably, PA can play distinct roles within each of these compartments. For example, in the nucleus it behaves as a mitogen, influencing gene expression regulation, and in the Golgi membrane it plays a role in membrane trafficking. Here, we discuss how a biophysical experimental approach enabled PA behavior to be described in the context of a lipid bilayer and to what extent various physicochemical conditions may modulate the functional properties of this lipid. Understanding these aspects would help to unravel specific mechanisms of PA-driven membrane transformations and protein recruitment and thus would lead to a clearer picture of the biological role of PA.

Global Metabolic Responses to Salt Stress in Fifteen Species

PubMed Central

Pollak, Georg R.; Kuehne, Andreas; Sauer, Uwe

2016-01-01

Cells constantly adapt to unpredictably changing extracellular solute concentrations. A cornerstone of the cellular osmotic stress response is the metabolic supply of energy and building blocks to mount appropriate defenses. Yet, the extent to which osmotic stress impinges on the metabolic network remains largely unknown. Moreover, it is mostly unclear which, if any, of the metabolic responses to osmotic stress are conserved among diverse organisms or confined to particular groups of species. Here we investigate the global metabolic responses of twelve bacteria, two yeasts and two human cell lines exposed to sustained hyperosmotic salt stress by measuring semiquantitative levels of hundreds of cellular metabolites using nontargeted metabolomics. Beyond the accumulation of osmoprotectants, we observed significant changes of numerous metabolites in all species. Global metabolic responses were predominantly species-specific, yet individual metabolites were characteristically affected depending on species’ taxonomy, natural habitat, envelope structure or salt tolerance. Exploiting the breadth of our dataset, the correlation of individual metabolite response magnitudes across all species implicated lower glycolysis, tricarboxylic acid cycle, branched-chain amino acid metabolism and heme biosynthesis to be generally important for salt tolerance. Thus, our findings place the global metabolic salt stress response into a phylogenetic context and provide insights into the cellular phenotype associated with salt tolerance. PMID:26848578

Digital Single-Cell Analysis of Plant Organ Development Using 3DCellAtlas[OPEN

PubMed Central

Montenegro-Johnson, Thomas D.; Stamm, Petra; Strauss, Soeren; Topham, Alexander T.; Tsagris, Michail; Wood, Andrew T.A.; Smith, Richard S.; Bassel, George W.

2015-01-01

Diverse molecular networks underlying plant growth and development are rapidly being uncovered. Integrating these data into the spatial and temporal context of dynamic organ growth remains a technical challenge. We developed 3DCellAtlas, an integrative computational pipeline that semiautomatically identifies cell types and quantifies both 3D cellular anisotropy and reporter abundance at single-cell resolution across whole plant organs. Cell identification is no less than 97.8% accurate and does not require transgenic lineage markers or reference atlases. Cell positions within organs are defined using an internal indexing system generating cellular level organ atlases where data from multiple samples can be integrated. Using this approach, we quantified the organ-wide cell-type-specific 3D cellular anisotropy driving Arabidopsis thaliana hypocotyl elongation. The impact ethylene has on hypocotyl 3D cell anisotropy identified the preferential growth of endodermis in response to this hormone. The spatiotemporal dynamics of the endogenous DELLA protein RGA, expansin gene EXPA3, and cell expansion was quantified within distinct cell types of Arabidopsis roots. A significant regulatory relationship between RGA, EXPA3, and growth was present in the epidermis and endodermis. The use of single-cell analyses of plant development enables the dynamics of diverse regulatory networks to be integrated with 3D organ growth. PMID:25901089

Low Expression of lncRNA-GAS5 Is Implicated in Human Primary Varicose Great Saphenous Veins

PubMed Central

Yuan, Tian-You; Wang, Shi-Yi; Feng, Jing; Wang, Jing; Liu, Yuan; Wu, Ya-Han; Ma, Xiu-E; Ge, Jin; Cui, Ying-Yu; Jiang, Xiao-Yan

2015-01-01

The cellular mechanisms of primary varicose great saphenous veins (GSVs) involve inflammation, apoptosis, and proliferation of local cells and extracellular matrix degradation. Long non-coding RNAs (lncRNAs) play important roles in these cellular processes; however, which and how lncRNAs related to these mechanisms take effect on GSVs remain unclear. By screening lncRNAs that might experience changes in GSV varicosities, we selected the lower expressed lncRNA-GAS5 (growth arrest specific transcript 5) for functional assessments. Silencing of lncRNA-GAS5 promoted cell proliferation and migration, and cell cycle of the human saphenous vein smooth muscle cells (HSVSMCs), whereas overexpressing it inhibited these cellular behaviors and reduced apoptosis of HSVSMCs. RNA pull-down experiment revealed a direct bind of lncRNA-GAS5 to a Ca2+-dependent RNA-binding protein, Annexin A2. Further experiments showed that silencing of Annexin A2 reduced the HSVSMCs proliferation and vice versa. In the context of lncRNA-GAS5 knockdown, silencing of Annexin A2 reduced the proliferation of HSVSMCs while overexpression of Annexin A2 increased the proliferation. Thus, the low expression of lncRNA-GAS5 may facilitate HSVSMCs proliferation and migration through Annexin A2 and thereby the pathogenesis of GSV varicosities. PMID:25806802

A scalable strategy for high-throughput GFP tagging of endogenous human proteins.

PubMed

Leonetti, Manuel D; Sekine, Sayaka; Kamiyama, Daichi; Weissman, Jonathan S; Huang, Bo

2016-06-21

A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context.

SOX2 as a New Regulator of HPV16 Transcription.

PubMed

Martínez-Ramírez, Imelda; Del-Castillo-Falconi, Víctor; Mitre-Aguilar, Irma B; Amador-Molina, Alfredo; Carrillo-García, Adela; Langley, Elizabeth; Zentella-Dehesa, Alejandro; Soto-Reyes, Ernesto; García-Carrancá, Alejandro; Herrera, Luis A; Lizano, Marcela

2017-07-05

Persistent infections with high-risk human papillomavirus (HPV) constitute the main risk factor for cervical cancer development. HPV16 is the most frequent type associated to squamous cell carcinomas (SCC), followed by HPV18. The long control region (LCR) in the HPV genome contains the replication origin and sequences recognized by cellular transcription factors (TFs) controlling viral transcription. Altered expression of E6 and E7 viral oncogenes, modulated by the LCR, causes modifications in cellular pathways such as proliferation, leading to malignant transformation. The aim of this study was to identify specific TFs that could contribute to the modulation of high-risk HPV transcriptional activity, related to the cellular histological origin. We identified sex determining region Y (SRY)-box 2 (SOX2) response elements present in HPV16-LCR. SOX2 binding to the LCR was demonstrated by in vivo and in vitro assays. The overexpression of this TF repressed HPV16-LCR transcriptional activity, as shown through reporter plasmid assays and by the down-regulation of endogenous HPV oncogenes. Site-directed mutagenesis revealed that three putative SOX2 binding sites are involved in the repression of the LCR activity. We propose that SOX2 acts as a transcriptional repressor of HPV16-LCR, decreasing the expression of E6 and E7 oncogenes in a SCC context.

DIGE Proteome Analysis Reveals Suitability of Ischemic Cardiac In Vitro Model for Studying Cellular Response to Acute Ischemia and Regeneration

PubMed Central

Haas, Sina; Jahnke, Heinz-Georg; Moerbt, Nora; von Bergen, Martin; Aharinejad, Seyedhossein; Andrukhova, Olena; Robitzki, Andrea A.

2012-01-01

Proteomic analysis of myocardial tissue from patient population is suited to yield insights into cellular and molecular mechanisms taking place in cardiovascular diseases. However, it has been limited by small sized biopsies and complicated by high variances between patients. Therefore, there is a high demand for suitable model systems with the capability to simulate ischemic and cardiotoxic effects in vitro, under defined conditions. In this context, we established an in vitro ischemia/reperfusion cardiac disease model based on the contractile HL-1 cell line. To identify pathways involved in the cellular alterations induced by ischemia and thereby defining disease-specific biomarkers and potential target structures for new drug candidates we used fluorescence 2D-difference gel electrophoresis. By comparing spot density changes in ischemic and reperfusion samples we detected several protein spots that were differentially abundant. Using MALDI-TOF/TOF-MS and ESI-MS the proteins were identified and subsequently grouped by functionality. Most prominent were changes in apoptosis signalling, cell structure and energy-metabolism. Alterations were confirmed by analysis of human biopsies from patients with ischemic cardiomyopathy. With the establishment of our in vitro disease model for ischemia injury target identification via proteomic research becomes independent from rare human material and will create new possibilities in cardiac research. PMID:22384053

[Neurotransmitter mechanisms of context-dependent behavior].

PubMed

D'iakonova, V E

2012-01-01

The influence of context on behavioral choice is well known. Context can refer to behavioral state of an animal and to external factors such as season, presence of other individuals or food availability. How external and internal factors influencing decision-making are translated at the cellular level? I review the recent neuroethological data that strongly suggest that context reflects in content of neuroactive substances (neurotransmitters, modulators, hormones) that present in the extraneuronal milieu, while heterochemical neuronal microenvironment in its turn impacts motor program selection.

Biotechnologies as a Context for Enhancing Junior High-School Students' Ability to Ask Meaningful Questions about Abstract Biological Processes.

ERIC Educational Resources Information Center

Olsher, G.; Dreyfus, A.

1999-01-01

Suggests a new approach to teaching about biochemical cellular processes by stimulating student interest in those biochemical processes that allowed for the outcomes of modern biotechnologies. Discusses the development of students' ability to ask meaningful questions about intra-cellular processes, and the resulting meaningful learning of relevant…

A Cell-Line-Specific Atlas of PARP-Mediated Protein Asp/Glu-ADP-Ribosylation in Breast Cancer.

PubMed

Zhen, Yuanli; Zhang, Yajie; Yu, Yonghao

2017-11-21

PARP1 plays a critical role in regulating many biological processes linked to cellular stress responses. Although DNA strand breaks are potent stimuli of PARP1 enzymatic activity, the context-dependent mechanism regulating PARP1 activation and signaling is poorly understood. We performed global characterization of the PARP1-dependent, Asp/Glu-ADP-ribosylated proteome in a panel of cell lines originating from benign breast epithelial cells, as well as common subtypes of breast cancer. From these analyses, we identified 503 specific ADP-ribosylation sites on 322 proteins. Despite similar expression levels, PARP1 is differentially activated in these cell lines under genotoxic conditions, which generates signaling outputs with substantial heterogeneity. By comparing protein abundances and ADP-ribosylation levels, we could dissect cell-specific PARP1 targets that are driven by unique expression patterns versus cell-specific regulatory mechanisms of PARylation. Intriguingly, PARP1 modifies many proteins in a cell-specific manner, including those involved in transcriptional regulation, mRNA metabolism, and protein translation. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Biologically active LIL proteins built with minimal chemical diversity

PubMed Central

Heim, Erin N.; Marston, Jez L.; Federman, Ross S.; Edwards, Anne P. B.; Karabadzhak, Alexander G.; Petti, Lisa M.; Engelman, Donald M.; DiMaio, Daniel

2015-01-01

We have constructed 26-amino acid transmembrane proteins that specifically transform cells but consist of only two different amino acids. Most proteins are long polymers of amino acids with 20 or more chemically distinct side-chains. The artificial transmembrane proteins reported here are the simplest known proteins with specific biological activity, consisting solely of an initiating methionine followed by specific sequences of leucines and isoleucines, two hydrophobic amino acids that differ only by the position of a methyl group. We designate these proteins containing leucine (L) and isoleucine (I) as LIL proteins. These proteins functionally interact with the transmembrane domain of the platelet-derived growth factor β-receptor and specifically activate the receptor to transform cells. Complete mutagenesis of these proteins identified individual amino acids required for activity, and a protein consisting solely of leucines, except for a single isoleucine at a particular position, transformed cells. These surprisingly simple proteins define the minimal chemical diversity sufficient to construct proteins with specific biological activity and change our view of what can constitute an active protein in a cellular context. PMID:26261320

E2F transcription factors and digestive system malignancies: how much do we know?

PubMed

Evangelou, Konstantinos; Havaki, Sophia; Kotsinas, Athanassios

2014-08-07

The E2F proteins comprise a family of 8 members that function as transcription factors. They are key targets of the retinoblastoma protein (RB) and were initially divided into groups of activators and repressors. Accumulating data suggest that there is no specific role for each individual E2F member. Instead, each E2F can exert a variety of cellular effects, some of which represent opposing ones. For instance, specific E2Fs can activate transcription and repression, promote or hamper cell proliferation, augment or inhibit apoptosis, all being dependent on the cellular context. This complexity reflects the importance that these transcription factors have on a cell's fate. Thus, delineating the specific role for each E2F member in specific malignancies, although not easy, is a challenging and continuously pursued task, especially in view of potential E2F targeted therapies. Therefore, several reviews are continuously trying to evaluate available data on E2F status in various malignancies. Such reviews have attempted to reach a consensus, often in the simplistic form of oncogenes or tumor suppressor genes for the E2Fs. However they frequently miss spatial and temporal alterations of these factors during tumor development, which should also be considered in conjunction with the status of the regulatory networks that these factors participate in. In the current ''Letter to the Editor'', we comment on the flaws, misinterpretations and omissions in one such review article published recently in the World Journal of Gastroenterology regarding the role of E2Fs in digestive system malignancies.

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

Cyclophilin A as a potential genetic adjuvant to improve HIV-1 Gag DNA vaccine immunogenicity by eliciting broad and long-term Gag-specific cellular immunity in mice.

PubMed

Hou, Jue; Zhang, Qicheng; Liu, Zheng; Wang, Shuhui; Li, Dan; Liu, Chang; Liu, Ying; Shao, Yiming

2016-01-01

Previous research has shown that host Cyclophilin A (CyPA) can promote dendritic cell maturation and the subsequent innate immune response when incorporated into an HIV-1 Gag protein to circumvent the resistance of dendritic cells to HIV-1 infection. This led us to hypothesize that CyPA may improve HIV-1 Gag-specific vaccine immunogenicity via binding with Gag antigen. The adjuvant effect of CyPA was evaluated using a DNA vaccine with single or dual expression cassettes. Mouse studies indicated that CyPA specifically and markedly promoted HIV-1 Gag-specific cellular immunity but not an HIV-1 Env-specific cellular response. The Gag/CyPA dual expression cassettes stimulated a greater Gag-specific cellular immune response, than Gag immunization alone. Furthermore, CyPA induced a broad Gag-specific T cell response and strong cellular immunity that lasted up to 5 months. In addition, CyPA skewed to cellular rather than humoral immunity. To investigate the mechanisms of the adjuvant effect, site-directed mutagenesis in CyPA, including active site residues H54Q and F60A resulted in mutants that were co-expressed with Gag in dual cassettes. The immune response to this vaccine was analyzed in vivo. Interestingly, the wild type CyPA markedly increased Gag cellular immunity, but the H54Q and F60A mutants drastically reduced CyPA adjuvant activation. Therefore, we suggest that the adjuvant effect of CyPA was based on Gag-CyPA-specific interactions. Herein, we report that Cyclophilin A can augment HIV-1 Gag-specific cellular immunity as a genetic adjuvant in multiplex DNA immunization strategies, and that activity of this adjuvant is specific, broad, long-term, and based on Gag-CyPA interaction.

Calcium-sensing receptor (CaSR): pharmacological properties and signaling pathways.

PubMed

Conigrave, Arthur D; Ward, Donald T

2013-06-01

In this article we consider the mechanisms by which the calcium-sensing receptor (CaSR) induces its cellular responses via the control (activation or inhibition) of signaling pathways. We consider key features of CaSR-mediated signaling including its control of the heterotrimeric G-proteins Gq/11, Gi/o and G12/13 and the downstream consequences recognizing that very few CaSR-mediated cell phenomena have been fully described. We also consider the manner in which the CaSR contributes to the formation of specific signaling scaffolds via peptide recognition sequences in its intracellular C-terminal along with the origins of its high level of cooperativity, particularly for Ca(2+)o, and its remarkable resistance to desensitization. We also consider the nature of the mechanisms by which the CaSR controls oscillatory and sustained Ca(2+)i mobilizing responses and inhibits or elevates cyclic adenosine monophosphate (cAMP) levels dependent on the cellular and signaling context. Finally, we consider the diversity of the receptor's ligands, ligand binding sites and broader compartment-dependent physiological roles leading to the identification of pronounced ligand-biased signaling for agonists including Sr(2+) and modulators including l-amino acids and the clinically effective calcimimetic cinacalcet. We note the implications of these findings for the development of new designer drugs that might target the CaSR in pathophysiological contexts beyond those established for the treatment of disorders of calcium metabolism. Copyright © 2013 Elsevier Ltd. All rights reserved.

COMP-1 promotes competitive advantage of nematode sperm

PubMed Central

Hansen, Jody M; Chavez, Daniela R; Stanfield, Gillian M

2015-01-01

Competition among sperm to fertilize oocytes is a ubiquitous feature of sexual reproduction as well as a profoundly important aspect of sexual selection. However, little is known about the cellular mechanisms sperm use to gain competitive advantage or how these mechanisms are regulated genetically. In this study, we utilize a forward genetic screen in Caenorhabditis elegans to identify a gene, comp-1, whose function is specifically required in competitive contexts. We show that comp-1 functions in sperm to modulate their migration through and localization within the reproductive tract, thereby promoting their access to oocytes. Contrary to previously described models, comp-1 mutant sperm show no defects in size or velocity, thereby defining a novel pathway for preferential usage. Our results indicate not only that sperm functional traits can influence the outcome of sperm competition, but also that these traits can be modulated in a context-dependent manner depending on the presence of competing sperm. DOI: http://dx.doi.org/10.7554/eLife.05423.001 PMID:25789512

The NOTCH1-autophagy interaction: Regulating self-eating for survival.

PubMed

Sarin, Apurva; Marcel, Nimi

2017-02-01

T-cell subsets in the mammalian immune system use varied mechanisms for survival, a demand imposed by the diverse and dynamic niches that they function in. In a recent study, we showed that survival of natural T-regulatory cells (Tregs) was determined by spatially regulated NOTCH1 activity signaling leading to the activation of macroautophagy/autophagy. While this interaction was revealed in experimental conditions of limited nutrient availability in vitro, the consequences of this interaction were confirmed in the context of immune physiology. Consistently, disrupting NOTCH signaling or the autophagy cascade was deleterious to Tregs. At the molecular level, ligand-activated NOTCH1, which is enriched outside the nucleus in Tregs, was detected in complexes that included specific molecular intermediates controlling the progression of autophagy. Mitochondria were a prominent cellular target, with organelle remodeling and function dependent on NOTCH1 signaling to autophagy. It is tempting to speculate that the link between autophagy and the developmental regulator NOTCH1 identified in this work may be conserved in other biological contexts.

Discovery of stimulation-responsive immune enhancers with CRISPR activation

PubMed Central

Simeonov, Dimitre R.; Gowen, Benjamin G.; Boontanrart, Mandy; Roth, Theodore L.; Gagnon, John D.; Mumbach, Maxwell R.; Satpathy, Ansuman T.; Lee, Youjin; Bray, Nicolas L.; Chan, Alice Y.; Lituiev, Dmytro S.; Nguyen, Michelle L.; Gate, Rachel E.; Subramaniam, Meena; Li, Zhongmei; Woo, Jonathan M.; Mitros, Therese; Ray, Graham J.; Curie, Gemma L.; Naddaf, Nicki; Chu, Julia S.; Ma, Hong; Boyer, Eric; Van Gool, Frederic; Huang, Hailiang; Liu, Ruize; Tobin, Victoria R.; Schumann, Kathrin; Daly, Mark J.; Farh, Kyle K; Ansel, K. Mark; Ye, Chun J.; Greenleaf, William J.; Anderson, Mark S.; Bluestone, Jeffrey A.; Chang, Howard Y.; Corn, Jacob E.; Marson, Alexander

2017-01-01

The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues1–3. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption4–6, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa)7 to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (TH17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs. PMID:28854172

Discovery of stimulation-responsive immune enhancers with CRISPR activation.

PubMed

Simeonov, Dimitre R; Gowen, Benjamin G; Boontanrart, Mandy; Roth, Theodore L; Gagnon, John D; Mumbach, Maxwell R; Satpathy, Ansuman T; Lee, Youjin; Bray, Nicolas L; Chan, Alice Y; Lituiev, Dmytro S; Nguyen, Michelle L; Gate, Rachel E; Subramaniam, Meena; Li, Zhongmei; Woo, Jonathan M; Mitros, Therese; Ray, Graham J; Curie, Gemma L; Naddaf, Nicki; Chu, Julia S; Ma, Hong; Boyer, Eric; Van Gool, Frederic; Huang, Hailiang; Liu, Ruize; Tobin, Victoria R; Schumann, Kathrin; Daly, Mark J; Farh, Kyle K; Ansel, K Mark; Ye, Chun J; Greenleaf, William J; Anderson, Mark S; Bluestone, Jeffrey A; Chang, Howard Y; Corn, Jacob E; Marson, Alexander

2017-09-07

The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (T H 17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.

Discovery of stimulation-responsive immune enhancers with CRISPR activation

NASA Astrophysics Data System (ADS)

Simeonov, Dimitre R.; Gowen, Benjamin G.; Boontanrart, Mandy; Roth, Theodore L.; Gagnon, John D.; Mumbach, Maxwell R.; Satpathy, Ansuman T.; Lee, Youjin; Bray, Nicolas L.; Chan, Alice Y.; Lituiev, Dmytro S.; Nguyen, Michelle L.; Gate, Rachel E.; Subramaniam, Meena; Li, Zhongmei; Woo, Jonathan M.; Mitros, Therese; Ray, Graham J.; Curie, Gemma L.; Naddaf, Nicki; Chu, Julia S.; Ma, Hong; Boyer, Eric; van Gool, Frederic; Huang, Hailiang; Liu, Ruize; Tobin, Victoria R.; Schumann, Kathrin; Daly, Mark J.; Farh, Kyle K.; Ansel, K. Mark; Ye, Chun J.; Greenleaf, William J.; Anderson, Mark S.; Bluestone, Jeffrey A.; Chang, Howard Y.; Corn, Jacob E.; Marson, Alexander

2017-09-01

The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (TH17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.

Impact of Diesel Exhaust Particles on Th2 Response in the Lung in Asthmatic Mice

PubMed Central

Inoue, Ken-ichiro; Koike, Eiko; Yanagisawa, Rie; Takano, Hirohisa

2008-01-01

Although it has been accepted that pulmonary exposure to diesel exhaust particles (DEP), representative constituents in particulate matter of mass median aerodynamic diameter < or 2.5 µm (PM2.5), exacerbates murine allergic asthma, the in vivo effects of DEP on their cellular events in the context of allergen-specific Th response have never been examined. The aim of this study is to elucidate whether in vivo repetitive exposure to DEP combined with allergen (ovalbumin) facilitate allergen-specific Th response in the lung using a simple ex vivo assay system. As a result, repetitive pulmonary exposure to DEP in vivo, if combined with allergen, amplifies ex vivo allergen-specific Th2 response in the lung compared to that to allergen alone, characterized by high levels of interleukin (IL)-4 and IL-5. The result suggests that in asthmatic subjects, DEP promote Th2-prone milieu in the lung, which additively/synergistically augment asthma pathophysiology in vivo. PMID:19015755

Systems analysis of arrestin pathway functions.

PubMed

Maudsley, Stuart; Siddiqui, Sana; Martin, Bronwen

2013-01-01

To fully appreciate the diversity and specificity of complex cellular signaling events, such as arrestin-mediated signaling from G protein-coupled receptor activation, a complex systems-level investigation currently appears to be the best option. A rational combination of transcriptomics, proteomics, and interactomics, all coherently integrated with applied next-generation bioinformatics, is vital for the future understanding of the development, translation, and expression of GPCR-mediated arrestin signaling events in physiological contexts. Through a more nuanced, systems-level appreciation of arrestin-mediated signaling, the creation of arrestin-specific molecular response "signatures" should be made simple and ultimately amenable to drug discovery processes. Arrestin-based signaling paradigms possess important aspects, such as its specific temporal kinetics and ability to strongly affect transcriptional activity, that make it an ideal test bed for next-generation of drug discovery bioinformatic approaches such as multi-parallel dose-response analysis, data texturization, and latent semantic indexing-based natural language data processing and feature extraction. Copyright © 2013 Elsevier Inc. All rights reserved.

In Vitro Assays for Mouse Müller Cell Phenotyping Through microRNA Profiling in the Damaged Retina.

PubMed

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

2018-01-01

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

Functional cis-regulatory modules encoded by mouse-specific endogenous retrovirus

PubMed Central

Sundaram, Vasavi; Choudhary, Mayank N. K.; Pehrsson, Erica; Xing, Xiaoyun; Fiore, Christopher; Pandey, Manishi; Maricque, Brett; Udawatta, Methma; Ngo, Duc; Chen, Yujie; Paguntalan, Asia; Ray, Tammy; Hughes, Ava; Cohen, Barak A.; Wang, Ting

2017-01-01

Cis-regulatory modules contain multiple transcription factor (TF)-binding sites and integrate the effects of each TF to control gene expression in specific cellular contexts. Transposable elements (TEs) are uniquely equipped to deposit their regulatory sequences across a genome, which could also contain cis-regulatory modules that coordinate the control of multiple genes with the same regulatory logic. We provide the first evidence of mouse-specific TEs that encode a module of TF-binding sites in mouse embryonic stem cells (ESCs). The majority (77%) of the individual TEs tested exhibited enhancer activity in mouse ESCs. By mutating individual TF-binding sites within the TE, we identified a module of TF-binding motifs that cooperatively enhanced gene expression. Interestingly, we also observed the same motif module in the in silico constructed ancestral TE that also acted cooperatively to enhance gene expression. Our results suggest that ancestral TE insertions might have brought in cis-regulatory modules into the mouse genome. PMID:28348391

Cell-based therapeutics: the next pillar of medicine.

PubMed

Fischbach, Michael A; Bluestone, Jeffrey A; Lim, Wendell A

2013-04-03

Two decades ago, the pharmaceutical industry-long dominated by small-molecule drugs-was revolutionized by the the advent of biologics. Today, biomedicine sits on the cusp of a new revolution: the use of microbial and human cells as versatile therapeutic engines. Here, we discuss the promise of this "third pillar" of therapeutics in the context of current scientific, regulatory, economic, and perceptual challenges. History suggests that the advent of cellular medicines will require the development of a foundational cellular engineering science that provides a systematic framework for safely and predictably altering and regulating cellular behaviors.

Fluorine-containing nanoemulsions for MRI cell tracking

PubMed Central

Janjic, Jelena M.; Ahrens, Eric T.

2009-01-01

In this article we review the chemistry and nanoemulsion formulation of perfluorocarbons used for in vivo 19F MRI cell tracking. In this application, cells of interest are labeled in culture using a perfluorocarbon nanoemulsion. Labeled cells are introduced into a subject and tracked using 19F MRI or NMR spectroscopy. In the same imaging session, a high-resolution, conventional (1H) image can be used to place the 19F-labeled cells into anatomical context. Perfluorocarbon-based 19F cell tracking is a useful technology because of the high specificity for labeled cells, ability to quantify cell accumulations, and biocompatibility. This technology can be widely applied to studies of inflammation, cellular regenerative medicine, and immunotherapy. PMID:19920872

The Radiata and the evolutionary origins of the bilaterian body plan

NASA Technical Reports Server (NTRS)

Martindale, Mark Q.; Finnerty, John R.; Henry, Jonathan Q.

2002-01-01

The apparent conservation of cellular and molecular developmental mechanisms observed in a handful of bilaterian metazoans has spawned a "race" to reconstruct the bilaterian ancestor. Knowledge of this ancestor would permit us to reconstruct the evolutionary changes that have occurred along specific bilaterian lineages. However, comparisons among extant bilaterians provide an unnecessarily limited view of the ancestral bilaterian. Since the original bilaterians are believed by many to be derived from a radially symmetrical ancestor, additional evidence might be obtained by examining present-day radially symmetrical animals. We briefly review pertinent features of the body plans of the extant radial eumetazoan phyla, the Cnidaria, and Ctenophora, in the context of revealing potential evolutionary links to the bilaterians.

Different toxic effects of YTX in tumor K-562 and lymphoblastoid cell lines

PubMed Central

Fernández-Araujo, Andrea; Sánchez, Jon A.; Alfonso, Amparo; Vieytes, Mercedes R.; Botana, Luis M.

2015-01-01

Yessotoxin (YTX) modulates cellular phosphodiesterases (PDEs). In this regard, opposite effects had been described in the tumor model K-562 cell line and fresh human lymphocytes in terms of cell viability, cyclic adenosine 3',5'-cyclic monophosphate (cAMP) production and protein expression after YTX treatment. Studies in depth of the pathways activated by YTX in K-562 cell line, have demonstrated the activation of two different cell death types, apoptosis, and autophagy after 24 and 48 h of treatment, respectively. Furthermore, the key role of type 4A PDE (PDE4A) in both pathways activated by YTX was demonstrated. Therefore, taking into account the differences between cellular lines and fresh cells, a study of cell death pathways activated by YTX in a non-tumor cell line with mitotic activity, was performed. The cellular model used was the lymphoblastoid cell line that represents a non-tumor model with normal apoptotic and mitotic machinery. In this context, cell viability and cell proliferation, expression of proteins involved in cell death activated by YTX and mitochondrial mass, were studied after the incubation with the toxin. Opposite to the tumor model, no cell death activation was observed in lymphoblastoid cell line in the presence of YTX. In this sense, variations in apoptosis hallmarks were not detected in the lymphoblastoid cell line after YTX incubation, whereas this type I of programmed cell death was observed in K-562 cells. On the other hand, autophagy cell death was triggered in this cellular line, while other autophagic process is suggested in lymphoblastoid cells. These YTX effects are related to PDE4A in both cellular lines. In addition, while cell death is triggered in K-562 cells after YTX treatment, in lymphoblastoid cells the toxin stops cellular proliferation. These results point to YTX as a specific toxic compound of tumor cells, since in the non-tumor lymphoblastoid cell line, no cell death hallmarks are observed. PMID:26136685

On the holistic approach in cellular and cancer biology: nonlinearity, complexity, and quasi-determinism of the dynamic cellular network.

PubMed

Waliszewski, P; Molski, M; Konarski, J

1998-06-01

A keystone of the molecular reductionist approach to cellular biology is a specific deductive strategy relating genotype to phenotype-two distinct categories. This relationship is based on the assumption that the intermediary cellular network of actively transcribed genes and their regulatory elements is deterministic (i.e., a link between expression of a gene and a phenotypic trait can always be identified, and evolution of the network in time is predetermined). However, experimental data suggest that the relationship between genotype and phenotype is nonbijective (i.e., a gene can contribute to the emergence of more than just one phenotypic trait or a phenotypic trait can be determined by expression of several genes). This implies nonlinearity (i.e., lack of the proportional relationship between input and the outcome), complexity (i.e. emergence of the hierarchical network of multiple cross-interacting elements that is sensitive to initial conditions, possesses multiple equilibria, organizes spontaneously into different morphological patterns, and is controlled in dispersed rather than centralized manner), and quasi-determinism (i.e., coexistence of deterministic and nondeterministic events) of the network. Nonlinearity within the space of the cellular molecular events underlies the existence of a fractal structure within a number of metabolic processes, and patterns of tissue growth, which is measured experimentally as a fractal dimension. Because of its complexity, the same phenotype can be associated with a number of alternative sequences of cellular events. Moreover, the primary cause initiating phenotypic evolution of cells such as malignant transformation can be favored probabilistically, but not identified unequivocally. Thermodynamic fluctuations of energy rather than gene mutations, the material traits of the fluctuations alter both the molecular and informational structure of the network. Then, the interplay between deterministic chaos, complexity, self-organization, and natural selection drives formation of malignant phenotype. This concept offers a novel perspective for investigation of tumorigenesis without invalidating current molecular findings. The essay integrates the ideas of the sciences of complexity in a biological context.

Analysis of Alternative Pre-RNA Splicing in the Mouse Retina Using a Fluorescent Reporter.

PubMed

Murphy, Daniel; Kolandaivelu, Saravanan; Ramamurthy, Visvanathan; Stoilov, Peter

2016-01-01

In vivo alternative splicing is controlled in a tissue and cell type specific manner. Often individual cellular components of complex tissues will express different splicing programs. Thus, when studying splicing in multicellular organisms it is critical to determine the exon inclusion levels in individual cells positioned in the context of their native tissue or organ. Here we describe how a fluorescent splicing reporter in combination with in vivo electroporation can be used to visualize alternative splicing in individual cells within mature tissues. In a test case we show how the splicing of a photoreceptor specific exon can be visualized within the mouse retina. The retina was chosen as an example of a complex tissue that is fragile and whose cells cannot be studied in culture. With minor modifications to the injection and electroporation procedure, the protocol we outline can be applied to other tissues and organs.

Epidermal growth factor receptor and variant III targeted immunotherapy

PubMed Central

Congdon, Kendra L.; Gedeon, Patrick C.; Suryadevara, Carter M.; Caruso, Hillary G.; Cooper, Laurence J.N.; Heimberger, Amy B.; Sampson, John H.

2014-01-01

Immunotherapeutic approaches to cancer have shown remarkable promise. A critical barrier to successfully executing such immune-mediated interventions is the selection of safe yet immunogenic targets. As patient deaths have occurred when tumor-associated antigens shared by normal tissue have been targeted by strong cellular immunotherapeutic platforms, route of delivery, target selection and the immune-mediated approach undertaken must work together to maximize efficacy with safety. Selected tumor-specific targets can spare potential toxicity to normal tissue; however, they are far less common than tumor-associated antigens and may not be present on all patients. In the context of immunotherapy for high-grade glioma, 2 of the most prominently studied antigens are the tumor-associated epidermal growth factor receptor and its tumor-specific genetic deletion variant III. In this review, we will summarize the immune-mediated strategies employed against these targets as well as the caveats particular to these approaches. PMID:25342601

Partial promoter substitutions generating transcriptional sentinels of diverse signaling pathways in embryonic stem cells and mice

PubMed Central

Serup, Palle; Gustavsen, Carsten; Klein, Tino; Potter, Leah A.; Lin, Robert; Mullapudi, Nandita; Wandzioch, Ewa; Hines, Angela; Davis, Ashley; Bruun, Christine; Engberg, Nina; Petersen, Dorthe R.; Peterslund, Janny M. L.; MacDonald, Raymond J.; Grapin-Botton, Anne; Magnuson, Mark A.; Zaret, Kenneth S.

2012-01-01

SUMMARY Extracellular signals in development, physiology, homeostasis and disease often act by regulating transcription. Herein we describe a general method and specific resources for determining where and when such signaling occurs in live animals and for systematically comparing the timing and extent of different signals in different cellular contexts. We used recombinase-mediated cassette exchange (RMCE) to test the effect of successively deleting conserved genomic regions of the ubiquitously active Rosa26 promoter and substituting the deleted regions for regulatory sequences that respond to diverse extracellular signals. We thereby created an allelic series of embryonic stem cells and mice, each containing a signal-responsive sentinel with different fluorescent reporters that respond with sensitivity and specificity to retinoic acids, bone morphogenic proteins, activin A, Wnts or Notch, and that can be adapted to any pathway that acts via DNA elements. PMID:22888097

Sex differences in the developing brain as a source of inherent risk.

PubMed

McCarthy, Margaret M

2016-12-01

Brain development diverges in males and females in response to androgen production by the fetal testis. This sexual differentiation of the brain occurs during a sensitive window and induces enduring neuroanatomical and physiological changes that profoundly impact behavior. What we know about the contribution of sex chromosomes is still emerging, highlighting the need to integrate multiple factors into understanding sex differences, including the importance of context. The cellular mechanisms are best modeled in rodents and have provided both unifying principles and surprising specifics. Markedly distinct signaling pathways direct differentiation in specific brain regions, resulting in mosaicism of relative maleness, femaleness, and sameness through-out the brain, while canalization both exaggerates and constrains sex differences. Non-neuronal cells and inflammatory mediators are found in greater number and at higher levels in parts of male brains. This higher baseline of inflammation is speculated to increase male vulnerability to developmental neuropsychiatric disorders that are triggered by inflammation.

Chitosan based hydrogels: characteristics and pharmaceutical applications

PubMed Central

Ahmadi, F.; Oveisi, Z.; Samani, S. Mohammadi; Amoozgar, Z.

2015-01-01

Hydrogel scaffolds serve as semi synthetic or synthetic extra cellular matrix to provide an amenable environment for cellular adherence and cellular remodeling in three dimensional structures mimicking that of natural cellular environment. Additionally, hydrogels have the capacity to carry small molecule drugs and/or proteins, growth factors and other necessary components for cell growth and differentiation. In the context of drug delivery, hydrogels can be utilized to localize drugs, increase drugs concentration at the site of action and consequently reduce off-targeted side effects. The current review aims to describe and classify hydrogels and their methods of production. The main highlight is chitosan-based hydrogels as biocompatible and medically relevant hydrogels for drug delivery. PMID:26430453

Dynamic maps of UV damage formation and repair for the human genome

PubMed Central

Hu, Jinchuan; Adebali, Ogun; Adar, Sheera; Sancar, Aziz

2017-01-01

Formation and repair of UV-induced DNA damage in human cells are affected by cellular context. To study factors influencing damage formation and repair genome-wide, we developed a highly sensitive single-nucleotide resolution damage mapping method [high-sensitivity damage sequencing (HS–Damage-seq)]. Damage maps of both cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts [(6-4)PPs] from UV-irradiated cellular and naked DNA revealed that the effect of transcription factor binding on bulky adducts formation varies, depending on the specific transcription factor, damage type, and strand. We also generated time-resolved UV damage maps of both CPDs and (6-4)PPs by HS–Damage-seq and compared them to the complementary repair maps of the human genome obtained by excision repair sequencing to gain insight into factors that affect UV-induced DNA damage and repair and ultimately UV carcinogenesis. The combination of the two methods revealed that, whereas UV-induced damage is virtually uniform throughout the genome, repair is affected by chromatin states, transcription, and transcription factor binding, in a manner that depends on the type of DNA damage. PMID:28607063

Correlative Microscopy of Vitreous Sections Provides Insights into BAR-Domain Organization In Situ.

PubMed

Bharat, Tanmay A M; Hoffmann, Patrick C; Kukulski, Wanda

2018-04-10

Electron microscopy imaging of macromolecular complexes in their native cellular context is limited by the inherent difficulty to acquire high-resolution tomographic data from thick cells and to specifically identify elusive structures within crowded cellular environments. Here, we combined cryo-fluorescence microscopy with electron cryo-tomography of vitreous sections into a coherent correlative microscopy workflow, ideal for detection and structural analysis of elusive protein assemblies in situ. We used this workflow to address an open question on BAR-domain coating of yeast plasma membrane compartments known as eisosomes. BAR domains can sense or induce membrane curvature, and form scaffold-like membrane coats in vitro. Our results demonstrate that in cells, the BAR protein Pil1 localizes to eisosomes of varying membrane curvature. Sub-tomogram analysis revealed a dense protein coat on curved eisosomes, which was not present on shallow eisosomes, indicating that while BAR domains can assemble at shallow membranes in vivo, scaffold formation is tightly coupled to curvature generation. Copyright © 2018 MRC Laboratory of Molecular Biology. Published by Elsevier Ltd.. All rights reserved.

Dynamic maps of UV damage formation and repair for the human genome.

PubMed

Hu, Jinchuan; Adebali, Ogun; Adar, Sheera; Sancar, Aziz

2017-06-27

Formation and repair of UV-induced DNA damage in human cells are affected by cellular context. To study factors influencing damage formation and repair genome-wide, we developed a highly sensitive single-nucleotide resolution damage mapping method [high-sensitivity damage sequencing (HS-Damage-seq)]. Damage maps of both cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts [(6-4)PPs] from UV-irradiated cellular and naked DNA revealed that the effect of transcription factor binding on bulky adducts formation varies, depending on the specific transcription factor, damage type, and strand. We also generated time-resolved UV damage maps of both CPDs and (6-4)PPs by HS-Damage-seq and compared them to the complementary repair maps of the human genome obtained by excision repair sequencing to gain insight into factors that affect UV-induced DNA damage and repair and ultimately UV carcinogenesis. The combination of the two methods revealed that, whereas UV-induced damage is virtually uniform throughout the genome, repair is affected by chromatin states, transcription, and transcription factor binding, in a manner that depends on the type of DNA damage.

Sequence context and crosslinking mechanism affect the efficiency of in vivo capture of a protein-protein interaction

PubMed Central

Lancia, Jody K.; Nwokoye, Adaora; Dugan, Amanda; Joiner, Cassandra; Pricer, Rachel; Mapp, Anna K.

2014-01-01

Protein-protein interactions (PPIs) are essential for implementing cellular processes and thus methods for the discovery and study of PPIs are highly desirable. An emerging method for capturing PPIs in their native cellular environment is in vivo covalent chemical capture, a method that uses nonsense suppression to site specifically incorporate photoactivable unnatural amino acids in living cells. However, in one study we found that this method did not capture a PPI for which there was abundant functional evidence, a complex formed between the transcriptional activator Gal4 and its repressor protein Gal80. Here we describe the factors that influence the success of covalent chemical capture and show that the innate reactivity of the two unnatural amino acids utilized, (p-benzoylphenylalanine (pBpa) and p-azidophenylalanine (pAzpa)), plays a profound role in the capture of Gal80 by Gal4. Based upon these data, guidelines are outlined for the successful use of in vivo photo-crosslinking to capture novel PPIs and to characterize the interfaces. PMID:24037947

Tissue- and cell-type–specific manifestations of heteroplasmic mtDNA 3243A>G mutation in human induced pluripotent stem cell-derived disease model

PubMed Central

Hämäläinen, Riikka H.; Manninen, Tuula; Koivumäki, Hanna; Kislin, Mikhail; Otonkoski, Timo; Suomalainen, Anu

2013-01-01

Mitochondrial DNA (mtDNA) mutations manifest with vast clinical heterogeneity. The molecular basis of this variability is mostly unknown because the lack of model systems has hampered mechanistic studies. We generated induced pluripotent stem cells from patients carrying the most common human disease mutation in mtDNA, m.3243A>G, underlying mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. During reprogramming, heteroplasmic mtDNA showed bimodal segregation toward homoplasmy, with concomitant changes in mtDNA organization, mimicking mtDNA bottleneck during epiblast specification. Induced pluripotent stem cell–derived neurons and various tissues derived from teratomas manifested cell-type specific respiratory chain (RC) deficiency patterns. Similar to MELAS patient tissues, complex I defect predominated. Upon neuronal differentiation, complex I specifically was sequestered in perinuclear PTEN-induced putative kinase 1 (PINK1) and Parkin-positive autophagosomes, suggesting active degradation through mitophagy. Other RC enzymes showed normal mitochondrial network distribution. Our data show that cellular context actively modifies RC deficiency manifestation in MELAS and that autophagy is a significant component of neuronal MELAS pathogenesis. PMID:24003133

Impact of labile metal nanoparticles on cellular homeostasis. Current developments in imaging, synthesis and applications.

PubMed

Chevallet, Mireille; Veronesi, Giulia; Fuchs, Alexandra; Mintz, Elisabeth; Michaud-Soret, Isabelle; Deniaud, Aurélien

2017-06-01

The use of nanomaterials is constantly increasing in electronics, cosmetics, food additives, and is emerging in advanced biomedical applications such as theranostics, bio-imaging and therapeutics. However their safety raises concerns and requires appropriate methods to analyze their fate in vivo. In this review, we describe the current knowledge about the toxicity of labile metal (ZnO, CuO and Ag) nanoparticles (NPs) both at the organism and cellular levels, and describe the pathways that are triggered to maintain cellular homeostasis. We also describe advanced elemental imaging approaches to analyze intracellular NP fate. Finally, we open the discussion by presenting recent developments in terms of synthesis and applications of Ag and CuO NPs. Labile metal nanoparticles (MeNPs) release metal ions that trigger a cellular response involving biomolecules binding to the ions followed by regulation of the redox balance. In addition, specific mechanisms are set up by the cell in response to physiological ions such as Cu(I) and Zn(II). Among all types of NPs, labile MeNPs induce the strongest inflammatory responses which are most probably due to the combined effects of the NPs and of its released ions. Interestingly, recent developments in imaging technologies enable the intracellular visualization of both the NPs and their ions and promise new insights into nanoparticle fate and toxicity. The exponential use of nanotechnologies associated with the difficulties of assessing their impact on health and the environment has prompted scientists to develop novel methodologies to characterize these nanoobjects in a biological context. Copyright © 2016 Elsevier B.V. All rights reserved.

CORSEN, a new software dedicated to microscope-based 3D distance measurements: mRNA-mitochondria distance, from single-cell to population analyses.

PubMed

Jourdren, Laurent; Delaveau, Thierry; Marquenet, Emelie; Jacq, Claude; Garcia, Mathilde

2010-07-01

Recent improvements in microscopy technology allow detection of single molecules of RNA, but tools for large-scale automatic analyses of particle distributions are lacking. An increasing number of imaging studies emphasize the importance of mRNA localization in the definition of cell territory or the biogenesis of cell compartments. CORSEN is a new tool dedicated to three-dimensional (3D) distance measurements from imaging experiments especially developed to access the minimal distance between RNA molecules and cellular compartment markers. CORSEN includes a 3D segmentation algorithm allowing the extraction and the characterization of the cellular objects to be processed--surface determination, aggregate decomposition--for minimal distance calculations. CORSEN's main contribution lies in exploratory statistical analysis, cell population characterization, and high-throughput assays that are made possible by the implementation of a batch process analysis. We highlighted CORSEN's utility for the study of relative positions of mRNA molecules and mitochondria: CORSEN clearly discriminates mRNA localized to the vicinity of mitochondria from those that are translated on free cytoplasmic polysomes. Moreover, it quantifies the cell-to-cell variations of mRNA localization and emphasizes the necessity for statistical approaches. This method can be extended to assess the evolution of the distance between specific mRNAs and other cellular structures in different cellular contexts. CORSEN was designed for the biologist community with the concern to provide an easy-to-use and highly flexible tool that can be applied for diverse distance quantification issues.

A Highly Sensitive Chemiluminometric Assay for Real-Time Detection of Biological Hydrogen Peroxide Formation.

PubMed

Zhu, Hong; Jia, Zhenquan; Trush, Michael A; Li, Y Robert

2016-05-01

Hydrogen peroxide (H 2 O 2 ) is a major reactive oxygen species (ROS) produced by various cellular sources, especially mitochondria. At high levels, H 2 O 2 causes oxidative stress, leading to cell injury, whereas at low concentrations, this ROS acts as an important second messenger to participate in cellular redox signaling. Detection and measurement of the levels or rates of production of cellular H 2 O 2 are instrumental in studying the biological effects of this major ROS. While a number of assays have been developed over the past decades for detecting and/or quantifying biological H 2 O 2 formation, none has been shown to be perfect. Perhaps there is no perfect assay for sensitively and accurately quantifying H 2 O 2 as well as other ROS in cells, wherein numerous potential reactants are present to interfere with the reliable measurement of the specific ROS. In this context, each assay has its own advantages and intrinsic limitations. This article describes a highly sensitive assay for real-time detection of H 2 O 2 formation in cultured cells and isolated mitochondria. This assay is based on the luminol/horseradish peroxidase-dependent chemiluminescence that is inhibitable by catalase. The article discusses the usefulness and shortcomings of this chemiluminometric assay in detecting biological H 2 O 2 formation induced by beta-lapachone redox cycling with both cells and isolated mitochondria.

Optical cell stimulation for neuronal excitation (Conference Presentation)

NASA Astrophysics Data System (ADS)

Johannsmeier, Sonja; Heeger, Patrick; Terakawa, Mitsuhiro; Heisterkamp, Alexander; Ripken, Tammo; Heinemann, Dag

2017-02-01

Optical manipulation of cellular functions represents a growing field in biomedical sciences. The possibility to modulate specific targets with high spatial and temporal precision in a contactless manner allows a broad range of applications. Here, we present a study on stimulation of neuronal cells by optical means. As a long-term objective, we seek to improve the performance of current electric neurostimulation, especially in the context of cochlear implants. Firstly, we tested a gold nanoparticle mediated approach to modulate transmembrane conductivity by irradiation using a picosecond pulsed Nd:YAG laser at 532 nm for 40 ms in a neuroblastoma cell line (N2A) and primary murine neurons. The light absorption leads to a rapid temperature increase of the gold nanoparticles, which can induce an increased permeabilisation of the cellular membrane. Calcium transients were recorded as an indicator of neuronal activity. Although calcium signals were reliably detected upon laser irradiation, the temporal behavior did not resemble action potentials. The origin of these signals was investigated by an inhibitor study. These results indicate calcium induced calcium release (CICR) as the major source of the calcium transients. Consecutively, we tested alternative approaches for cell stimulation, such as glutamate release and optogenetics, and evaluated the potential of these methods for the application in a cochlear implant. Compared to the gold nanoparticle approach, both techniques induce less cellular stress and reliably produce action potentials.

Nitrosothiol signaling and protein nitrosation in cell death.

PubMed

Iyer, Anand Krishnan V; Rojanasakul, Yon; Azad, Neelam

2014-11-15

Nitric oxide, a reactive free radical, is an important signaling molecule that can lead to a plethora of cellular effects affecting homeostasis. A well-established mechanism by which NO manifests its effect on cellular functions is the post-translational chemical modification of cysteine thiols in substrate proteins by a process known as S-nitrosation. Studies that investigate regulation of cellular functions through NO have increasingly established S-nitrosation as the primary modulatory mechanism in their respective systems. There has been a substantial increase in the number of reports citing various candidate proteins undergoing S-nitrosation, which affects cell-death and -survival pathways in a number of tissues including heart, lung, brain and blood. With an exponentially growing list of proteins being identified as substrates for S-nitrosation, it is important to assimilate this information in different cell/tissue systems in order to gain an overall view of protein regulation of both individual proteins and a class of protein substrates. This will allow for broad mapping of proteins as a function of S-nitrosation, and help delineate their global effects on pathophysiological responses including cell death and survival. This information will not only provide a much better understanding of overall functional relevance of NO in the context of various disease states, it will also facilitate the generation of novel therapeutics to combat specific diseases that are driven by NO-mediated S-nitrosation. Copyright © 2014 Elsevier Inc. All rights reserved.

Kinase Substrate Sensor (KISS), a Mammalian In Situ Protein Interaction Sensor*

PubMed Central

Lievens, Sam; Gerlo, Sarah; Lemmens, Irma; De Clercq, Dries J. H.; Risseeuw, Martijn D. P.; Vanderroost, Nele; De Smet, Anne-Sophie; Ruyssinck, Elien; Chevet, Eric; Van Calenbergh, Serge; Tavernier, Jan

2014-01-01

Probably every cellular process is governed by protein-protein interaction (PPIs), which are often highly dynamic in nature being modulated by in- or external stimuli. Here we present KISS, for KInase Substrate Sensor, a mammalian two-hybrid approach designed to map intracellular PPIs and some of the dynamic features they exhibit. Benchmarking experiments indicate that in terms of sensitivity and specificity KISS is on par with other binary protein interaction technologies while being complementary with regard to the subset of PPIs it is able to detect. We used KISS to evaluate interactions between different types of proteins, including transmembrane proteins, expressed at their native subcellular location. In situ analysis of endoplasmic reticulum stress-induced clustering of the endoplasmic reticulum stress sensor ERN1 and ligand-dependent β-arrestin recruitment to GPCRs illustrated the method's potential to study functional PPI modulation in complex cellular processes. Exploring its use as a tool for in cell evaluation of pharmacological interference with PPIs, we showed that reported effects of known GPCR antagonists and PPI inhibitors are properly recapitulated. In a three-hybrid setup, KISS was able to map interactions between small molecules and proteins. Taken together, we established KISS as a sensitive approach for in situ analysis of protein interactions and their modulation in a changing cellular context or in response to pharmacological challenges. PMID:25154561

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).

Metabolomics strategy for the mapping of volatile exometabolome from Saccharomyces spp. widely used in the food industry based on comprehensive two-dimensional gas chromatography.

PubMed

Martins, Cátia; Brandão, Tiago; Almeida, Adelaide; Rocha, Sílvia M

2017-05-01

Saccharomyces spp. are widely used in the food and beverages industries. Their cellular excreted metabolites are important for general quality of products and can contribute to product differentiation. This exploratory study presents a metabolomics strategy for the comprehensive mapping of cellular metabolites of two yeast species, Saccharomyces cerevisiae and S. pastorianus (both collected in an industrial context) through a multidimensional chromatography platform. Solid-phase microextraction was used as a sample preparation method. The yeast viability, a specific technological quality parameter, was also assessed. This untargeted analysis allowed the putative identification of 525 analytes, distributed over 14 chemical families, the origin of which may be explained through the pathways network associated with yeasts metabolism. The expression of the different metabolic pathways was similar for both species, event that seems to be yeast genus dependent. Nevertheless, these species showed different growth rates, which led to statistically different metabolites content. This was the first in-depth approach that characterizes the headspace content of S. cerevisiae and S. pastorianus species cultures. The combination of a sample preparation method capable of providing released volatile metabolites directly from yeast culture headspace with comprehensive two-dimensional gas chromatography was successful in uncovering a specific metabolomic pattern for each species. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Stem Cells from Dental Pulp: What Epigenetics Can Do with Your Tooth

PubMed Central

Rodas-Junco, Beatriz A.; Canul-Chan, Michel; Rojas-Herrera, Rafael A.; De-la-Peña, Clelia; Nic-Can, Geovanny I.

2017-01-01

Adult stem cells have attracted scientific attention because they are able to self-renew and differentiate into several specialized cell types. In this context, human dental tissue-derived mesenchymal stem cells (hDT-MSCs) have emerged as a possible solution for repairing or regenerating damaged tissues. These cells can be isolated from primary teeth that are naturally replaced, third molars, or other dental tissues and exhibit self-renewal, a high proliferative rate and a great multilineage potential. However, the cellular and molecular mechanisms that determine lineage specification are still largely unknown. It is known that a change in cell fate requires the deletion of existing transcriptional programs, followed by the establishment of a new developmental program to give rise to a new cell lineage. Increasing evidence indicates that chromatin structure conformation can influence cell fate. In this way, reversible chemical modifications at the DNA or histone level, and combinations thereof can activate or inactivate cell-type-specific gene sequences, giving rise to an alternative cell fates. On the other hand, miRNAs are starting to emerge as a possible player in establishing particular somatic lineages. In this review, we discuss two new and promising research fields in medicine and biology, epigenetics and stem cells, by summarizing the properties of hDT-MSCs and highlighting the recent findings on epigenetic contributions to the regulation of cellular differentiation. PMID:29270128

Colicin Killing: Foiled Cell Defense and Hijacked Cell Functions

NASA Astrophysics Data System (ADS)

de Zamaroczy, Miklos; Chauleau, Mathieu

The study of bacteriocins, notably those produced by E. coli (and named colicins), was initiated in 1925 by Gratia, who first discovered "un remarquable exemple d'antagonisme entre deux souches de colibacilles". Since this innovating observation, the production of toxic exoproteins has been widely reported in all major lineages of Eubacteria and in Archaebacteria. Bacteriocins belong to the most abundant and most diverse group of these bacterial defense systems. Paradoxically, these antimicrobial cytotoxins are actually powerful weapons in the intense battle for bacterial survival. They are also biotechnologically useful since several bacteriocins are used as preservatives in the food industry or as antibiotics or as potential antitumor agents in human health care. Most colicins kill bacteria in one of two ways. The first type is those that form pores in the phospholipid bilayer of the inner membrane. They are active immediately after their translocation across the outer membrane. The translocation pathway requires generally either the BtuB receptor and the Tol (OmpF/TolABQR) complex, or the FepA, FhuA, or Cir receptor and the Ton (TonB/ExbBD) system. The second type of colicins encodes specific endonuclease activities that target DNA, rRNA, or tRNAs in the cytoplasm. To be active, these colicins require translocation across both the outer and inner membranes. The molecular mechanisms implicated in the complex cascade of interactions, required for the transfers of colicin molecules from the extracellular medium through the different "cellular compartments" (outer membrane, periplasm, inner membrane, and cytoplasm), are still incompletely understood. It is clear, however, that the colicins "hijack" specific cellular functions to facilitate access to their target. In this chapter, following a general presentation of colicin biology, we describe, compare, and update several of the concepts related to colicin toxicity and discuss recent, often unexpected findings, which help to advance our understanding of the molecular events governing colicin import. In particular, our review includes the following: (1) Structural data on the tripartite interaction of a colicin with the outer membrane receptor and the translocation machinery, (2) Comparison of the normal cellular functions of the Tol and Ton systems of the inner membrane with their "hijacked" roles during colicin import, (3) An analysis of the interaction of a nuclease-type colicin with its cognate immunity protein in the context of the immunity of producer cells, and of the dissociation of this complex in the context of the attack of the colicin on target cells, (4) Information on the endoproteolytic cleavage, which presumably accompanies the penetration of nuclease-type colicins into the cytoplasm. The new data presented here provides further insight into cellular functions "hijacked" or "borrowed" by colicins to permit their entry into target cells.

Cellular and synaptic network defects in autism

PubMed Central

Peça, João; Feng, Guoping

2012-01-01

Many candidate genes are now thought to confer susceptibility to autism spectrum disorder (ASD). Here we review four interrelated complexes, each composed of multiple families of genes that functionally coalesce on common cellular pathways. We illustrate a common thread in the organization of glutamatergic synapses and suggest a link between genes involved in Tuberous Sclerosis Complex, Fragile X syndrome, Angelman syndrome and several synaptic ASD candidate genes. When viewed in this context, progress in deciphering the molecular architecture of cellular protein-protein interactions together with the unraveling of synaptic dysfunction in neural networks may prove pivotal to advancing our understanding of ASDs. PMID:22440525

Cell-Based Therapeutics: The Next Pillar of Medicine

PubMed Central

Fischbach, Michael A.; Bluestone, Jeffrey A.; Lim, Wendell A.

2013-01-01

Two decades ago, the pharmaceutical industry—long dominated by small-molecule drugs—was revolutionized by the the advent of biologics. Today, biomedicine sits on the cusp of a new revolution: the use of microbial and human cells as versatile therapeutic engines. Here, we discuss the promise of this “third pillar” of therapeutics in the context of current scientific, regulatory, economic, and perceptual challenges. History suggests that the advent of cellular medicines will require the development of a foundational cellular engineering science that provides a systematic framework for safely and predictably altering and regulating cellular behaviors. PMID:23552369

Total cellular glycomics allows characterizing cells and streamlining the discovery process for cellular biomarkers.

PubMed

Fujitani, Naoki; Furukawa, Jun-ichi; Araki, Kayo; Fujioka, Tsuyoshi; Takegawa, Yasuhiro; Piao, Jinhua; Nishioka, Taiki; Tamura, Tomohiro; Nikaido, Toshio; Ito, Makoto; Nakamura, Yukio; Shinohara, Yasuro

2013-02-05

Although many of the frequently used pluripotency biomarkers are glycoconjugates, a glycoconjugate-based exploration of novel cellular biomarkers has proven difficult due to technical difficulties. This study reports a unique approach for the systematic overview of all major classes of oligosaccharides in the cellular glycome. The proposed method enabled mass spectrometry-based structurally intensive analyses, both qualitatively and quantitatively, of cellular N- and O-linked glycans derived from glycoproteins, glycosaminoglycans, and glycosphingolipids, as well as free oligosaccharides of human embryonic stem cells (hESCs), induced pluripotent stem cells (hiPSCs), and various human cells derived from normal and carcinoma cells. Cellular total glycomes were found to be highly cell specific, demonstrating their utility as unique cellular descriptors. Structures of glycans of all classes specifically observed in hESCs and hiPSCs tended to be immature in general, suggesting the presence of stem cell-specific glycosylation spectra. The current analysis revealed the high similarity of the total cellular glycome between hESCs and hiPSCs, although it was suggested that hESCs are more homogeneous than hiPSCs from a glycomic standpoint. Notably, this study enabled a priori identification of known pluripotency biomarkers such as SSEA-3, -4, and -5 and Tra-1-60/81, as well as a panel of glycans specifically expressed by hESCs and hiPSCs.

Treatment Analysis in a Cancer Stem Cell Context Using a Tumor Growth Model Based on Cellular Automata.

PubMed

Monteagudo, Ángel; Santos, José

2015-01-01

Cancer can be viewed as an emergent behavior in terms of complex system theory and artificial life, Cellular Automata (CA) being the tool most used for studying and characterizing the emergent behavior. Different approaches with CA models were used to model cancer growth. The use of the abstract model of acquired cancer hallmarks permits the direct modeling at cellular level, where a cellular automaton defines the mitotic and apoptotic behavior of cells, and allows for an analysis of different dynamics of the cellular system depending on the presence of the different hallmarks. A CA model based on the presence of hallmarks in the cells, which includes a simulation of the behavior of Cancer Stem Cells (CSC) and their implications for the resultant growth behavior of the multicellular system, was employed. This modeling of cancer growth, in the avascular phase, was employed to analyze the effect of cancer treatments in a cancer stem cell context. The model clearly explains why, after treatment against non-stem cancer cells, the regrowth capability of CSCs generates a faster regrowth of tumor behavior, and also shows that a continuous low-intensity treatment does not favor CSC proliferation and differentiation, thereby allowing an unproblematic control of future tumor regrowth. The analysis performed indicates that, contrary to the current attempts at CSC control, trying to make CSC proliferation more difficult is an important point to consider, especially in the immediate period after a standard treatment for controlling non-stem cancer cell proliferation.

In silico methods for co-transcriptional RNA secondary structure prediction and for investigating alternative RNA structure expression.

PubMed

Meyer, Irmtraud M

2017-05-01

RNA transcripts are the primary products of active genes in any living organism, including many viruses. Their cellular destiny not only depends on primary sequence signals, but can also be determined by RNA structure. Recent experimental evidence shows that many transcripts can be assigned more than a single functional RNA structure throughout their cellular life and that structure formation happens co-transcriptionally, i.e. as the transcript is synthesised in the cell. Moreover, functional RNA structures are not limited to non-coding transcripts, but can also feature in coding transcripts. The picture that now emerges is that RNA structures constitute an additional layer of information that can be encoded in any RNA transcript (and on top of other layers of information such as protein-context) in order to exert a wide range of functional roles. Moreover, different encoded RNA structures can be expressed at different stages of a transcript's life in order to alter the transcript's behaviour depending on its actual cellular context. Similar to the concept of alternative splicing for protein-coding genes, where a single transcript can yield different proteins depending on cellular context, it is thus appropriate to propose the notion of alternative RNA structure expression for any given transcript. This review introduces several computational strategies that my group developed to detect different aspects of RNA structure expression in vivo. Two aspects are of particular interest to us: (1) RNA secondary structure features that emerge during co-transcriptional folding and (2) functional RNA structure features that are expressed at different times of a transcript's life and potentially mutually exclusive. Copyright © 2017. Published by Elsevier Inc.

Pollen tube energetics: respiration, fermentation and the race to the ovule

PubMed Central

Rounds, Caleb M.; Winship, Lawrence J.; Hepler, Peter K.

2011-01-01

Background Pollen tubes grow by transferring chemical energy from stored cellular starch and newly assimilated sugars into ATP. This drives myriad processes essential for cell elongation, directly or through the creation of ion gradients. Respiration plays a central role in generating and regulating this energy flow and thus in the success of plant reproduction. Pollen tubes are easily grown in vitro and have become an excellent model for investigating the contributions of respiration to plant cellular growth and morphogenesis at the molecular, biochemical and physiological levels. Scope In recent decades, pollen tube research has become increasingly focused on the molecular mechanisms involved in cellular processes. Yet, effective growth and development requires an intact, integrated set of cellular processes, all supplied with a constant flow of energy. Here we bring together information from the current and historical literature concerning respiration, fermentation and mitochondrial physiology in pollen tubes, and assess the significance of more recent molecular and genetic investigations in a physiological context. Conclusions The rapid growth of the pollen tube down the style has led to the evolution of high rates of pollen tube respiration. Respiration rates in lily predict a total energy turnover of 40–50 fmol ATP s−1 per pollen grain. Within this context we examine the energetic requirements of cell wall synthesis, osmoregulation, actin dynamics and cyclosis. At present, we can only estimate the amount of energy required, because data from growing pollen tubes are not available. In addition to respiration, we discuss fermentation and mitochondrial localization. We argue that the molecular pathways need to be examined within the physiological context to understand better the mechanisms that control tip growth in pollen tubes. PMID:22476489

A Simple Method for Visualization of Locus-Specific H4K20me1 Modifications in Living Caenorhabditis elegans Single Cells.

PubMed

Shinkai, Yoichi; Kuramochi, Masahiro; Doi, Motomichi

2018-05-03

Recently, advances in next-generation sequencing technologies have enabled genome-wide analyses of epigenetic modifications; however, it remains difficult to analyze the states of histone modifications at a single-cell resolution in living multicellular organisms because of the heterogeneity within cellular populations. Here we describe a simple method to visualize histone modifications on the specific sequence of target locus at a single-cell resolution in living Caenorhabditis elegans , by combining the LacO/LacI system and a genetically-encoded H4K20me1-specific probe, "mintbody". We demonstrate that Venus-labeled mintbody and mTurquoise2-labeled LacI can co-localize on an artificial chromosome carrying both the target locus and LacO sequences, where H4K20me1 marks the target locus. We demonstrate that our visualization method can precisely detect H4K20me1 depositions on the her-1 gene sequences on the artificial chromosome, to which the dosage compensation complex binds to regulate sex determination. The degree of H4K20me1 deposition on the her-1 sequences on the artificial chromosome correlated strongly with sex, suggesting that, using the artificial chromosome, this method can reflect context-dependent changes of H4K20me1 on endogenous genomes. Furthermore, we demonstrate live imaging of H4K20me1 depositions on the artificial chromosome. Combined with ChIP assays, this mintbody-LacO/LacI visualization method will enable analysis of developmental and context-dependent alterations of locus-specific histone modifications in specific cells and elucidation of the underlying molecular mechanisms. Copyright © 2018, G3: Genes, Genomes, Genetics.

Cellular bone matrices: viable stem cell-containing bone graft substitutes

PubMed Central

Skovrlj, Branko; Guzman, Javier Z.; Al Maaieh, Motasem; Cho, Samuel K.; Iatridis, James C.; Qureshi, Sheeraz A.

2015-01-01

BACKGROUND CONTEXT Advances in the field of stem cell technology have stimulated the development and increased use of allogenic bone grafts containing live mesenchymal stem cells (MSCs), also known as cellular bone matrices (CBMs). It is estimated that CBMs comprise greater than 17% of all bone grafts and bone graft substitutes used. PURPOSE To critically evaluate CBMs, specifically their technical specifications, existing published data supporting their use, US Food and Drug Administration (FDA) regulation, cost, potential pitfalls, and other aspects pertaining to their use. STUDY DESIGN Areview of literature. METHODS A series of Ovid, Medline, and Pubmed-National Library of Medicine/National Institutes of Health (www.ncbi.nlm.nih.gov) searches were performed. Only articles in English journals or published with English language translations were included. Level of evidence of the selected articles was assessed. Specific technical information on each CBM was obtained by direct communication from the companies marketing the individual products. RESULTS Five different CBMs are currently available for use in spinal fusion surgery. There is a wide variation between the products with regard to the average donor age at harvest, total cellular concentration, percentage of MSCs, shelf life, and cell viability after defrosting. Three retrospective studies evaluating CBMs and fusion have shown fusion rates ranging from 90.2% to 92.3%, and multiple industry-sponsored trials are underway. No independent studies evaluating spinal fusion rates with the use of CBMs exist. All the commercially available CBMs claim to meet the FDA criteria under Section 361, 21 CFR Part 1271, and are not undergoing FDA premarket review. The CBMs claim to provide viable MSCs and are offered at a premium cost. Numerous challenges exist in regard to MSCs’ survival, function, osteoblastic potential, and cytokine production once implanted into the intended host. CONCLUSIONS Cellular bone matrices may be a promising bone augmentation technology in spinal fusion surgery. Although CBMs appear to be safe for use as bone graft substitutes, their efficacy in spinal fusion surgery remains highly inconclusive. Large, nonindustry sponsored studies evaluating the efficacy of CBMs are required. Without results from such studies, surgeons must be made aware of the potential pitfalls of CBMs in spinal fusion surgery. With the currently available data, there is insufficient evidence to support the use of CBMs as bone graft substitutes in spinal fusion surgery. PMID:24929059

Recontextualising Cellular Respiration in Upper Secondary Biology Education. Characteristics and Practicability of a Learning and Teaching Strategy

ERIC Educational Resources Information Center

Wierdsma, Menno; Knippels, Marie-Christine; van Oers, Bert; Boersma, Kerst

2016-01-01

Since concepts may have different meanings in different contexts, students have to learn to recontextualise them, i.e. to adapt their meanings to a new context. It is unclear, however, what characteristics a learning and teaching strategy for recontextualising should have. The study aims to develop such a learning and teaching strategy for…

A detailed view on sulphur metabolism at the cellular and whole-plant level illustrates challenges in metabolite flux analyses.

PubMed

Rennenberg, Heinz; Herschbach, Cornelia

2014-11-01

Understanding the dynamics of physiological process in the systems biology era requires approaches at the genome, transcriptome, proteome, and metabolome levels. In this context, metabolite flux experiments have been used in mapping metabolite pathways and analysing metabolic control. In the present review, sulphur metabolism was taken to illustrate current challenges of metabolic flux analyses. At the cellular level, restrictions in metabolite flux analyses originate from incomplete knowledge of the compartmentation network of metabolic pathways. Transport of metabolites through membranes is usually not considered in flux experiments but may be involved in controlling the whole pathway. Hence, steady-state and snapshot readings need to be expanded to time-course studies in combination with compartment-specific metabolite analyses. Because of species-specific differences, differences between tissues, and stress-related responses, the quantitative significance of different sulphur sinks has to be elucidated; this requires the development of methods for whole-sulphur metabolome approaches. Different cell types can contribute to metabolite fluxes to different extents at the tissue and organ level. Cell type-specific analyses are needed to characterize these contributions. Based on such approaches, metabolite flux analyses can be expanded to the whole-plant level by considering long-distance transport and, thus, the interaction of roots and the shoot in metabolite fluxes. However, whole-plant studies need detailed empirical and mathematical modelling that have to be validated by experimental analyses. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Frodo proteins: modulators of Wnt signaling in vertebrate development.

PubMed

Brott, Barbara K; Sokol, Sergei Y

2005-09-01

The Frodo/dapper (Frd) proteins are recently discovered signaling adaptors, which functionally and physically interact with Wnt and Nodal signaling pathways during vertebrate development. The Frd1 and Frd2 genes are expressed in dynamic patterns in early embryos, frequently in cells undergoing epithelial-mesenchymal transition. The Frd proteins function in multiple developmental processes, including mesoderm and neural tissue specification, early morphogenetic cell movements, and organogenesis. Loss-of-function studies using morpholino antisense oligonucleotides demonstrate that the Frd proteins regulate Wnt signal transduction in a context-dependent manner and may be involved in Nodal signaling. The identification of Frd-associated factors and cellular targets of the Frd proteins should shed light on the molecular mechanisms underlying Frd functions in embryonic development and in cancer.

Roles for Hedgehog signaling in adult organ homeostasis and repair

PubMed Central

Petrova, Ralitsa; Joyner, Alexandra L.

2014-01-01

The hedgehog (HH) pathway is well known for its mitogenic and morphogenic functions during development, and HH signaling continues in discrete populations of cells within many adult mammalian tissues. Growing evidence indicates that HH regulates diverse quiescent stem cell populations, but the exact roles that HH signaling plays in adult organ homeostasis and regeneration remain poorly understood. Here, we review recently identified functions of HH in modulating the behavior of tissue-specific adult stem and progenitor cells during homeostasis, regeneration and disease. We conclude that HH signaling is a key factor in the regulation of adult tissue homeostasis and repair, acting via multiple different routes to regulate distinct cellular outcomes, including maintenance of plasticity, in a context-dependent manner. PMID:25183867

Lysine Deacetylases and Regulated Glycolysis in Macrophages.

PubMed

Shakespear, Melanie R; Iyer, Abishek; Cheng, Catherine Youting; Das Gupta, Kaustav; Singhal, Amit; Fairlie, David P; Sweet, Matthew J

2018-06-01

Regulated cellular metabolism has emerged as a fundamental process controlling macrophage functions, but there is still much to uncover about the precise signaling mechanisms involved. Lysine acetylation regulates the activity, stability, and/or localization of metabolic enzymes, as well as inflammatory responses, in macrophages. Two protein families, the classical zinc-dependent histone deacetylases (HDACs) and the NAD-dependent HDACs (sirtuins, SIRTs), mediate lysine deacetylation. We describe here mechanisms by which classical HDACs and SIRTs directly regulate specific glycolytic enzymes, as well as evidence that links these protein deacetylases to the regulation of glycolysis-related genes. In these contexts, we discuss HDACs and SIRTs as key control points for regulating immunometabolism and inflammatory outputs from macrophages. Copyright © 2018 Elsevier Ltd. All rights reserved.

Non-coding RNA networks in cancer.

PubMed

Anastasiadou, Eleni; Jacob, Leni S; Slack, Frank J

2018-01-01

Thousands of unique non-coding RNA (ncRNA) sequences exist within cells. Work from the past decade has altered our perception of ncRNAs from 'junk' transcriptional products to functional regulatory molecules that mediate cellular processes including chromatin remodelling, transcription, post-transcriptional modifications and signal transduction. The networks in which ncRNAs engage can influence numerous molecular targets to drive specific cell biological responses and fates. Consequently, ncRNAs act as key regulators of physiological programmes in developmental and disease contexts. Particularly relevant in cancer, ncRNAs have been identified as oncogenic drivers and tumour suppressors in every major cancer type. Thus, a deeper understanding of the complex networks of interactions that ncRNAs coordinate would provide a unique opportunity to design better therapeutic interventions.

Lipids, lysosomes, and autophagy

PubMed Central

2016-01-01

Lipids are essential components of a cell providing energy substrates for cellular processes, signaling intermediates, and building blocks for biological membranes. Lipids are constantly recycled and redistributed within a cell. Lysosomes play an important role in this recycling process that involves the recruitment of lipids to lysosomes via autophagy or endocytosis for their degradation by lysosomal hydrolases. The catabolites produced are redistributed to various cellular compartments to support basic cellular function. Several studies demonstrated a bidirectional relationship between lipids and lysosomes that regulate autophagy. While lysosomal degradation pathways regulate cellular lipid metabolism, lipids also regulate lysosome function and autophagy. In this review, we focus on this bidirectional relationship in the context of dietary lipids and provide an overview of recent evidence of how lipid-overload lipotoxicity, as observed in obesity and metabolic syndrome, impairs lysosomal function and autophagy that may eventually lead to cellular dysfunction or cell death. PMID:27330054

Immobilized Cell Research

DTIC Science & Technology

1990-10-31

specifically with the biotech nologi cal side of cellular immobilization, there aje aspects of this research that have importance in other fields. 20 C...meetings dealt lem facing the Navy. The techniques reviewed here specifically with the biotechnological side of cellular im- should be of particular...phenomena. types of organisms, and the many techniques used to compare cellular physiologies. Undoubtedly, any tech- Why Use Immobilized Cells in

Global analysis of bacterial transcription factors to predict cellular target processes.

PubMed

Doerks, Tobias; Andrade, Miguel A; Lathe, Warren; von Mering, Christian; Bork, Peer

2004-03-01

Whole-genome sequences are now available for >100 bacterial species, giving unprecedented power to comparative genomics approaches. We have applied genome-context methods to predict target processes that are regulated by transcription factors (TFs). Of 128 orthologous groups of proteins annotated as TFs, to date, 36 are functionally uncharacterized; in our analysis we predict a probable cellular target process or biochemical pathway for half of these functionally uncharacterized TFs.

Survey statistics of automated segmentations applied to optical imaging of mammalian cells.

PubMed

Bajcsy, Peter; Cardone, Antonio; Chalfoun, Joe; Halter, Michael; Juba, Derek; Kociolek, Marcin; Majurski, Michael; Peskin, Adele; Simon, Carl; Simon, Mylene; Vandecreme, Antoine; Brady, Mary

2015-10-15

The goal of this survey paper is to overview cellular measurements using optical microscopy imaging followed by automated image segmentation. The cellular measurements of primary interest are taken from mammalian cells and their components. They are denoted as two- or three-dimensional (2D or 3D) image objects of biological interest. In our applications, such cellular measurements are important for understanding cell phenomena, such as cell counts, cell-scaffold interactions, cell colony growth rates, or cell pluripotency stability, as well as for establishing quality metrics for stem cell therapies. In this context, this survey paper is focused on automated segmentation as a software-based measurement leading to quantitative cellular measurements. We define the scope of this survey and a classification schema first. Next, all found and manually filteredpublications are classified according to the main categories: (1) objects of interests (or objects to be segmented), (2) imaging modalities, (3) digital data axes, (4) segmentation algorithms, (5) segmentation evaluations, (6) computational hardware platforms used for segmentation acceleration, and (7) object (cellular) measurements. Finally, all classified papers are converted programmatically into a set of hyperlinked web pages with occurrence and co-occurrence statistics of assigned categories. The survey paper presents to a reader: (a) the state-of-the-art overview of published papers about automated segmentation applied to optical microscopy imaging of mammalian cells, (b) a classification of segmentation aspects in the context of cell optical imaging, (c) histogram and co-occurrence summary statistics about cellular measurements, segmentations, segmented objects, segmentation evaluations, and the use of computational platforms for accelerating segmentation execution, and (d) open research problems to pursue. The novel contributions of this survey paper are: (1) a new type of classification of cellular measurements and automated segmentation, (2) statistics about the published literature, and (3) a web hyperlinked interface to classification statistics of the surveyed papers at https://isg.nist.gov/deepzoomweb/resources/survey/index.html.

Crossing boundaries: the importance of cellular membranes in industrial biotechnology.

PubMed

Jezierska, Sylwia; Van Bogaert, Inge N A

2017-05-01

How small molecules cross cellular membranes is an often overlooked issue in an industrial microbiology and biotechnology context. This is to a large extent governed by the technical difficulties to study these transport systems or by the lack of knowledge on suitable efflux pumps. This review emphasizes the importance of microbial cellular membranes in industrial biotechnology by highlighting successful strategies of membrane engineering towards more resistant and hence better performing microorganisms, as well as transporter and other engineering strategies for increased efflux of primary and secondary metabolites. Furthermore, the benefits and limitations of eukaryotic subcellular compartmentalization are discussed, as well as the biotechnological potential of membrane vesicles.

The HUPO proteomics standards initiative--overcoming the fragmentation of proteomics data.

PubMed

Hermjakob, Henning

2006-09-01

Proteomics is a key field of modern biomolecular research, with many small and large scale efforts producing a wealth of proteomics data. However, the vast majority of this data is never exploited to its full potential. Even in publicly funded projects, often the raw data generated in a specific context is analysed, conclusions are drawn and published, but little attention is paid to systematic documentation, archiving, and public access to the data supporting the scientific results. It is often difficult to validate the results stated in a particular publication, and even simple global questions like "In which cellular contexts has my protein of interest been observed?" can currently not be answered with realistic effort, due to a lack of standardised reporting and collection of proteomics data. The Proteomics Standards Initiative (PSI), a work group of the Human Proteome Organisation (HUPO), defines community standards for data representation in proteomics to facilitate systematic data capture, comparison, exchange and verification. In this article we provide an overview of PSI organisational structure, activities, and current results, as well as ways to get involved in the broad-based, open PSI process.

Dynamic interactions between 14-3-3 proteins and phosphoproteins regulate diverse cellular processes

PubMed Central

2004-01-01

14-3-3 proteins exert an extraordinarily widespread influence on cellular processes in all eukaryotes. They operate by binding to specific phosphorylated sites on diverse target proteins, thereby forcing conformational changes or influencing interactions between their targets and other molecules. In these ways, 14-3-3s ‘finish the job’ when phosphorylation alone lacks the power to drive changes in the activities of intracellular proteins. By interacting dynamically with phosphorylated proteins, 14-3-3s often trigger events that promote cell survival – in situations from preventing metabolic imbalances caused by sudden darkness in leaves to mammalian cell-survival responses to growth factors. Recent work linking specific 14-3-3 isoforms to genetic disorders and cancers, and the cellular effects of 14-3-3 agonists and antagonists, indicate that the cellular complement of 14-3-3 proteins may integrate the specificity and strength of signalling through to different cellular responses. PMID:15167810

Structural basis for genome wide recognition of 5-bp GC motifs by SMAD transcription factors.

PubMed

Martin-Malpartida, Pau; Batet, Marta; Kaczmarska, Zuzanna; Freier, Regina; Gomes, Tiago; Aragón, Eric; Zou, Yilong; Wang, Qiong; Xi, Qiaoran; Ruiz, Lidia; Vea, Angela; Márquez, José A; Massagué, Joan; Macias, Maria J

2017-12-12

Smad transcription factors activated by TGF-β or by BMP receptors form trimeric complexes with Smad4 to target specific genes for cell fate regulation. The CAGAC motif has been considered as the main binding element for Smad2/3/4, whereas Smad1/5/8 have been thought to preferentially bind GC-rich elements. However, chromatin immunoprecipitation analysis in embryonic stem cells showed extensive binding of Smad2/3/4 to GC-rich cis-regulatory elements. Here, we present the structural basis for specific binding of Smad3 and Smad4 to GC-rich motifs in the goosecoid promoter, a nodal-regulated differentiation gene. The structures revealed a 5-bp consensus sequence GGC(GC)|(CG) as the binding site for both TGF-β and BMP-activated Smads and for Smad4. These 5GC motifs are highly represented as clusters in Smad-bound regions genome-wide. Our results provide a basis for understanding the functional adaptability of Smads in different cellular contexts, and their dependence on lineage-determining transcription factors to target specific genes in TGF-β and BMP pathways.

Multi-Compartmentalisation in the MAPK Signalling Pathway Contributes to the Emergence of Oscillatory Behaviour and to Ultrasensitivity

PubMed Central

Shuaib, Aban; Hartwell, Adam; Kiss-Toth, Endre; Holcombe, Mike

2016-01-01

Signal transduction through the Mitogen Activated Protein Kinase (MAPK) pathways is evolutionarily highly conserved. Many cells use these pathways to interpret changes to their environment and respond accordingly. The pathways are central to triggering diverse cellular responses such as survival, apoptosis, differentiation and proliferation. Though the interactions between the different MAPK pathways are complex, nevertheless, they maintain a high level of fidelity and specificity to the original signal. There are numerous theories explaining how fidelity and specificity arise within this complex context; spatio-temporal regulation of the pathways and feedback loops are thought to be very important. This paper presents an agent based computational model addressing multi-compartmentalisation and how this influences the dynamics of MAPK cascade activation. The model suggests that multi-compartmentalisation coupled with periodic MAPK kinase (MAPKK) activation may be critical factors for the emergence of oscillation and ultrasensitivity in the system. Finally, the model also establishes a link between the spatial arrangements of the cascade components and temporal activation mechanisms, and how both contribute to fidelity and specificity of MAPK mediated signalling. PMID:27243235

Cellular Immune Response to Cytomegalovirus Infection After Renal Transplantation

PubMed Central

Linnemann, Calvin C.; Kauffman, Carol A.; First, M. Roy; Schiff, Gilbert M.; Phair, John P.

1978-01-01

A prospective study of 15 patients who received renal transplants defined the effect of renal transplantation on the cellular immune response to cytomegalovirus infection. Of 15 patients, 14 developed cytomegalovirus infection, usually in the first 2 months after transplantation, and all infections were accompanied by a normal humoral immune response. After the initiation of immunosuppressive therapy and transplantation, there was a general depression of lymphocyte transformation, as reflected in the response to phytohemagglutinin, accompanied by a specific defect in cellular immunity, as indicated by lymphocyte transformation to cytomegalovirus antigen. Eleven patients had cellular immunity to cytomegalovirus before transplantation, and all of these became negative in the first month after transplantation. In subsequent months, only 6 of the 14 study patients with cytomegalovirus infection developed specific cellular immune responses to cytomegalovirus. This occurred most often in patients who had severe febrile illnesses in association with infection. The specific cellular immune response which developed in the posttransplant period did not persist in three of the patients. This study demonstrates the dissociation of the humoral and cellular immune response to cytomegalovirus infection in renal transplant patients and indicates the importance of the loss of cellular immunity in the appearance of infection. Previously infected patients lost their cell-mediated immunity and had reactivation infections despite the presence of serum antibody. PMID:215541

Inflammatory responses to secondary organic aerosols (SOA) generated from biogenic and anthropogenic precursors

NASA Astrophysics Data System (ADS)

Tuet, Wing Y.; Chen, Yunle; Fok, Shierly; Champion, Julie A.; Ng, Nga L.

2017-09-01

Cardiopulmonary health implications resulting from exposure to secondary organic aerosols (SOA), which comprise a significant fraction of ambient particulate matter (PM), have received increasing interest in recent years. In this study, alveolar macrophages were exposed to SOA generated from the photooxidation of biogenic and anthropogenic precursors (isoprene, α-pinene, β-caryophyllene, pentadecane, m-xylene, and naphthalene) under different formation conditions (RO2 + HO2 vs. RO2 + NO dominant, dry vs. humid). Various cellular responses were measured, including reactive oxygen and nitrogen species (ROS/RNS) production and secreted levels of cytokines, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). SOA precursor identity and formation condition affected all measured responses in a hydrocarbon-specific manner. With the exception of naphthalene SOA, cellular responses followed a trend where TNF-α levels reached a plateau with increasing IL-6 levels. ROS/RNS levels were consistent with relative levels of TNF-α and IL-6, due to their respective inflammatory and anti-inflammatory effects. Exposure to naphthalene SOA, whose aromatic-ring-containing products may trigger different cellular pathways, induced higher levels of TNF-α and ROS/RNS than suggested by the trend. Distinct cellular response patterns were identified for hydrocarbons whose photooxidation products shared similar chemical functionalities and structures, which suggests that the chemical structure (carbon chain length and functionalities) of photooxidation products may be important for determining cellular effects. A positive nonlinear correlation was also detected between ROS/RNS levels and previously measured DTT (dithiothreitol) activities for SOA samples. In the context of ambient samples collected during summer and winter in the greater Atlanta area, all laboratory-generated SOA produced similar or higher levels of ROS/RNS and DTT activities. These results suggest that the health effects of SOA are important considerations for understanding the health implications of ambient aerosols.

Mechanistic considerations on the wavelength-dependent variations of UVR genotoxicity and mutagenesis in skin: the discrimination of UVA-signature from UV-signature mutation.

PubMed

Ikehata, Hironobu

2018-05-31

Ultraviolet radiation (UVR) predominantly induces UV-signature mutations, C → T and CC → TT base substitutions at dipyrimidine sites, in the cellular and skin genome. I observed in our in vivo mutation studies of mouse skin that these UVR-specific mutations show a wavelength-dependent variation in their sequence-context preference. The C → T mutation occurs most frequently in the 5'-TCG-3' sequence regardless of the UVR wavelength, but is recovered more preferentially there as the wavelength increases, resulting in prominent occurrences exclusively in the TCG sequence in the UVA wavelength range, which I will designate as a "UVA signature" in this review. The preference of the UVB-induced C → T mutation for the sequence contexts shows a mixed pattern of UVC- and UVA-induced mutations, and a similar pattern is also observed for natural sunlight, in which UVB is the most genotoxic component. In addition, the CC → TT mutation hardly occurs at UVA1 wavelengths, although it is detected rarely but constantly in the UVC and UVB ranges. This wavelength-dependent variation in the sequence-context preference of the UVR-specific mutations could be explained by two different photochemical mechanisms of cyclobutane pyrimidine dimer (CPD) formation. The UV-signature mutations observed in the UVC and UVB ranges are known to be caused mainly by CPDs produced through the conventional singlet/triplet excitation of pyrimidine bases after the direct absorption of the UVC/UVB photon energy in those bases. On the other hand, a novel photochemical mechanism through the direct absorption of the UVR energy to double-stranded DNA, which is called "collective excitation", has been proposed for the UVA-induced CPD formation. The UVA photons directly absorbed by DNA produce CPDs with a sequence context preference different from that observed for CPDs caused by the UVC/UVB-mediated singlet/triplet excitation, causing CPD formation preferentially at thymine-containing dipyrimidine sites and probably also preferably at methyl CpG-associated dipyrimidine sites, which include the TCG sequence. In this review, I present a mechanistic consideration on the wavelength-dependent variation of the sequence context preference of the UVR-specific mutations and rationalize the proposition of the UVA-signature mutation, in addition to the UV-signature mutation.

In search of cellular control: signal transduction in context

NASA Technical Reports Server (NTRS)

Ingber, D.

1998-01-01

The field of molecular cell biology has experienced enormous advances over the last century by reducing the complexity of living cells into simpler molecular components and binding interactions that are amenable to rigorous biochemical analysis. However, as our tools become more powerful, there is a tendency to define mechanisms by what we can measure. The field is currently dominated by efforts to identify the key molecules and sequences that mediate the function of critical receptors, signal transducers, and molecular switches. Unfortunately, these conventional experimental approaches ignore the importance of supramolecular control mechanisms that play a critical role in cellular regulation. Thus, the significance of individual molecular constituents cannot be fully understood when studied in isolation because their function may vary depending on their context within the structural complexity of the living cell. These higher-order regulatory mechanisms are based on the cell's use of a form of solid-state biochemistry in which molecular components that mediate biochemical processing and signal transduction are immobilized on insoluble cytoskeletal scaffolds in the cytoplasm and nucleus. Key to the understanding of this form of cellular regulation is the realization that chemistry is structure and hence, recognition of the the importance of architecture and mechanics for signal integration and biochemical control. Recent work that has unified chemical and mechanical signaling pathways provides a glimpse of how this form of higher-order cellular control may function and where paths may lie in the future.

A reporter model to visualize imprinting stability at the Dlk1 locus during mouse development and in pluripotent cells.

PubMed

Swanzey, Emily; Stadtfeld, Matthias

2016-11-15

Genomic imprinting results in the monoallelic expression of genes that encode important regulators of growth and proliferation. Dysregulation of imprinted genes, such as those within the Dlk1-Dio3 locus, is associated with developmental syndromes and specific diseases. Our ability to interrogate causes of imprinting instability has been hindered by the absence of suitable model systems. Here, we describe a Dlk1 knock-in reporter mouse that enables single-cell visualization of allele-specific expression and prospective isolation of cells, simultaneously. We show that this 'imprinting reporter mouse' can be used to detect tissue-specific Dlk1 expression patterns in developing embryos. We also apply this system to pluripotent cell culture and demonstrate that it faithfully indicates DNA methylation changes induced upon cellular reprogramming. Finally, the reporter system reveals the role of elevated oxygen levels in eroding imprinted Dlk1 expression during prolonged culture and in vitro differentiation. The possibility to study allele-specific expression in different contexts makes our reporter system a useful tool to dissect the regulation of genomic imprinting in normal development and disease. © 2016. Published by The Company of Biologists Ltd.

[Visualization and Functional Regulation of Live Cell Proteins Based on Labeling Probe Design].

PubMed

Mizukami, Shin; Kikuchi, Kazuya

2016-01-01

  There are several approaches to understanding the physiological roles of biomolecules: (1) by observing the localization or activities of biomolecules (based on microscopic imaging experiments with fluorescent proteins or fluorescent probes) and (2) by investigating the cellular response via activation or suppression of functions of the target molecule (by using inhibitors, antagonists, siRNAs, etc.). In this context, protein-labeling technology serves as a powerful tool that can be used in various experiments, such as for fluorescence imaging of target proteins. Recently, we developed a protein-labeling technology that uses a mutant β-lactamase (a bacterial hydrolase) as the tag protein. In this protein-labeling technology, also referred to as the BL-tag technology, various β-lactam compounds were used as specific ligands that were covalently labeled to the tag. One major advantage of this labeling technology is that various functions can be carried out by suitably designing both the functional moieties such as the fluorophore and the β-lactam ligand structure. In this review, we briefly introduce the BL-tag technology and describe our future outlook for this technology, such as in fluorescence imaging of biomolecules and functional regulation of cellular proteins in living cells.

Correlated Light and Electron Microscopy/Electron Tomography of Mitochondria In Situ

PubMed Central

Perkins, Guy A.; Sun, Mei G.; Frey, Terrence G.

2009-01-01

Three-dimensional light microscopy and three-dimensional electron microscopy (electron tomography) separately provide very powerful tools to study cellular structure and physiology, including the structure and physiology of mitochondria. Fluorescence microscopy allows one to study processes in live cells with specific labels and stains that follow the movement of labeled proteins and changes within cellular compartments but does not have sufficient resolution to define the ultrastructure of intracellular organelles such as mitochondria. Electron microscopy and electron tomography provide the highest resolution currently available to study mitochondrial ultrastructure but cannot follow processes in living cells. We describe the combination of these two techniques in which fluorescence confocal microscopy is used to study structural and physiologic changes in mitochondria within apoptotic HeLa cells to define the apoptotic timeframe. Cells can then be selected at various stages of the apoptotic timeframe for examination at higher resolution by electron microscopy and electron tomography. This is a form of “virtual” 4-dimensional electron microscopy that has revealed interesting structural changes in the mitochondria of HeLa cells during apoptosis. The same techniques can be applied, with modification, to study other dynamic processes within cells in other experimental contexts. PMID:19348881

Cellular Magnesium Matrix Foam Composites for Mechanical Damping Applications

NASA Astrophysics Data System (ADS)

Shunmugasamy, Vasanth Chakravarthy; Mansoor, Bilal; Gupta, Nikhil

2016-01-01

The damping characteristics of metal alloys and metal matrix composites are relevant to the automotive, aerospace, and marine structures. Use of lightweight materials can help in increasing payload capacity and in decreasing fuel consumption. Lightweight composite materials possessing high damping capabilities that can be designed as structural members can greatly benefit in addressing these needs. In this context, the damping properties of lightweight metals such as aluminum and magnesium and their respective composites have been studied in the existing literature. This review focuses on analyzing the damping properties of aluminum and magnesium alloys and their cellular composites. The damping properties of various lightweight alloys and composites are compared on the basis of their density to understand the potential for weight saving in structural applications. Magnesium alloys are observed to possess better damping properties in comparison to aluminum. However, aluminum matrix syntactic foams reinforced with silicon carbide hollow particles possess a damping capacity and density comparable to magnesium alloy. By using the data presented in the study, composites with specific compositions and properties can be selected for a given application. In addition, the comparison of the results helps in identifying the areas where attention needs to be focused to address the future needs.

Multifunctional enzymes from reduced genomes - model proteins for simple primordial metabolism?

PubMed

Seelig, Burckhard

2017-08-01

Billions of years of evolution have yielded today's complex metabolic networks driven by efficient and highly specialized enzymes. In contrast, the metabolism of the earliest cellular life forms was likely much simpler with only a few enzymes of comparatively low activity. It has been speculated that these early enzymes had low specificities and in turn were able to perform multiple functions. In this issue of Molecular Microbiology, Ferla et al. describe examples of enzymes that catalyze chemically distinct reactions while using the same active site. Most importantly, the authors demonstrated that the comparatively weak activities of these multifunctional enzymes are each physiologically relevant. These findings contrast with simply promiscuous enzyme activities, which have been described numerous times but are not physiologically relevant. Ferla et al. elegantly combined initial bioinformatics searches for enzyme candidates with sound kinetic measurements, evolutionary considerations and even structural discussions. The phenomenon of multifunctionality appears to be a mechanism for bacteria with reduced genomes to compensate for their lack of certain enzymes. In the broader context of evolution, these organisms could be considered living model systems to study features of long-extinct early cellular life. © 2017 John Wiley & Sons Ltd.

Emerging connections between RNA and autophagy.

PubMed

Frankel, Lisa B; Lubas, Michal; Lund, Anders H

2017-01-02

Macroautophagy/autophagy is a key catabolic process, essential for maintaining cellular homeostasis and survival through the removal and recycling of unwanted cellular material. Emerging evidence has revealed intricate connections between the RNA and autophagy research fields. While a majority of studies have focused on protein, lipid and carbohydrate catabolism via autophagy, accumulating data supports the view that several types of RNA and associated ribonucleoprotein complexes are specifically recruited to phagophores (precursors to autophagosomes) and subsequently degraded in the lysosome/vacuole. Moreover, recent studies have revealed a substantial number of novel autophagy regulators with RNA-related functions, indicating roles for RNA and associated proteins not only as cargo, but also as regulators of this process. In this review, we discuss widespread evidence of RNA catabolism via autophagy in yeast, plants and animals, reviewing the molecular mechanisms and biological importance in normal physiology, stress and disease. In addition, we explore emerging evidence of core autophagy regulation mediated by RNA-binding proteins and noncoding RNAs, and point to gaps in our current knowledge of the connection between RNA and autophagy. Finally, we discuss the pathological implications of RNA-protein aggregation, primarily in the context of neurodegenerative disease.

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.

Interactions between genetic variation and cellular environment in skeletal muscle gene expression.

PubMed

Taylor, D Leland; Knowles, David A; Scott, Laura J; Ramirez, Andrea H; Casale, Francesco Paolo; Wolford, Brooke N; Guan, Li; Varshney, Arushi; Albanus, Ricardo D'Oliveira; Parker, Stephen C J; Narisu, Narisu; Chines, Peter S; Erdos, Michael R; Welch, Ryan P; Kinnunen, Leena; Saramies, Jouko; Sundvall, Jouko; Lakka, Timo A; Laakso, Markku; Tuomilehto, Jaakko; Koistinen, Heikki A; Stegle, Oliver; Boehnke, Michael; Birney, Ewan; Collins, Francis S

2018-01-01

From whole organisms to individual cells, responses to environmental conditions are influenced by genetic makeup, where the effect of genetic variation on a trait depends on the environmental context. RNA-sequencing quantifies gene expression as a molecular trait, and is capable of capturing both genetic and environmental effects. In this study, we explore opportunities of using allele-specific expression (ASE) to discover cis-acting genotype-environment interactions (GxE)-genetic effects on gene expression that depend on an environmental condition. Treating 17 common, clinical traits as approximations of the cellular environment of 267 skeletal muscle biopsies, we identify 10 candidate environmental response expression quantitative trait loci (reQTLs) across 6 traits (12 unique gene-environment trait pairs; 10% FDR per trait) including sex, systolic blood pressure, and low-density lipoprotein cholesterol. Although using ASE is in principle a promising approach to detect GxE effects, replication of such signals can be challenging as validation requires harmonization of environmental traits across cohorts and a sufficient sampling of heterozygotes for a transcribed SNP. Comprehensive discovery and replication will require large human transcriptome datasets, or the integration of multiple transcribed SNPs, coupled with standardized clinical phenotyping.

Single-Cell and Single-Molecule Analysis of Gene Expression Regulation.

PubMed

Vera, Maria; Biswas, Jeetayu; Senecal, Adrien; Singer, Robert H; Park, Hye Yoon

2016-11-23

Recent advancements in single-cell and single-molecule imaging technologies have resolved biological processes in time and space that are fundamental to understanding the regulation of gene expression. Observations of single-molecule events in their cellular context have revealed highly dynamic aspects of transcriptional and post-transcriptional control in eukaryotic cells. This approach can relate transcription with mRNA abundance and lifetimes. Another key aspect of single-cell analysis is the cell-to-cell variability among populations of cells. Definition of heterogeneity has revealed stochastic processes, determined characteristics of under-represented cell types or transitional states, and integrated cellular behaviors in the context of multicellular organisms. In this review, we discuss novel aspects of gene expression of eukaryotic cells and multicellular organisms revealed by the latest advances in single-cell and single-molecule imaging technology.

Simulation Based Optimization of Complex Monolithic Composite Structures Using Cellular Core Technology

NASA Astrophysics Data System (ADS)

Hickmott, Curtis W.

Cellular core tooling is a new technology which has the capability to manufacture complex integrated monolithic composite structures. This novel tooling method utilizes thermoplastic cellular cores as inner tooling. The semi-rigid nature of the cellular cores makes them convenient for lay-up, and under autoclave temperature and pressure they soften and expand providing uniform compaction on all surfaces including internal features such as ribs and spar tubes. This process has the capability of developing fully optimized aerospace structures by reducing or eliminating assembly using fasteners or bonded joints. The technology is studied in the context of evaluating its capabilities, advantages, and limitations in developing high quality structures. The complex nature of these parts has led to development of a model using the Finite Element Analysis (FEA) software Abaqus and the plug-in COMPRO Common Component Architecture (CCA) provided by Convergent Manufacturing Technologies. This model utilizes a "virtual autoclave" technique to simulate temperature profiles, resin flow paths, and ultimately deformation from residual stress. A model has been developed simulating the temperature profile during curing of composite parts made with the cellular core technology. While modeling of composites has been performed in the past, this project will look to take this existing knowledge and apply it to this new manufacturing method capable of building more complex parts and develop a model designed specifically for building large, complex components with a high degree of accuracy. The model development has been carried out in conjunction with experimental validation. A double box beam structure was chosen for analysis to determine the effects of the technology on internal ribs and joints. Double box beams were manufactured and sectioned into T-joints for characterization. Mechanical behavior of T-joints was performed using the T-joint pull-off test and compared to traditional tooling methods. Components made with the cellular core tooling method showed an improved strength at the joints. It is expected that this knowledge will help optimize the processing of complex, integrated structures and benefit applications in aerospace where lighter, structurally efficient components would be advantageous.

Polyomavirus-Specific Cellular Immunity: From BK-Virus-Specific Cellular Immunity to BK-Virus-Associated Nephropathy?

PubMed Central

Dekeyser, Manon; François, Hélène; Beaudreuil, Séverine; Durrbach, Antoine

2015-01-01

In renal transplantation, BK-virus (BKV)-associated nephropathy has emerged as a major complication, with a prevalence of 1–10% and graft loss in >50% of cases. BKV is a member of the polyomavirus family and rarely induces apparent clinical disease in the general population. However, replication of polyomaviruses, associated with significant organ disease, is observed in patients with acquired immunosuppression. Monitoring of specific immunity combined with viral load could be used to individually assess the risk of viral reactivation and virus control. We review the current knowledge on BKV-specific cellular immunity and, more specifically, in immunocompromised patients. In the future, immune-based therapies could allow us to treat and prevent BKV-associated nephropathy. PMID:26136745

Deregulation upon DNA damage revealed by joint analysis of context-specific perturbation data

PubMed Central

2011-01-01

Background Deregulation between two different cell populations manifests itself in changing gene expression patterns and changing regulatory interactions. Accumulating knowledge about biological networks creates an opportunity to study these changes in their cellular context. Results We analyze re-wiring of regulatory networks based on cell population-specific perturbation data and knowledge about signaling pathways and their target genes. We quantify deregulation by merging regulatory signal from the two cell populations into one score. This joint approach, called JODA, proves advantageous over separate analysis of the cell populations and analysis without incorporation of knowledge. JODA is implemented and freely available in a Bioconductor package 'joda'. Conclusions Using JODA, we show wide-spread re-wiring of gene regulatory networks upon neocarzinostatin-induced DNA damage in Human cells. We recover 645 deregulated genes in thirteen functional clusters performing the rich program of response to damage. We find that the clusters contain many previously characterized neocarzinostatin target genes. We investigate connectivity between those genes, explaining their cooperation in performing the common functions. We review genes with the most extreme deregulation scores, reporting their involvement in response to DNA damage. Finally, we investigate the indirect impact of the ATM pathway on the deregulated genes, and build a hypothetical hierarchy of direct regulation. These results prove that JODA is a step forward to a systems level, mechanistic understanding of changes in gene regulation between different cell populations. PMID:21693013

Deregulation upon DNA damage revealed by joint analysis of context-specific perturbation data.

PubMed

Szczurek, Ewa; Markowetz, Florian; Gat-Viks, Irit; Biecek, Przemysław; Tiuryn, Jerzy; Vingron, Martin

2011-06-21

Deregulation between two different cell populations manifests itself in changing gene expression patterns and changing regulatory interactions. Accumulating knowledge about biological networks creates an opportunity to study these changes in their cellular context. We analyze re-wiring of regulatory networks based on cell population-specific perturbation data and knowledge about signaling pathways and their target genes. We quantify deregulation by merging regulatory signal from the two cell populations into one score. This joint approach, called JODA, proves advantageous over separate analysis of the cell populations and analysis without incorporation of knowledge. JODA is implemented and freely available in a Bioconductor package 'joda'. Using JODA, we show wide-spread re-wiring of gene regulatory networks upon neocarzinostatin-induced DNA damage in Human cells. We recover 645 deregulated genes in thirteen functional clusters performing the rich program of response to damage. We find that the clusters contain many previously characterized neocarzinostatin target genes. We investigate connectivity between those genes, explaining their cooperation in performing the common functions. We review genes with the most extreme deregulation scores, reporting their involvement in response to DNA damage. Finally, we investigate the indirect impact of the ATM pathway on the deregulated genes, and build a hypothetical hierarchy of direct regulation. These results prove that JODA is a step forward to a systems level, mechanistic understanding of changes in gene regulation between different cell populations.

TRAF2 multitasking in TNF receptor-induced signaling to NF-κB, MAP kinases and cell death.

PubMed

Borghi, Alice; Verstrepen, Lynn; Beyaert, Rudi

2016-09-15

Tumor Necrosis Factor (TNF) is a potent inflammatory cytokine that exerts its functions through the activation of two distinct receptors, TNFR1 and TNFR2. Both receptors can activate canonical NF-κB and JNK MAP kinase signaling, while TNFR2 can also activate non-canonical NF-κB signaling, leading to numerous changes in gene expression that drive inflammation, cell proliferation and cell survival. On the other hand, TNFR1 also activates signaling pathways leading to cell death by either apoptosis or necroptosis, depending on the cellular context. A key player in TNFR1- and TNFR2-induced signaling is the RING finger protein TRAF2, which is recruited to both receptors upon their stimulation. TRAF2 exerts multiple receptor-specific functions but also mediates cross-talk between TNFR1 and TNFR2, dictating the outcome of TNF stimulation. In this review, we provide an overview of the positive and negative regulatory role of TRAF2 in different TNFR1 and TNFR2 signaling pathways. We discuss the underlying molecular mechanism of action, distinguishing between TRAF2 scaffold and E3 ubiquitin ligase functions, and the regulation of TRAF2 by specific post-translational modifications. Finally, we elaborate on some possible strategies to modulate TRAF2 function in the context of therapeutic targeting in autoimmunity and cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Primary response against cytomegalovirus during antiviral prophylaxis with valganciclovir, in solid organ transplant recipients

PubMed Central

La Rosa, Corinna; Limaye, Ajit P.; Krishnan, Aparna; Blumstein, Gideon; Longmate, Jeff; Diamond, Don J.

2012-01-01

Antiviral prophylaxis has proved successful for prevention of cytomegalovirus (CMV) disease in solid organ transplant (SOT) patients; though emerging data suggest that antiviral agents interfere with immunity, and may inhibit immune-priming. In this context, we investigated levels and phenotype of primary CMV-specific immune responses that developed during antiviral prophylaxis in a cohort of CMV seronegative recipients (R−) of a SOT from a seropositive donor (D+). We longitudinally monitored CMV viral load, antibodies and levels of the negative immuno-modulator IL-10. PBMC were stimulated with CMV-specific peptide libraries to measure CD137 activation marker on CMV-specific T-cells and levels of PD-1 receptor, which is overexpressed on exhausted T-cells. Unexpectedly, the majority (13/18) of D+R− patients who developed a primary CMV response showed early post-transplant CMV-specific responses, though levels of PD-1 on CMV-specific T-cells remained elevated throughout prophylaxis. A strong inverse association was found between levels of plasma IL-10 and CMV-specific cellular immune responses. Our study suggests that during prophylaxis, subclinical CMV infection might have occurred in the D+R− patients, and primary CMV-specific responses were detected early post-transplant when levels of plasma IL-10 were low. Extended prophylaxis or antiviral treatment did not appear to suppress CMV-specific antibodies or T-cells, which however showed exhaustion phenotypes. PMID:21672050

The interplay between HIF-1 and calcium signalling in cancer.

PubMed

Azimi, Iman

2018-04-01

The interplay between hypoxia-inducible factor-1 (HIF-1) and calcium in cancer has begun to be unravelled with recent findings demonstrating the relationships between the two in different cancer types. This is an area of significance considering the crucial roles of both HIF-1 and calcium signalling in cancer progression and metastasis. This review summarises the experimental evidence of the crosstalk between HIF-1 and specific calcium channels, pumps and regulators in the context of cancer. HIF-1 as a master regulator of hypoxic transcriptional responses, mediates transcription of several calcium modulators. On the other hand, specific calcium channels and pumps regulate HIF-1 activity through controlling its transcription, translation, stabilisation, or nuclear translocation. Identifying the interplay between HIF-1 and components of the calcium signal will give new insights into mechanisms underlying cellular responses to physiological and pathophysiological cues, and may provide novel and more efficient therapeutic strategies for the control of cancer progression. Copyright © 2018 Elsevier Ltd. All rights reserved.

Wnt-Lrp5 Signaling Regulates Fatty Acid Metabolism in the Osteoblast

PubMed Central

Frey, Julie L.; Li, Zhu; Ellis, Jessica M.; Zhang, Qian; Farber, Charles R.; Aja, Susan; Wolfgang, Michael J.; Clemens, Thomas L.

2015-01-01

The Wnt coreceptors Lrp5 and Lrp6 are essential for normal postnatal bone accrual and osteoblast function. In this study, we identify a previously unrecognized skeletal function unique to Lrp5 that enables osteoblasts to oxidize fatty acids. Mice lacking the Lrp5 coreceptor specifically in osteoblasts and osteocytes exhibit the expected reductions in postnatal bone mass but also exhibit an increase in body fat with corresponding reductions in energy expenditure. Conversely, mice expressing a high bone mass mutant Lrp5 allele are leaner with reduced plasma triglyceride and free fatty acid levels. In this context, Wnt-initiated signals downstream of Lrp5, but not the closely related Lrp6 coreceptor, regulate the activation of β-catenin and thereby induce the expression of key enzymes required for fatty acid β-oxidation. These results suggest that Wnt-Lrp5 signaling regulates basic cellular activities beyond those associated with fate specification and differentiation in bone and that the skeleton influences global energy homeostasis via mechanisms independent of osteocalcin and glucose metabolism. PMID:25802278

Gas1 extends the range of Hedgehog action by facilitating its signaling

PubMed Central

Martinelli, David C.; Fan, Chen-Ming

2007-01-01

Cellular signaling initiated by Hedgehog binding to Patched1 has profound importance in mammalian embryogenesis, genetic disease, and cancer. Hedgehog acts as a morphogen to specify distinctive cell fates using different concentration thresholds, but our knowledge of how the concentration gradient is interpreted into the activity gradient is incomplete. The membrane protein Growth Arrest-Specific Gene 1 (GAS1) was thought to be a negative regulator of the Hedgehog concentration gradient. Here, we report unexpected genetic evidence that Gas1 positively regulates Hedgehog signaling in multiple developmental contexts, an effect particularly noticeable at regions where Hedgehog acts at low concentration. Using a combination of in vitro cell culture and in ovo electroporation assays, we demonstrate that GAS1 acts cooperatively with Patched1 for Hedgehog binding and enhances signaling activity in a cell-autonomous manner. Our data support a model in which GAS1 helps transform the Hedgehog protein gradient into the observed activity gradient. We propose that Gas1 is an evolutionarily novel, vertebrate-specific Hedgehog pathway regulator. PMID:17504940

Sex differences in the developing brain as a source of inherent risk

PubMed Central

McCarthy, Margaret M.

2016-01-01

Brain development diverges in males and females in response to androgen production by the fetal testis. This sexual differentiation of the brain occurs during a sensitive window and induces enduring neuroanatomical and physiological changes that profoundly impact behavior. What we know about the contribution of sex chromosomes is still emerging, highlighting the need to integrate multiple factors into understanding sex differences, including the importance of context. The cellular mechanisms are best modeled in rodents and have provided both unifying principles and surprising specifics. Markedly distinct signaling pathways direct differentiation in specific brain regions, resulting in mosaicism of relative maleness, femaleness, and sameness through-out the brain, while canalization both exaggerates and constrains sex differences. Non-neuronal cells and inflammatory mediators are found in greater number and at higher levels in parts of male brains. This higher baseline of inflammation is speculated to increase male vulnerability to developmental neuropsychiatric disorders that are triggered by inflammation. PMID:28179808

Trial Watch

PubMed Central

Vacchelli, Erika; Eggermont, Alexander; Fridman, Wolf Hervé; Galon, Jérôme; Tartour, Eric; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

2013-01-01

Adoptive cell transfer (ACT) represents a prominent form of immunotherapy against malignant diseases. ACT is conceptually distinct from dendritic cell-based approaches (which de facto constitute cellular vaccines) and allogeneic transplantation (which can be employed for the therapy of hematopoietic tumors) as it involves the isolation of autologous lymphocytes exhibiting antitumor activity, their expansion/activation ex vivo and their reintroduction into the patient. Re-infusion is most often performed in the context of lymphodepleting regimens (to minimize immunosuppression by host cells) and combined with immunostimulatory interventions, such as the administration of Toll-like receptor agonists. Autologous cells that are suitable for ACT protocols can be isolated from tumor-infiltrating lymphocytes or generated by engineering their circulating counterparts for the expression of transgenic tumor-specific T-cell receptors. Importantly, lymphocytes can be genetically modified prior to re-infusion for increasing their persistence in vivo, boosting antitumor responses and minimizing side effects. Moreover, recent data indicate that exhausted antitumor T lymphocytes may be rejuvenated in vitro by exposing them to specific cytokine cocktails, a strategy that might considerably improve the clinical success of ACT. Following up the Trial Watch that we published on this topic in the third issue of OncoImmunology (May 2012), here we summarize the latest developments in ACT-related research, covering both high-impact studies that have been published during the last 13 months and clinical trials that have been initiated in the same period to assess the antineoplastic profile of this form of cellular immunotherapy. PMID:23762803

Caprine PrP variants harboring Asp-146, His-154 and Gln-211 alleles display reduced convertibility upon interaction with pathogenic murine prion protein in scrapie infected cells.

PubMed

Kanata, Eirini; Arsenakis, Minas; Sklaviadis, Theodoros

2016-09-02

Scrapie, the prion disease of sheep and goats, is a devastating malady of small ruminants. Due to its infectious nature, epidemic outbreaks may occur in flocks/herds consisting of highly susceptible animals. Field studies identified scrapie-protective caprine PrP variants, harboring specific single amino acid changes (Met-142, Arg-143, Asp-146, Ser-146, His-154, Gln-211 and Lys-222). Their effects are under further evaluation, and aim to determine the most protective allele. We assessed some of these variants (Asp-146, His-154, Gln-211 and Lys-222), after their exogenous expression as murine-caprine chimeras in a scrapie- infected murine cell line. We report that exogenously expressed PrPs undergo conformational conversion upon interaction with the endogenous pathological murine prion protein (PrP SC ), which results in the detection of goat-specific and partially PK-resistant moieties. These moieties display a PK-resistance pattern distinct from the one detected in natural goat scrapie cases. Within this cellular model, distinct conformational conversion potentials were assigned to the tested variants. Molecules carrying the Asp-146, His-154 and Gln-211 alleles showed significantly lower conversion levels compared to wild type, confirming their protective effects against scrapie. Although we utilized a heterologous conversion system, this is to our knowledge, the first study of caprine PrP variants in a cellular context of scrapie, that confirms the protective effects of some of the studied alleles.

AntiJen: a quantitative immunology database integrating functional, thermodynamic, kinetic, biophysical, and cellular data

PubMed Central

Toseland, Christopher P; Clayton, Debra J; McSparron, Helen; Hemsley, Shelley L; Blythe, Martin J; Paine, Kelly; Doytchinova, Irini A; Guan, Pingping; Hattotuwagama, Channa K; Flower, Darren R

2005-01-01

AntiJen is a database system focused on the integration of kinetic, thermodynamic, functional, and cellular data within the context of immunology and vaccinology. Compared to its progenitor JenPep, the interface has been completely rewritten and redesigned and now offers a wider variety of search methods, including a nucleotide and a peptide BLAST search. In terms of data archived, AntiJen has a richer and more complete breadth, depth, and scope, and this has seen the database increase to over 31,000 entries. AntiJen provides the most complete and up-to-date dataset of its kind. While AntiJen v2.0 retains a focus on both T cell and B cell epitopes, its greatest novelty is the archiving of continuous quantitative data on a variety of immunological molecular interactions. This includes thermodynamic and kinetic measures of peptide binding to TAP and the Major Histocompatibility Complex (MHC), peptide-MHC complexes binding to T cell receptors, antibodies binding to protein antigens and general immunological protein-protein interactions. The database also contains quantitative specificity data from position-specific peptide libraries and biophysical data, in the form of diffusion co-efficients and cell surface copy numbers, on MHCs and other immunological molecules. The uses of AntiJen include the design of vaccines and diagnostics, such as tetramers, and other laboratory reagents, as well as helping parameterize the bioinformatic or mathematical in silico modeling of the immune system. The database is accessible from the URL: . PMID:16305757

Trial Watch: Adoptive cell transfer for anticancer immunotherapy.

PubMed

Vacchelli, Erika; Eggermont, Alexander; Fridman, Wolf Hervé; Galon, Jérôme; Tartour, Eric; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

2013-05-01

Adoptive cell transfer (ACT) represents a prominent form of immunotherapy against malignant diseases. ACT is conceptually distinct from dendritic cell-based approaches (which de facto constitute cellular vaccines) and allogeneic transplantation (which can be employed for the therapy of hematopoietic tumors) as it involves the isolation of autologous lymphocytes exhibiting antitumor activity, their expansion/activation ex vivo and their reintroduction into the patient. Re-infusion is most often performed in the context of lymphodepleting regimens (to minimize immunosuppression by host cells) and combined with immunostimulatory interventions, such as the administration of Toll-like receptor agonists. Autologous cells that are suitable for ACT protocols can be isolated from tumor-infiltrating lymphocytes or generated by engineering their circulating counterparts for the expression of transgenic tumor-specific T-cell receptors. Importantly, lymphocytes can be genetically modified prior to re-infusion for increasing their persistence in vivo, boosting antitumor responses and minimizing side effects. Moreover, recent data indicate that exhausted antitumor T lymphocytes may be rejuvenated in vitro by exposing them to specific cytokine cocktails, a strategy that might considerably improve the clinical success of ACT. Following up the Trial Watch that we published on this topic in the third issue of OncoImmunology (May 2012), here we summarize the latest developments in ACT-related research, covering both high-impact studies that have been published during the last 13 months and clinical trials that have been initiated in the same period to assess the antineoplastic profile of this form of cellular immunotherapy.

Dysregulation of the Mitochondrial Unfolded Protein Response Induces Non-Apoptotic Dopaminergic Neurodegeneration in C. elegans Models of Parkinson's Disease

PubMed Central

Martinez, Bryan A.; Petersen, Daniel A.; Gaeta, Anthony L.

2017-01-01

Due to environmental insult or innate genetic deficiency, protein folding environments of the mitochondrial matrix are prone to dysregulation, prompting the activation of a specific organellar stress-response mechanism, the mitochondrial unfolded protein response (UPRMT). In Caenorhabditis elegans, mitochondrial damage leads to nuclear translocation of the ATFS-1 transcription factor to activate the UPRMT. After short-term acute stress has been mitigated, the UPRMT is eventually suppressed to restore homeostasis to C. elegans hermaphrodites. In contrast, and reflective of the more chronic nature of progressive neurodegenerative disorders such as Parkinson's disease (PD), here, we report the consequences of prolonged, cell-autonomous activation of the UPRMT in C. elegans dopaminergic neurons. We reveal that neuronal function and integrity decline rapidly with age, culminating in activity-dependent, non-apoptotic cell death. In a PD-like context wherein transgenic nematodes express the Lewy body constituent protein α-synuclein (αS), we not only find that this protein and its PD-associated disease variants have the capacity to induce the UPRMT, but also that coexpression of αS and ATFS-1-associated dysregulation of the UPRMT synergistically potentiate dopaminergic neurotoxicity. This genetic interaction is in parallel to mitophagic pathways dependent on the C. elegans PINK1 homolog, which is necessary for cellular resistance to chronic malfunction of the UPRMT. Given the increasingly recognized role of mitochondrial quality control in neurodegenerative diseases, these studies illustrate, for the first time, an insidious aspect of mitochondrial signaling in which the UPRMT pathway, under disease-associated, context-specific dysregulation, exacerbates disruption of dopaminergic neurons in vivo, resulting in the neurodegeneration characteristic of PD. SIGNIFICANCE STATEMENT Disruptions or alterations in the activation of pathways that regulate mitochondrial quality control have been linked to neurodegenerative diseases due in part to the central role of mitochondria in metabolism, ROS regulation, and proteostasis. The extent to which these pathways, including the mitochondrial unfolded protein response (UPRMT) and mitophagy, are active may predict severity and progression of these disorders, as well as sensitivity to compounding stressors. Furthermore, therapeutic strategies that aim to induce these pathways may benefit from increased study into cellular responses that arise from long-term or ectopic stimulation, especially in neuronal compartments. By demonstrating the detrimental consequences of prolonged cellular activation of the UPRMT, we provide evidence that this pathway is not a universally beneficial mechanism because dysregulation has neurotoxic consequences. PMID:29030433

Receptor Tyrosine Kinases in Drosophila Development

PubMed Central

Sopko, Richelle; Perrimon, Norbert

2013-01-01

Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases. PMID:23732470

Long non-coding RNAs involved in autophagy regulation

PubMed Central

Yang, Lixian; Wang, Hanying; Shen, Qi; Feng, Lifeng; Jin, Hongchuan

2017-01-01

Autophagy degrades non-functioning or damaged proteins and organelles to maintain cellular homeostasis in a physiological or pathological context. Autophagy can be protective or detrimental, depending on its activation status and other conditions. Therefore, autophagy has a crucial role in a myriad of pathophysiological processes. From the perspective of autophagy-related (ATG) genes, the molecular dissection of autophagy process and the regulation of its level have been largely unraveled. However, the discovery of long non-coding RNAs (lncRNAs) provides a new paradigm of gene regulation in almost all important biological processes, including autophagy. In this review, we highlight recent advances in autophagy-associated lncRNAs and their specific autophagic targets, as well as their relevance to human diseases such as cancer, cardiovascular disease, diabetes and cerebral ischemic stroke. PMID:28981093

Synthesis and aggregation properties of dissymmetric phytanyl-gemini surfactants for use as improved DNA transfection vectors.

PubMed

Wang, Haitang; Wettig, Shawn D

2011-01-14

Improvements in transfection efficiency are required in order to make the goal of cellular gene delivery by non-viral vectors realizable. Novel derivatives of gemini surfactants having dissymmetric tail groups have been designed specifically as a means to improve DNA transfection; the micelle and interfacial properties are reported herein. The effect of these substitutions on the aggregation properties of the gemini surfactants is discussed in the context of results for the m-3-m gemini series, previously reported in the literature. Phytanyl substitution results in lower cmc and higher micelle ionization. In addition, the phytanyl substituted gemini surfactants form vesicles at room temperature. Preliminary in vitro transfection assays showed the phytanyl substituted gemini surfactants to be more efficient transfection vectors as compared to symmetric gemini surfactants.

Two copies of mthmg1, encoding a novel mitochondrial HMG-like protein, delay accumulation of mitochondrial DNA deletions in Podospora anserina.

PubMed

Dequard-Chablat, Michelle; Allandt, Cynthia

2002-08-01

In the filamentous fungus Podospora anserina, two degenerative processes which result in growth arrest are associated with mitochondrial genome (mitochondrial DNA [mtDNA]) instability. Senescence is correlated with mtDNA rearrangements and amplification of specific regions (senDNAs). Premature death syndrome is characterized by the accumulation of specific mtDNA deletions. This accumulation is due to indirect effects of the AS1-4 mutation, which alters a cytosolic ribosomal protein gene. The mthmg1 gene has been identified as a double-copy suppressor of premature death. It greatly delays premature death and the accumulation of deletions when it is present in two copies in an ASI-4 context. The duplication of mthmg1 has no significant effect on the wild-type life span or on senDNA patterns. In anAS1+ context, deletion of the mthmg1 gene alters germination, growth, and fertility and reduces the life span. The deltamthmg1 senescent strains display a particular senDNA pattern. This deletion is lethal in an AS1-4 context. According to its physical properties (very basic protein with putative mitochondrial targeting sequence and HMG-type DNA-binding domains) and the cellular localization of an mtHMG1-green fluorescent protein fusion, mtHMG1 appears to be a mitochondrial protein possibly associated with mtDNA. It is noteworthy that it is the first example of a protein combining the two DNA-binding domains, AT-hook motif and HMG-1 boxes. It may be involved in the stability and/or transmission of the mitochondrial genome. To date, no structural homologues have been found in other organisms. However, mtHMG1 displays functional similarities with the Saccharomyces cerevisiae mitochondrial HMG-box protein Abf2.

Calcium homeostasis and organelle function in the pathogenesis of obesity and diabetes

PubMed Central

Arruda, Ana Paula; Hotamisligil, Gökhan S.

2015-01-01

Summary A number of chronic metabolic pathologies, including obesity, diabetes, cardiovascular disease, asthma, and cancer cluster together to present the greatest threat to human health. As research in this field has advanced, it has become clear that unresolved metabolic inflammation, organelle dysfunction, and other cellular and metabolic stresses underlie the development of these chronic metabolic diseases. However, the relationship between these systems and pathological mechanisms is poorly understood. Here, we will discuss the role of cellular Ca2+ homeostasis as a critical mechanism integrating the myriad of cellular and subcellular dysfunctional networks found in metabolic tissues such as liver and adipose tissue in the context of metabolic disease particularly in obesity and diabetes. PMID:26190652

Therapeutic cloning and cellular reprogramming.

PubMed

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

2012-01-01

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

Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to Detect CD19-Specific T Cells in Clinical Trials

PubMed Central

Jena, Bipulendu; Maiti, Sourindra; Huls, Helen; Singh, Harjeet; Lee, Dean A.; Champlin, Richard E.; Cooper, Laurence J. N.

2013-01-01

Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19+ tumor targets. This clone can be used to detect CD19-specific CAR+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy. PMID:23469246

A fresh look at the male-specific region of the human Y chromosome.

PubMed

Jangravi, Zohreh; Alikhani, Mehdi; Arefnezhad, Babak; Sharifi Tabar, Mehdi; Taleahmad, Sara; Karamzadeh, Razieh; Jadaliha, Mahdieh; Mousavi, Seyed Ahmad; Ahmadi Rastegar, Diba; Parsamatin, Pouria; Vakilian, Haghighat; Mirshahvaladi, Shahab; Sabbaghian, Marjan; Mohseni Meybodi, Anahita; Mirzaei, Mehdi; Shahhoseini, Maryam; Ebrahimi, Marzieh; Piryaei, Abbas; Moosavi-Movahedi, Ali Akbar; Haynes, Paul A; Goodchild, Ann K; Nasr-Esfahani, Mohammad Hossein; Jabbari, Esmaiel; Baharvand, Hossein; Sedighi Gilani, Mohammad Ali; Gourabi, Hamid; Salekdeh, Ghasem Hosseini

2013-01-04

The Chromosome-centric Human Proteome Project (C-HPP) aims to systematically map the entire human proteome with the intent to enhance our understanding of human biology at the cellular level. This project attempts simultaneously to establish a sound basis for the development of diagnostic, prognostic, therapeutic, and preventive medical applications. In Iran, current efforts focus on mapping the proteome of the human Y chromosome. The male-specific region of the Y chromosome (MSY) is unique in many aspects and comprises 95% of the chromosome's length. The MSY continually retains its haploid state and is full of repeated sequences. It is responsible for important biological roles such as sex determination and male fertility. Here, we present the most recent update of MSY protein-encoding genes and their association with various traits and diseases including sex determination and reversal, spermatogenesis and male infertility, cancers such as prostate cancers, sex-specific effects on the brain and behavior, and graft-versus-host disease. We also present information available from RNA sequencing, protein-protein interaction, post-translational modification of MSY protein-coding genes and their implications in biological systems. An overview of Human Y chromosome Proteome Project is presented and a systematic approach is suggested to ensure that at least one of each predicted protein-coding gene's major representative proteins will be characterized in the context of its major anatomical sites of expression, its abundance, and its functional relevance in a biological and/or medical context. There are many technical and biological issues that will need to be overcome in order to accomplish the full scale mapping.

Distinct Domains of CheA Confer Unique Functions in Chemotaxis and Cell Length in Azospirillum brasilense Sp7.

PubMed

Gullett, Jessica M; Bible, Amber; Alexandre, Gladys

2017-07-01

Chemotaxis is the movement of cells in response to gradients of diverse chemical cues. Motile bacteria utilize a conserved chemotaxis signal transduction system to bias their motility and navigate through a gradient. A central regulator of chemotaxis is the histidine kinase CheA. This cytoplasmic protein interacts with membrane-bound receptors, which assemble into large polar arrays, to propagate the signal. In the alphaproteobacterium Azospirillum brasilense , Che1 controls transient increases in swimming speed during chemotaxis, but it also biases the cell length at division. However, the exact underlying molecular mechanisms for Che1-dependent control of multiple cellular behaviors are not known. Here, we identify specific domains of the CheA1 histidine kinase implicated in modulating each of these functions. We show that CheA1 is produced in two isoforms: a membrane-anchored isoform produced as a fusion with a conserved seven-transmembrane domain of unknown function (TMX) at the N terminus and a soluble isoform similar to prototypical CheA. Site-directed and deletion mutagenesis combined with behavioral assays confirm the role of CheA1 in chemotaxis and implicate the TMX domain in mediating changes in cell length. Fluorescence microscopy further reveals that the membrane-anchored isoform is distributed around the cell surface while the soluble isoform localizes at the cell poles. Together, the data provide a mechanism for the role of Che1 in controlling multiple unrelated cellular behaviors via acquisition of a new domain in CheA1 and production of distinct functional isoforms. IMPORTANCE Chemotaxis provides a significant competitive advantage to bacteria in the environment, and this function has been transferred laterally multiple times, with evidence of functional divergence in different genomic contexts. The molecular principles that underlie functional diversification of chemotaxis in various genomic contexts are unknown. Here, we provide a molecular mechanism by which a single CheA protein controls two unrelated functions: chemotaxis and cell length. Acquisition of this multifunctionality is seemingly a recent evolutionary event. The findings illustrate a mechanism by which chemotaxis function may be co-opted to regulate additional cellular functions. Copyright © 2017 American Society for Microbiology.

Distinct Domains of CheA Confer Unique Functions in Chemotaxis and Cell Length in Azospirillum brasilense Sp7

PubMed Central

Gullett, Jessica M.

2017-01-01

ABSTRACT Chemotaxis is the movement of cells in response to gradients of diverse chemical cues. Motile bacteria utilize a conserved chemotaxis signal transduction system to bias their motility and navigate through a gradient. A central regulator of chemotaxis is the histidine kinase CheA. This cytoplasmic protein interacts with membrane-bound receptors, which assemble into large polar arrays, to propagate the signal. In the alphaproteobacterium Azospirillum brasilense, Che1 controls transient increases in swimming speed during chemotaxis, but it also biases the cell length at division. However, the exact underlying molecular mechanisms for Che1-dependent control of multiple cellular behaviors are not known. Here, we identify specific domains of the CheA1 histidine kinase implicated in modulating each of these functions. We show that CheA1 is produced in two isoforms: a membrane-anchored isoform produced as a fusion with a conserved seven-transmembrane domain of unknown function (TMX) at the N terminus and a soluble isoform similar to prototypical CheA. Site-directed and deletion mutagenesis combined with behavioral assays confirm the role of CheA1 in chemotaxis and implicate the TMX domain in mediating changes in cell length. Fluorescence microscopy further reveals that the membrane-anchored isoform is distributed around the cell surface while the soluble isoform localizes at the cell poles. Together, the data provide a mechanism for the role of Che1 in controlling multiple unrelated cellular behaviors via acquisition of a new domain in CheA1 and production of distinct functional isoforms. IMPORTANCE Chemotaxis provides a significant competitive advantage to bacteria in the environment, and this function has been transferred laterally multiple times, with evidence of functional divergence in different genomic contexts. The molecular principles that underlie functional diversification of chemotaxis in various genomic contexts are unknown. Here, we provide a molecular mechanism by which a single CheA protein controls two unrelated functions: chemotaxis and cell length. Acquisition of this multifunctionality is seemingly a recent evolutionary event. The findings illustrate a mechanism by which chemotaxis function may be co-opted to regulate additional cellular functions. PMID:28416707

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

PubMed Central

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

2016-01-01

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. PMID:27452732

The Potential Role of Senescence As a Modulator of Platelets and Tumorigenesis

PubMed Central

Valenzuela, Claudio A.; Quintanilla, Ricardo; Moore-Carrasco, Rodrigo; Brown, Nelson E.

2017-01-01

In addition to thrombus formation, alterations in platelet function are frequently observed in cancer patients. Importantly, both thrombus and tumor formation are influenced by age, although the mechanisms through which physiological aging modulates these processes remain poorly understood. In this context, the potential effects of senescent cells on platelet function represent pathophysiological mechanisms that deserve further exploration. Cellular senescence has traditionally been viewed as a barrier to tumorigenesis. However, far from being passive bystanders, senescent cells are metabolically active and able to secrete a variety of soluble and insoluble factors. This feature, known as the senescence-associated secretory phenotype (SASP), may provide senescent cells with the capacity to modify the tissue environment and, paradoxically, promote proliferation and neoplastic transformation of neighboring cells. In fact, the SASP-dependent ability of senescent cells to enhance tumorigenesis has been confirmed in cellular systems involving epithelial cells and fibroblasts, leaving open the question as to whether similar interactions can be extended to other cellular contexts. In this review, we discuss the diverse functions of platelets in tumorigenesis and suggest the possibility that senescent cells might also influence tumorigenesis through their ability to modulate the functional status of platelets through the SASP. PMID:28894697

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.

Heat Shock Protein 70 Family Members Interact with Crimean-Congo Hemorrhagic Fever Virus and Hazara Virus Nucleocapsid Proteins and Perform a Functional Role in the Nairovirus Replication Cycle.

PubMed

Surtees, Rebecca; Dowall, Stuart D; Shaw, Amelia; Armstrong, Stuart; Hewson, Roger; Carroll, Miles W; Mankouri, Jamel; Edwards, Thomas A; Hiscox, Julian A; Barr, John N

2016-10-15

The Nairovirus genus of the Bunyaviridae family contains serious human and animal pathogens classified within multiple serogroups and species. Of these serogroups, the Crimean-Congo hemorrhagic fever virus (CCHFV) serogroup comprises sole members CCHFV and Hazara virus (HAZV). CCHFV is an emerging zoonotic virus that causes often-fatal hemorrhagic fever in infected humans for which preventative or therapeutic strategies are not available. In contrast, HAZV is nonpathogenic to humans and thus represents an excellent model to study aspects of CCHFV biology under conditions of more-accessible biological containment. The three RNA segments that form the nairovirus genome are encapsidated by the viral nucleocapsid protein (N) to form ribonucleoprotein (RNP) complexes that are substrates for RNA synthesis and packaging into virus particles. We used quantitative proteomics to identify cellular interaction partners of CCHFV N and identified robust interactions with cellular chaperones. These interactions were validated using immunological methods, and the specific interaction between native CCHFV N and cellular chaperones of the HSP70 family was confirmed during live CCHFV infection. Using infectious HAZV, we showed for the first time that the nairovirus N-HSP70 association was maintained within both infected cells and virus particles, where N is assembled as RNPs. Reduction of active HSP70 levels in cells by the use of small-molecule inhibitors significantly reduced HAZV titers, and a model for chaperone function in the context of high genetic variability is proposed. These results suggest that chaperones of the HSP70 family are required for nairovirus replication and thus represent a genetically stable cellular therapeutic target for preventing nairovirus-mediated disease. Nairoviruses compose a group of human and animal viruses that are transmitted by ticks and associated with serious or fatal disease. One member is Crimean-Congo hemorrhagic fever virus (CCHFV), which is responsible for fatal human disease and is recognized as an emerging threat within Europe in response to climate change. No preventative or therapeutic strategies against nairovirus-mediated disease are currently available. Here we show that the N protein of CCHFV and the related Hazara virus interact with a cellular protein, HSP70, during both the intracellular and extracellular stages of the virus life cycle. The use of inhibitors that block HSP70 function reduces virus titers by up to 1,000-fold, suggesting that this interaction is important within the context of the nairovirus life cycle and may represent a potent target for antinairovirus therapies against which the virus cannot easily develop resistance. Copyright © 2016 Surtees et al.

Heterogeneity of Metazoan Cells and Beyond: To Integrative Analysis of Cellular Populations at Single-Cell Level.

PubMed

Barteneva, Natasha S; Vorobjev, Ivan A

2018-01-01

In this paper, we review some of the recent advances in cellular heterogeneity and single-cell analysis methods. In modern research of cellular heterogeneity, there are four major approaches: analysis of pooled samples, single-cell analysis, high-throughput single-cell analysis, and lately integrated analysis of cellular population at a single-cell level. Recently developed high-throughput single-cell genetic analysis methods such as RNA-Seq require purification step and destruction of an analyzed cell often are providing a snapshot of the investigated cell without spatiotemporal context. Correlative analysis of multiparameter morphological, functional, and molecular information is important for differentiation of more uniform groups in the spectrum of different cell types. Simplified distributions (histograms and 2D plots) can underrepresent biologically significant subpopulations. Future directions may include the development of nondestructive methods for dissecting molecular events in intact cells, simultaneous correlative cellular analysis of phenotypic and molecular features by hybrid technologies such as imaging flow cytometry, and further progress in supervised and non-supervised statistical analysis algorithms.

Ion transport across the biological membrane by computational protein design

NASA Astrophysics Data System (ADS)

Grigoryan, Gevorg

The cellular membrane is impermeable to most of the chemicals the cell needs to take in or discard to survive. Therefore, transporters-a class of transmembrane proteins tasked with shuttling cargo chemicals in and out of the cell-are essential to all cellular life. From existing crystal structures, we know transporters to be complex machines, exquisitely tuned for specificity and controllability. But how could membrane-bound life have evolved if it needed such complex machines to exist first? To shed light onto this question, we considered the task of designing a transporter de novo. As our guiding principle, we took the ``alternating-access model''-a conceptual mechanism stating that transporters work by rocking between two conformations, each exposing the cargo-binding site to either the intra- or the extra-cellular environment. A computational design framework was developed to encode an anti-parallel four-helix bundle that rocked between two alternative states to orchestrate the movement of Zn(II) ions across the membrane. The ensemble nature of both states was accounted for using a free energy-based approach, and sequences were chosen based on predicted formation of the targeted topology in the membrane and bi-stability. A single sequence was prepared experimentally and shown to function as a Zn(II) transporter in lipid vesicles. Further, transport was specific to Zn(II) ions and several control peptides supported the underlying design principles. This included a mutant designed to retain all properties but with reduced rocking, which showed greatly depressed transport ability. These results suggest that early transporters could have evolved in the context of simple topologies, to be later tuned by evolution for improved properties and controllability. Our study also serves as an important advance in computational protein design, showing the feasibility of designing functional membrane proteins and of tuning conformational landscapes for desired function. Alfred P. Sloan Foundation Research Fellowship.

NeuroRDF: semantic integration of highly curated data to prioritize biomarker candidates in Alzheimer's disease.

PubMed

Iyappan, Anandhi; Kawalia, Shweta Bagewadi; Raschka, Tamara; Hofmann-Apitius, Martin; Senger, Philipp

2016-07-08

Neurodegenerative diseases are incurable and debilitating indications with huge social and economic impact, where much is still to be learnt about the underlying molecular events. Mechanistic disease models could offer a knowledge framework to help decipher the complex interactions that occur at molecular and cellular levels. This motivates the need for the development of an approach integrating highly curated and heterogeneous data into a disease model of different regulatory data layers. Although several disease models exist, they often do not consider the quality of underlying data. Moreover, even with the current advancements in semantic web technology, we still do not have cure for complex diseases like Alzheimer's disease. One of the key reasons accountable for this could be the increasing gap between generated data and the derived knowledge. In this paper, we describe an approach, called as NeuroRDF, to develop an integrative framework for modeling curated knowledge in the area of complex neurodegenerative diseases. The core of this strategy lies in the usage of well curated and context specific data for integration into one single semantic web-based framework, RDF. This increases the probability of the derived knowledge to be novel and reliable in a specific disease context. This infrastructure integrates highly curated data from databases (Bind, IntAct, etc.), literature (PubMed), and gene expression resources (such as GEO and ArrayExpress). We illustrate the effectiveness of our approach by asking real-world biomedical questions that link these resources to prioritize the plausible biomarker candidates. Among the 13 prioritized candidate genes, we identified MIF to be a potential emerging candidate due to its role as a pro-inflammatory cytokine. We additionally report on the effort and challenges faced during generation of such an indication-specific knowledge base comprising of curated and quality-controlled data. Although many alternative approaches have been proposed and practiced for modeling diseases, the semantic web technology is a flexible and well established solution for harmonized aggregation. The benefit of this work, to use high quality and context specific data, becomes apparent in speculating previously unattended biomarker candidates around a well-known mechanism, further leveraged for experimental investigations.

33 CFR 183.516 - Cellular plastic used to encase fuel tanks.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 33 Navigation and Navigable Waters 2 2013-07-01 2013-07-01 false Cellular plastic used to encase....516 Cellular plastic used to encase fuel tanks. (a) Cellular plastic used to encase metallic fuel... water per square foot of cut surface, measure under Military Specification MIL P-21929B. (b) Non...

33 CFR 183.516 - Cellular plastic used to encase fuel tanks.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 33 Navigation and Navigable Waters 2 2014-07-01 2014-07-01 false Cellular plastic used to encase....516 Cellular plastic used to encase fuel tanks. (a) Cellular plastic used to encase metallic fuel... water per square foot of cut surface, measure under Military Specification MIL P-21929B. (b) Non...

33 CFR 183.516 - Cellular plastic used to encase fuel tanks.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 33 Navigation and Navigable Waters 2 2011-07-01 2011-07-01 false Cellular plastic used to encase....516 Cellular plastic used to encase fuel tanks. (a) Cellular plastic used to encase metallic fuel... water per square foot of cut surface, measure under Military Specification MIL P-21929B. (b) Non...

33 CFR 183.516 - Cellular plastic used to encase fuel tanks.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 33 Navigation and Navigable Waters 2 2012-07-01 2012-07-01 false Cellular plastic used to encase....516 Cellular plastic used to encase fuel tanks. (a) Cellular plastic used to encase metallic fuel... water per square foot of cut surface, measure under Military Specification MIL P-21929B. (b) Non...

AGCVIII Kinases: at the crossroads of cellular signaling

USDA-ARS?s Scientific Manuscript database

AGCVIII kinases regulate diverse developmental and cellular processes in plants. As putative mediators of secondary messengers, AGCVIII kinases potentially integrate developmental and environmental cues into specific cellular responses through substrate phosphorylation. Here we discuss the functiona...

Modeling the mechanics of cancer: effect of changes in cellular and extra-cellular mechanical properties.

PubMed

Katira, Parag; Bonnecaze, Roger T; Zaman, Muhammad H

2013-01-01

Malignant transformation, though primarily driven by genetic mutations in cells, is also accompanied by specific changes in cellular and extra-cellular mechanical properties such as stiffness and adhesivity. As the transformed cells grow into tumors, they interact with their surroundings via physical contacts and the application of forces. These forces can lead to changes in the mechanical regulation of cell fate based on the mechanical properties of the cells and their surrounding environment. A comprehensive understanding of cancer progression requires the study of how specific changes in mechanical properties influences collective cell behavior during tumor growth and metastasis. Here we review some key results from computational models describing the effect of changes in cellular and extra-cellular mechanical properties and identify mechanistic pathways for cancer progression that can be targeted for the prediction, treatment, and prevention of cancer.

Cancer metabolism in space and time: Beyond the Warburg effect.

PubMed

Danhier, Pierre; Bański, Piotr; Payen, Valéry L; Grasso, Debora; Ippolito, Luigi; Sonveaux, Pierre; Porporato, Paolo E

2017-08-01

Altered metabolism in cancer cells is pivotal for tumor growth, most notably by providing energy, reducing equivalents and building blocks while several metabolites exert a signaling function promoting tumor growth and progression. A cancer tissue cannot be simply reduced to a bulk of proliferating cells. Tumors are indeed complex and dynamic structures where single cells can heterogeneously perform various biological activities with different metabolic requirements. Because tumors are composed of different types of cells with metabolic activities affected by different spatial and temporal contexts, it is important to address metabolism taking into account cellular and biological heterogeneity. In this review, we describe this heterogeneity also in metabolic fluxes, thus showing the relative contribution of different metabolic activities to tumor progression according to the cellular context. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux. Copyright © 2017 Elsevier B.V. All rights reserved.

The Role of Transporters in the Toxicity of Nucleoside and Nucleotide Analogs

PubMed Central

Koczor, Christopher A; Torres, Rebecca A

2013-01-01

Introduction Two families of nucleoside analogs have been developed to treat viral infections and cancer, but these compounds can cause tissue and cell-specific toxicity related to their uptake and subcellular activity which are dictated by host enzymes and transporters. Cellular uptake of these compounds requires nucleoside transporters that share functional similarities but differ in substrate specificity. Tissue-specific cellular expression of these transporters enables nucleoside analogs to produce their tissue specific toxic effects, a limiting factor in the treatment of retroviruses and cancer. Areas Covered This review discusses the families of nucleoside transporters and how they mediate cellular uptake of nucleoside analogs. Specific focus is placed on examples of known cases of transporter-mediated cellular toxicity and classification of the toxicities resulting. Efflux transporters are also explored as a contributor to analog toxicity and cell-specific effects. Expert Opinion Efforts to modulate transporter uptake/clearance remain long-term goals of oncologists and virologists. Accordingly, subcellular approaches that either increase or decrease intracellular nucleoside analog concentrations are eagerly sought and include transporter inhibitors and targeting transporter expression. However, additional understanding of nucleoside transporter kinetics, tissue expression, and genetic polymorphisms are required to design better molecules and better therapies. PMID:22509856

Cellular, molecular, and epigenetic mechanisms in non-associative conditioning: implications for pain and memory.

PubMed

Rahn, Elizabeth J; Guzman-Karlsson, Mikael C; David Sweatt, J

2013-10-01

Sensitization is a form of non-associative conditioning in which amplification of behavioral responses can occur following presentation of an aversive or noxious stimulus. Understanding the cellular and molecular underpinnings of sensitization has been an overarching theme spanning the field of learning and memory as well as that of pain research. In this review we examine how sensitization, both in the context of learning as well as pain processing, shares evolutionarily conserved behavioral, cellular/synaptic, and epigenetic mechanisms across phyla. First, we characterize the behavioral phenomenon of sensitization both in invertebrates and vertebrates. Particular emphasis is placed on long-term sensitization (LTS) of withdrawal reflexes in Aplysia following aversive stimulation or injury, although additional invertebrate models are also covered. In the context of vertebrates, sensitization of mammalian hyperarousal in a model of post-traumatic stress disorder (PTSD), as well as mammalian models of inflammatory and neuropathic pain is characterized. Second, we investigate the cellular and synaptic mechanisms underlying these behaviors. We focus our discussion on serotonin-mediated long-term facilitation (LTF) and axotomy-mediated long-term hyperexcitability (LTH) in reduced Aplysia systems, as well as mammalian spinal plasticity mechanisms of central sensitization. Third, we explore recent evidence implicating epigenetic mechanisms in learning- and pain-related sensitization. This review illustrates the fundamental and functional overlay of the learning and memory field with the pain field which argues for homologous persistent plasticity mechanisms in response to sensitizing stimuli or injury across phyla. Copyright © 2013 Elsevier Inc. All rights reserved.

Model-based design of experiments for cellular processes.

PubMed

Chakrabarty, Ankush; Buzzard, Gregery T; Rundell, Ann E

2013-01-01

Model-based design of experiments (MBDOE) assists in the planning of highly effective and efficient experiments. Although the foundations of this field are well-established, the application of these techniques to understand cellular processes is a fertile and rapidly advancing area as the community seeks to understand ever more complex cellular processes and systems. This review discusses the MBDOE paradigm along with applications and challenges within the context of cellular processes and systems. It also provides a brief tutorial on Fisher information matrix (FIM)-based and Bayesian experiment design methods along with an overview of existing software packages and computational advances that support MBDOE application and adoption within the Systems Biology community. As cell-based products and biologics progress into the commercial sector, it is anticipated that MBDOE will become an essential practice for design, quality control, and production. Copyright © 2013 Wiley Periodicals, Inc.

Wise retained in the endoplasmic reticulum inhibits Wnt signaling by reducing cell surface LRP6.

PubMed

Guidato, Sonia; Itasaki, Nobue

2007-10-15

The Wnt signaling pathway is tightly regulated by extracellular and intracellular modulators. Wise was isolated as a secreted protein capable of interacting with the Wnt co-receptor LRP6. Studies in Xenopus embryos revealed that Wise either enhances or inhibits the Wnt pathway depending on the cellular context. Here we show that the cellular localization of Wise has distinct effects on the Wnt pathway readout. While secreted Wise either synergizes or inhibits the Wnt signals depending on the partner ligand, ER-retained Wise consistently blocks the Wnt pathway. ER-retained Wise reduces LRP6 on the cell surface, making cells less susceptible to the Wnt signal. This study provides a cellular mechanism for the action of Wise and introduces the modulation of cellular susceptibility to Wnt signals as a novel mechanism of the regulation of the Wnt pathway.

Discriminative segmentation of microscopic cellular images.

PubMed

Cheng, Li; Ye, Ning; Yu, Weimiao; Cheah, Andre

2011-01-01

Microscopic cellular images segmentation has become an important routine procedure in modern biological research, due to the rapid advancement of fluorescence probes and robotic microscopes in recent years. In this paper we advocate a discriminative learning approach for cellular image segmentation. In particular, three new features are proposed to capture the appearance, shape and context information, respectively. Experiments are conducted on three different cellular image datasets. Despite the significant disparity among these datasets, the proposed approach is demonstrated to perform reasonably well. As expected, for a particular dataset, some features turn out to be more suitable than others. Interestingly, we observe that a further gain can often be obtained on top of using the "good" features, by also retaining those features that perform poorly. This might be due to the complementary nature of these features, as well as the capacity of our approach to better integrate and exploit different sources of information.

Spatial reconstruction of single-cell gene expression

PubMed Central

Satija, Rahul; Farrell, Jeffrey A.; Gennert, David; Schier, Alexander F.; Regev, Aviv

2015-01-01

Spatial localization is a key determinant of cellular fate and behavior, but spatial RNA assays traditionally rely on staining for a limited number of RNA species. In contrast, single-cell RNA-seq allows for deep profiling of cellular gene expression, but established methods separate cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos, inferring a transcriptome-wide map of spatial patterning. We confirmed Seurat’s accuracy using several experimental approaches, and used it to identify a set of archetypal expression patterns and spatial markers. Additionally, Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems. PMID:25867923

Respiratory syncytial virus induced type I IFN production by pDC is regulated by RSV-infected airway epithelial cells, RSV-exposed monocytes and virus specific antibodies.

PubMed

Schijf, Marcel A; Lukens, Michael V; Kruijsen, Debby; van Uden, Nathalie O P; Garssen, Johan; Coenjaerts, Frank E J; Van't Land, Belinda; van Bleek, Grada M

2013-01-01

Innate immune responses elicited upon virus exposure are crucial for the effective eradication of viruses, the onset of adaptive immune responses and for establishing proper immune memory. Respiratory syncytial virus (RSV) is responsible for a high disease burden in neonates and immune compromised individuals, causing severe lower respiratory tract infections. During primary infections exuberant innate immune responses may contribute to disease severity. Furthermore, immune memory is often insufficient to protect during RSV re-exposure, which results in frequent symptomatic reinfections. Therefore, identifying the cell types and pattern recognition receptors (PRRs) involved in RSV-specific innate immune responses is necessary to understand incomplete immunity against RSV. We investigated the innate cellular response triggered upon infection of epithelial cells and peripheral blood mononuclear cells. We show that CD14(+) myeloid cells and epithelial cells are the major source of IL-8 and inflammatory cytokines, IL-6 and TNF-α, when exposed to live RSV Three routes of RSV-induced IFN-α production can be distinguished that depend on the cross-talk of different cell types and the presence or absence of virus specific antibodies, whereby pDC are the ultimate source of IFN-α. RSV-specific antibodies facilitate direct TLR7 access into endosomal compartments, while in the absence of antibodies, infection of monocytes or epithelial cells is necessary to provide an early source of type I interferons, required to engage the IFN-α,β receptor (IFNAR)-mediated pathway of IFN-α production by pDC. However, at high pDC density infection with RSV causes IFN-α production without the need for a second party cell. Our study shows that cellular context and immune status are factors affecting innate immune responses to RSV. These issues should therefore be addressed during the process of vaccine development and other interventions for RSV disease.

A Genetically Encoded Probe for Live-Cell Imaging of H4K20 Monomethylation.

PubMed

Sato, Yuko; Kujirai, Tomoya; Arai, Ritsuko; Asakawa, Haruhiko; Ohtsuki, Chizuru; Horikoshi, Naoki; Yamagata, Kazuo; Ueda, Jun; Nagase, Takahiro; Haraguchi, Tokuko; Hiraoka, Yasushi; Kimura, Akatsuki; Kurumizaka, Hitoshi; Kimura, Hiroshi

2016-10-09

Eukaryotic gene expression is regulated in the context of chromatin. Dynamic changes in post-translational histone modification are thought to play key roles in fundamental cellular functions such as regulation of the cell cycle, development, and differentiation. To elucidate the relationship between histone modifications and cellular functions, it is important to monitor the dynamics of modifications in single living cells. A genetically encoded probe called mintbody (modification-specific intracellular antibody), which is a single-chain variable fragment tagged with a fluorescent protein, has been proposed as a useful visualization tool. However, the efficacy of intracellular expression of antibody fragments has been limited, in part due to different environmental conditions in the cytoplasm compared to the endoplasmic reticulum where secreted proteins such as antibodies are folded. In this study, we have developed a new mintbody specific for histone H4 Lys20 monomethylation (H4K20me1). The specificity of the H4K20me1-mintbody in living cells was verified using yeast mutants and mammalian cells in which this target modification was diminished. Expression of the H4K20me1-mintbody allowed us to monitor the oscillation of H4K20me1 levels during the cell cycle. Moreover, dosage-compensated X chromosomes were visualized using the H4K20me1-mintbody in mouse and nematode cells. Using X-ray crystallography and mutational analyses, we identified critical amino acids that contributed to stabilization and/or proper folding of the mintbody. Taken together, these data provide important implications for future studies aimed at developing functional intracellular antibodies. Specifically, the H4K20me1-mintbody provides a powerful tool to track this particular histone modification in living cells and organisms. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Respiratory Syncytial Virus Induced Type I IFN Production by pDC Is Regulated by RSV-Infected Airway Epithelial Cells, RSV-Exposed Monocytes and Virus Specific Antibodies

PubMed Central

Schijf, Marcel A.; Lukens, Michael V.; Kruijsen, Debby; van Uden, Nathalie O. P.; Garssen, Johan; Coenjaerts, Frank E. J.; van’t Land, Belinda; van Bleek, Grada M.

2013-01-01

Innate immune responses elicited upon virus exposure are crucial for the effective eradication of viruses, the onset of adaptive immune responses and for establishing proper immune memory. Respiratory syncytial virus (RSV) is responsible for a high disease burden in neonates and immune compromised individuals, causing severe lower respiratory tract infections. During primary infections exuberant innate immune responses may contribute to disease severity. Furthermore, immune memory is often insufficient to protect during RSV re-exposure, which results in frequent symptomatic reinfections. Therefore, identifying the cell types and pattern recognition receptors (PRRs) involved in RSV-specific innate immune responses is necessary to understand incomplete immunity against RSV. We investigated the innate cellular response triggered upon infection of epithelial cells and peripheral blood mononuclear cells. We show that CD14+ myeloid cells and epithelial cells are the major source of IL-8 and inflammatory cytokines, IL-6 and TNF-α, when exposed to live RSV Three routes of RSV-induced IFN-α production can be distinguished that depend on the cross-talk of different cell types and the presence or absence of virus specific antibodies, whereby pDC are the ultimate source of IFN-α. RSV-specific antibodies facilitate direct TLR7 access into endosomal compartments, while in the absence of antibodies, infection of monocytes or epithelial cells is necessary to provide an early source of type I interferons, required to engage the IFN-α,β receptor (IFNAR)-mediated pathway of IFN-α production by pDC. However, at high pDC density infection with RSV causes IFN-α production without the need for a second party cell. Our study shows that cellular context and immune status are factors affecting innate immune responses to RSV. These issues should therefore be addressed during the process of vaccine development and other interventions for RSV disease. PMID:24303065

Germline disruption of Pten localization causes enhanced sex-dependent social motivation and increased glial production.

PubMed

Tilot, Amanda K; Gaugler, Mary K; Yu, Qi; Romigh, Todd; Yu, Wanfeng; Miller, Robert H; Frazier, Thomas W; Eng, Charis

2014-06-15

PTEN Hamartoma Tumor Syndrome (PHTS) is an autosomal-dominant genetic condition underlying a subset of autism spectrum disorder (ASD) with macrocephaly. Caused by germline mutations in PTEN, PHTS also causes increased risks of multiple cancers via dysregulation of the PI3K and MAPK signaling pathways. Conditional knockout models have shown that neural Pten regulates social behavior, proliferation and cell size. Although much is known about how the intracellular localization of PTEN regulates signaling in cancer cell lines, we know little of how PTEN localization influences normal brain physiology and behavior. To address this, we generated a germline knock-in mouse model of cytoplasm-predominant Pten and characterized its behavioral and cellular phenotypes. The homozygous Pten(m3m4) mice have decreased total Pten levels including a specific drop in nuclear Pten and exhibit region-specific increases in brain weight. The Pten(m3m4) model displays sex-specific increases in social motivation, poor balance and normal recognition memory-a profile reminiscent of some individuals with high functioning ASD. The cytoplasm-predominant protein caused cellular hypertrophy limited to the soma and led to increased NG2 cell proliferation and accumulation of glia. The animals also exhibit significant astrogliosis and microglial activation, indicating a neuroinflammatory phenotype. At the signaling level, Pten(m3m4) mice show brain region-specific differences in Akt activation. These results demonstrate that differing alterations to the same autism-linked gene can cause distinct behavioral profiles. The Pten(m3m4) model is the first murine model of inappropriately elevated social motivation in the context of normal cognition and may expand the range of autism-related behaviors replicated in animal models. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The third dimension bridges the gap between cell culture and live tissue.

PubMed

Pampaloni, Francesco; Reynaud, Emmanuel G; Stelzer, Ernst H K

2007-10-01

Moving from cell monolayers to three-dimensional (3D) cultures is motivated by the need to work with cellular models that mimic the functions of living tissues. Essential cellular functions that are present in tissues are missed by 'petri dish'-based cell cultures. This limits their potential to predict the cellular responses of real organisms. However, establishing 3D cultures as a mainstream approach requires the development of standard protocols, new cell lines and quantitative analysis methods, which include well-suited three-dimensional imaging techniques. We believe that 3D cultures will have a strong impact on drug screening and will also decrease the use of laboratory animals, for example, in the context of toxicity assays.

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

Context-specific adjustment of cognitive control: Transfer of adaptive control sets.

PubMed

Surrey, Caroline; Dreisbach, Gesine; Fischer, Rico

2017-11-01

Cognitive control protects processing of relevant information from interference by irrelevant information. The level of this processing selectivity can be flexibly adjusted to different control demands (e.g., frequency of conflict) associated with a certain context, leading to the formation of specific context-control associations. In the present study we investigated the robustness and transferability of the acquired context-control demands to new situations. In three experiments, we used a version of the context-specific proportion congruence (CSPC) paradigm, in which each context (e.g., location) is associated with a specific conflict frequency, determining high and low control demands. In a learning phase, associations between context and control demands were established. In a subsequent transfer block, stimulus-response mappings, whole task sets, or context-control demands changed. Results showed an impressive robustness of context-control associations, as context-specific adjustments of control from the learning phase were virtually unaffected by new stimuli and tasks in the transfer block. Only a change of the context-control demand eliminated the context-specific adjustment of control. These findings suggest that context-control associations that have proven to be adaptive in the past are continuously applied despite major changes in the task structure as long as the context-control associations remain the same.

Cell-type-specific role of ΔFosB in nucleus accumbens in modulating inter-male aggression.

PubMed

Aleyasin, Hossein; Flanigan, Meghan E; Golden, Sam A; Takahashi, Aki; Menard, Caroline; Pfau, Madeline L; Multer, Jacob; Pina, Jacqueline; McCabe, Kathryn A; Bhatti, Naemal; Hodes, Georgia E; Heshmati, Mitra; Neve, Rachael L; Nestler, Eric J; Heller, Elizabeth A; Russo, Scott J

2018-06-11

A growing number of studies implicate the brain's reward circuitry in aggressive behavior. However, the cellular and molecular mechanisms within brain reward regions that modulate the intensity of aggression as well as motivation for it have been underexplored. Here, we investigate the cell-type-specific influence of ΔFosB, a transcription factor known to regulate a range of reward and motivated behaviors, acting in the nucleus accumbens (NAc)-a key reward region-in male aggression in mice. We show that ΔFosB is specifically increased in dopamine D1 receptor (Drd1) expressing medium spiny neurons (D1-MSNs) in NAc after repeated aggressive encounters. Viral-mediated induction of ΔFosB selectively in D1-MSNs of NAc intensifies aggressive behavior, without affecting the preference for the aggression-paired context in a conditioned place preference (CPP) assay. In contrast, ΔFosB induction selectively in D2-MSNs reduces the time spent exploring the aggression-paired context during CPP without affecting the intensity of aggression per se. These data strongly support a dissociable cell-type-specific role for ΔFosB in the NAc in modulating aggression and aggression reward. Significance Statement: Aggressive behavior is associated with several neuropsychiatric disorders and can be disruptive for the individuals as well as their victims. Studies have shown a positive reinforcement mechanism underlying aggressive behavior that shares many common features with drug addiction. Here, we explore the cell-type-specific role of the addiction-associated transcription factor ΔFosB in the nucleus accumbens (NAc) in aggression. We found that ΔFosB expression promotes aggressive behavior, effects that are dissociable from its effects on aggression reward. This finding is a significant first step in identifying therapeutic targets for the reduction of aggressive behavior across a range of neuropsychiatric illnesses. Copyright © 2018 the authors.

Computing and Applying Atomic Regulons to Understand Gene Expression and Regulation

PubMed Central

Faria, José P.; Davis, James J.; Edirisinghe, Janaka N.; Taylor, Ronald C.; Weisenhorn, Pamela; Olson, Robert D.; Stevens, Rick L.; Rocha, Miguel; Rocha, Isabel; Best, Aaron A.; DeJongh, Matthew; Tintle, Nathan L.; Parrello, Bruce; Overbeek, Ross; Henry, Christopher S.

2016-01-01

Understanding gene function and regulation is essential for the interpretation, prediction, and ultimate design of cell responses to changes in the environment. An important step toward meeting the challenge of understanding gene function and regulation is the identification of sets of genes that are always co-expressed. These gene sets, Atomic Regulons (ARs), represent fundamental units of function within a cell and could be used to associate genes of unknown function with cellular processes and to enable rational genetic engineering of cellular systems. Here, we describe an approach for inferring ARs that leverages large-scale expression data sets, gene context, and functional relationships among genes. We computed ARs for Escherichia coli based on 907 gene expression experiments and compared our results with gene clusters produced by two prevalent data-driven methods: Hierarchical clustering and k-means clustering. We compared ARs and purely data-driven gene clusters to the curated set of regulatory interactions for E. coli found in RegulonDB, showing that ARs are more consistent with gold standard regulons than are data-driven gene clusters. We further examined the consistency of ARs and data-driven gene clusters in the context of gene interactions predicted by Context Likelihood of Relatedness (CLR) analysis, finding that the ARs show better agreement with CLR predicted interactions. We determined the impact of increasing amounts of expression data on AR construction and find that while more data improve ARs, it is not necessary to use the full set of gene expression experiments available for E. coli to produce high quality ARs. In order to explore the conservation of co-regulated gene sets across different organisms, we computed ARs for Shewanella oneidensis, Pseudomonas aeruginosa, Thermus thermophilus, and Staphylococcus aureus, each of which represents increasing degrees of phylogenetic distance from E. coli. Comparison of the organism-specific ARs showed that the consistency of AR gene membership correlates with phylogenetic distance, but there is clear variability in the regulatory networks of closely related organisms. As large scale expression data sets become increasingly common for model and non-model organisms, comparative analyses of atomic regulons will provide valuable insights into fundamental regulatory modules used across the bacterial domain. PMID:27933038

Viral Activation of Cellular Metabolism

PubMed Central

Sanchez, Erica L.; Lagunoff, Michael

2015-01-01

To ensure optimal environments for their replication and spread, viruses have evolved to alter many host cell pathways. In the last decade, metabolomic studies have shown that eukaryotic viruses induce large-scale alterations in host cellular metabolism. Most viruses examined to date induce aerobic glycolysis also known as the Warburg effect. Many viruses tested also induce fatty acid synthesis as well as glutaminolysis. These modifications of carbon source utilization by infected cells can increase available energy for virus replication and virion production, provide specific cellular substrates for virus particles and create viral replication niches while increasing infected cell survival. Each virus species also likely requires unique metabolic changes for successful spread and recent research has identified additional virus-specific metabolic changes induced by many virus species. A better understanding of the metabolic alterations required for each virus may lead to novel therapeutic approaches through targeted inhibition of specific cellular metabolic pathways. PMID:25812764

Opposite regulation of MDM2 and MDMX expression in acquisition of mesenchymal phenotype in benign and cancer cells.

PubMed

Slabáková, Eva; Kharaishvili, Gvantsa; Smějová, Monika; Pernicová, Zuzana; Suchánková, Tereza; Remšík, Ján; Lerch, Stanislav; Straková, Nicol; Bouchal, Jan; Král, Milan; Culig, Zoran; Kozubík, Alois; Souček, Karel

2015-11-03

Plasticity of cancer cells, manifested by transitions between epithelial and mesenchymal phenotypes, represents a challenging issue in the treatment of neoplasias. Both epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are implicated in the processes of metastasis formation and acquisition of stem cell-like properties. Mouse double minute (MDM) 2 and MDMX are important players in cancer progression, as they act as regulators of p53, but their function in EMT and metastasis may be contradictory. Here, we show that the EMT phenotype in multiple cellular models and in clinical prostate and breast cancer samples is associated with a decrease in MDM2 and increase in MDMX expression. Modulation of EMT-accompanying changes in MDM2 expression in benign and transformed prostate epithelial cells influences their migration capacity and sensitivity to docetaxel. Analysis of putative mechanisms of MDM2 expression control demonstrates that in the context of defective p53 function, MDM2 expression is regulated by EMT-inducing transcription factors Slug and Twist. These results provide an alternative context-specific role of MDM2 in EMT, cell migration, metastasis, and therapy resistance.

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

Ciomperlik, Jessica J.; Basta, Holly A.; Palmenberg, Ann C., E-mail: acpalmen@wisc.edu

Cardiovirus infections inhibit nucleocytoplasmic trafficking by Leader protein-induced phosphorylation of Phe/Gly-containing nucleoporins (Nups). Recombinant Leader from encephalomyocarditis virus, Theiler's murine encephalomyelitis virus and Saffold virus target the same subset of Nups, including Nup62 and Nup98, but not Nup50. Reporter cell lines with fluorescence mCherry markers for M9, RS and classical SV40 import pathways, as well as the Crm1-mediated export pathway, all responded to transfection with the full panel of Leader proteins, showing consequent cessation of path-specific active import/export. For this to happen, the Nups had to be presented in the context of intact nuclear pores and exposed to cytoplasmic extracts.more » The Leader phosphorylation cascade was not effective against recombinant Nup proteins. The findings support a model of Leader-dependent Nup phosphorylation with the purpose of disrupting Nup-transportin interactions. - Highlights: • Nup98, but not Nup50 becomes phosphorylated by cardiovirus Leader protein-dependent mechanisms. • At least four independent nucleocytoplasmic trafficking pathways are inhibited by this process. • Nups must be presented in a nuclear pore context for Leader-directed phosphorylation. • Leader, by itself, does not cause activation of cellular kinases.« less

Biological organization of the extraocular muscles.

PubMed

Spencer, Robert F; Porter, John D

2006-01-01

Extraocular muscle is fundamentally distinct from other skeletal muscles. Here, we review the biological organization of the extraocular muscles with the intent of understanding this novel muscle group in the context of oculomotor system function. The specific objectives of this review are threefold. The first objective is to understand the anatomic arrangement of the extraocular muscles and their compartmental or layered organization in the context of a new concept of orbital mechanics, the active pulley hypothesis. The second objective is to present an integrated view of the morphologic, cellular, and molecular differences between extraocular and the more traditional skeletal muscles. The third objective is to relate recent data from functional and molecular biology studies to the established extraocular muscle fiber types. Developmental mechanisms that may be responsible for the divergence of the eye muscles from a skeletal muscle prototype also are considered. Taken together, a multidisciplinary understanding of extraocular muscle biology in health and disease provides insights into oculomotor system function and malfunction. Moreover, because the eye muscles are selectively involved or spared in a variety of neuromuscular diseases, knowledge of their biology may improve current pathogenic models of and treatments for devastating systemic diseases.

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

PubMed Central

Kang, Hong-Jun; Vassilopoulos, Athanassios

2016-01-01

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context. PMID:26966987

Peroxotitanates for Biodelivery of Metals

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

Hobbs, David; Elvington, M.

2009-02-11

Metal-based drugs are largely undeveloped in pharmacology. One limiting factor is the systemic toxicity of metal-based compounds. A solid-phase, sequestratable delivery agent for local delivery of metals could reduce systemic toxicity, facilitating new drug development in this nascent area. Amorphous peroxotitanates (APT) are ion exchange materials with high affinity for several heavy metal ions, and have been proposed to deliver or sequester metal ions in biological contexts. In the current study, we tested a hypothesis that APT are able to deliver metals or metal compounds to cells. We exposed fibroblasts (L929) or monocytes (THP1) to metal-APT materials for 72 hmore » in vitro, then measured cellular mitochondrial activity (SDH-MTT method) to assess the biological impact of the metal-APT materials vs. metals or APT alone. APT alone did not significantly affect cellular mitochondrial activity, but all metal-APT materials suppressed the mitochondrial activity of fibroblasts (by 30-65% of controls). The concentration of metal-APT materials required to suppress cellular mitochondrial activity was below that required for metals alone, suggesting that simple extracellular release of the metals from the metal-APT materials was not the primary mechanism of mitochondrial suppression. In contrast to fibroblasts, no metal-APT material had a measurable effect on THP1 monocyte mitochondrial activity, despite potent suppression by metals alone. This latter result suggested that 'biodelivery' by metal-APT materials may be cell type-specific. Therefore, it appears that APT are plausible solid phase delivery agents of metals or metal compounds to some types of cells for potential therapeutic effect.« less

A model of how different biology experts explain molecular and cellular mechanisms.

PubMed

Trujillo, Caleb M; Anderson, Trevor R; Pelaez, Nancy J

2015-01-01

Constructing explanations is an essential skill for all science learners. The goal of this project was to model the key components of expert explanation of molecular and cellular mechanisms. As such, we asked: What is an appropriate model of the components of explanation used by biology experts to explain molecular and cellular mechanisms? Do explanations made by experts from different biology subdisciplines at a university support the validity of this model? Guided by the modeling framework of R. S. Justi and J. K. Gilbert, the validity of an initial model was tested by asking seven biologists to explain a molecular mechanism of their choice. Data were collected from interviews, artifacts, and drawings, and then subjected to thematic analysis. We found that biologists explained the specific activities and organization of entities of the mechanism. In addition, they contextualized explanations according to their biological and social significance; integrated explanations with methods, instruments, and measurements; and used analogies and narrated stories. The derived methods, analogies, context, and how themes informed the development of our final MACH model of mechanistic explanations. Future research will test the potential of the MACH model as a guiding framework for instruction to enhance the quality of student explanations. © 2015 C. M. Trujillo et al. CBE—Life Sciences Education © 2015 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Principles of Unconventional Myosin Function and Targeting

PubMed Central

Hartman, M. Amanda; Finan, Dina; Sivaramakrishnan, Sivaraj; Spudich, James A.

2016-01-01

Unconventional myosins are a superfamily of actin-based motors implicated in diverse cellular processes. In recent years, much progress has been made in describing their biophysical properties, and headway has been made into analyzing their cellular functions. Here, we focus on the principles that guide in vivo motor function and targeting to specific cellular locations. Rather than describe each motor comprehensively, we outline the major themes that emerge from research across the superfamily and use specific examples to illustrate each. In presenting the data in this format, we seek to identify open questions in each field as well as to point out commonalities between them. To advance our understanding of myosins’ roles in vivo, clearly we must identify their cellular cargoes and the protein complexes that regulate motor attachment to fully appreciate their functions on the cellular and developmental levels. PMID:21639800

Diversification of the muscle proteome through alternative splicing.

PubMed

Nakka, Kiran; Ghigna, Claudia; Gabellini, Davide; Dilworth, F Jeffrey

2018-03-06

Skeletal muscles express a highly specialized proteome that allows the metabolism of energy sources to mediate myofiber contraction. This muscle-specific proteome is partially derived through the muscle-specific transcription of a subset of genes. Surprisingly, RNA sequencing technologies have also revealed a significant role for muscle-specific alternative splicing in generating protein isoforms that give specialized function to the muscle proteome. In this review, we discuss the current knowledge with respect to the mechanisms that allow pre-mRNA transcripts to undergo muscle-specific alternative splicing while identifying some of the key trans-acting splicing factors essential to the process. The importance of specific splicing events to specialized muscle function is presented along with examples in which dysregulated splicing contributes to myopathies. Though there is now an appreciation that alternative splicing is a major contributor to proteome diversification, the emergence of improved "targeted" proteomic methodologies for detection of specific protein isoforms will soon allow us to better appreciate the extent to which alternative splicing modifies the activity of proteins (and their ability to interact with other proteins) in the skeletal muscle. In addition, we highlight a continued need to better explore the signaling pathways that contribute to the temporal control of trans-acting splicing factor activity to ensure specific protein isoforms are expressed in the proper cellular context. An understanding of the signal-dependent and signal-independent events driving muscle-specific alternative splicing has the potential to provide us with novel therapeutic strategies to treat different myopathies.

Building bridges between cellular and molecular structural biology.

PubMed

Patwardhan, Ardan; Brandt, Robert; Butcher, Sarah J; Collinson, Lucy; Gault, David; Grünewald, Kay; Hecksel, Corey; Huiskonen, Juha T; Iudin, Andrii; Jones, Martin L; Korir, Paul K; Koster, Abraham J; Lagerstedt, Ingvar; Lawson, Catherine L; Mastronarde, David; McCormick, Matthew; Parkinson, Helen; Rosenthal, Peter B; Saalfeld, Stephan; Saibil, Helen R; Sarntivijai, Sirarat; Solanes Valero, Irene; Subramaniam, Sriram; Swedlow, Jason R; Tudose, Ilinca; Winn, Martyn; Kleywegt, Gerard J

2017-07-06

The integration of cellular and molecular structural data is key to understanding the function of macromolecular assemblies and complexes in their in vivo context. Here we report on the outcomes of a workshop that discussed how to integrate structural data from a range of public archives. The workshop identified two main priorities: the development of tools and file formats to support segmentation (that is, the decomposition of a three-dimensional volume into regions that can be associated with defined objects), and the development of tools to support the annotation of biological structures.

An ancient defense system eliminates unfit cells from developing tissues during cell competition

PubMed Central

Meyer, S. N.; Amoyel, M.; Bergantiños, C.; de la Cova, C.; Schertel, C.; Basler, K.; Johnston, L. A.

2016-01-01

Developing tissues that contain mutant or compromised cells present risks to animal health. Accordingly, the appearance of a population of suboptimal cells in a tissue elicits cellular interactions that prevent their contribution to the adult. Here we report that this quality control process, cell competition, uses specific components of the evolutionarily ancient and conserved innate immune system to eliminate Drosophila cells perceived as unfit. We find that Toll-related receptors (TRRs) and the cytokine Spätzle (Spz) lead to NFκB-dependent apoptosis. Diverse “loser” cells require different TRRs and NFκB factors and activate distinct pro-death genes, implying that the particular response is stipulated by the competitive context. Our findings demonstrate a functional repurposing of components of TRRs and NFκB signaling modules in the surveillance of cell fitness during development. PMID:25477468

A Cell-Permeable Inhibitor to Trap Gαq Proteins in the Empty Pocket Conformation

PubMed Central

Schmitz, Anna-Lena; Schrage, Ramona; Gaffal, Evelyn; Charpentier, Thomas H.; Wiest, Johannes; Hiltensperger, Georg; Morschel, Julia; Hennen, Stephanie; Häußler, Daniela; Horn, Velten; Wenzel, Daniela; Grundmann, Manuel; Büllesbach, Katrin M.; Schröder, Ralf; Brewitz, H. Henning; Schmidt, Johannes; Gomeza, Jesús; Galés, Céline; Fleischmann, Bernd K.; Tüting, Thomas; Imhof, Diana; Tietze, Daniel; Gütschow, Michael; Holzgrabe, Ulrike; Sondek, John; Harden, T. Kendall; Mohr, Klaus; Kostenis, Evi

2015-01-01

SUMMARY In spite of the crucial role of heterotrimeric G proteins as molecular switches transmitting signals from G protein-coupled receptors, their selective manipulation with small molecule, cell-permeable inhibitors still remains an unmet challenge. Here, we report that the small molecule BIM-46187, previously classified as pan-G protein inhibitor, preferentially silences Gαq signaling in a cellular context-dependent manner. Investigations into its mode of action reveal that BIM traps Gαq in the empty pocket conformation by permitting GDP exit but interdicting GTP entry, a molecular mechanism not yet assigned to any other small molecule Gα inhibitor to date. Our data show that Gα proteins may be “frozen” pharmacologically in an intermediate conformation along their activation pathway and propose a pharmacological strategy to specifically silence Gα subclasses with cell-permeable inhibitors. PMID:25036778

Considerations for the rational design of a Chlamydia vaccine.

PubMed

Liang, Steven; Bulir, David; Kaushic, Charu; Mahony, James

2017-04-03

Chlamydia trachomatis is the leading cause of preventable blindness and the most common bacterial sexually transmitted infection. Remarkable progress in vaccine research over the past six decades has led to the advancement of novel C. trachomatis vaccine candidates into clinical trials. However, many questions regarding the role of specific cellular populations and molecular mechanisms in protective immunity against human C. trachomatis genital tract infections remain unanswered. Biomarkers of vaccine induced protective immunity are elusive in humans, while a cautionary message on the translatability of data obtained from current animal models has emanated from vaccine research and development efforts against other important human pathogens. In this commentary, we highlight recent advances in Chlamydia vaccine development and discuss their implications in the context of a rational approach to the design of a human C. trachomatis vaccine.

Inhalative nanomedicine--opportunities and challenges.

PubMed

Bur, Michael; Henning, Andreas; Hein, Stephanie; Schneider, Marc; Lehr, Claus-Michael

2009-07-01

Inhalation therapy is still limited by the low bioavailability of the administered drugs. Advantages of the pulmonary administration site like large resorption area, low enzymatic equipment, and circumvention of the first pass effect are set into perspective by the rigid barrier properties of the alveolar region. As a consequence, the systemic bioavailability of peptides and proteins is still relatively limited, even when administered by modern pharmaceutical aerosol technologies. In the context of advanced pulmonary drug therapy the use of nanoparticles as alternative to micronsized drug formulation could be of special interest, because nanoparticles seem to overcome some cellular barriers quite efficiently. Besides such outstanding permeation properties, nanoparticles may also hold promises to escape from pulmonary clearance mechanisms and to allow for cell-specific targeting within the lung. Such opportunities and challenges of inhalative nanomedicine are reviewed in this short review.

The advancement of chemical cross-linking and mass spectrometry for structural proteomics: from single proteins to protein interaction networks.

PubMed

Sinz, Andrea

2014-12-01

During the last 15 years, chemical cross-linking combined with mass spectrometry (MS) and computational modeling has advanced from investigating 3D-structures of isolated proteins to deciphering protein interaction networks. In this article, the author discusses the advent, the development and the current status of the chemical cross-linking/MS strategy in the context of recent technological developments. A direct way to probe in vivo protein-protein interactions is by site-specific incorporation of genetically encoded photo-reactive amino acids or by non-directed incorporation of photo-reactive amino acids. As the chemical cross-linking/MS approach allows the capture of transient and weak interactions, it has the potential to become a routine technique for unraveling protein interaction networks in their natural cellular environment.

Toward Multiscale Models of Cyanobacterial Growth: A Modular Approach

PubMed Central

Westermark, Stefanie; Steuer, Ralf

2016-01-01

Oxygenic photosynthesis dominates global primary productivity ever since its evolution more than three billion years ago. While many aspects of phototrophic growth are well understood, it remains a considerable challenge to elucidate the manifold dependencies and interconnections between the diverse cellular processes that together facilitate the synthesis of new cells. Phototrophic growth involves the coordinated action of several layers of cellular functioning, ranging from the photosynthetic light reactions and the electron transport chain, to carbon-concentrating mechanisms and the assimilation of inorganic carbon. It requires the synthesis of new building blocks by cellular metabolism, protection against excessive light, as well as diurnal regulation by a circadian clock and the orchestration of gene expression and cell division. Computational modeling allows us to quantitatively describe these cellular functions and processes relevant for phototrophic growth. As yet, however, computational models are mostly confined to the inner workings of individual cellular processes, rather than describing the manifold interactions between them in the context of a living cell. Using cyanobacteria as model organisms, this contribution seeks to summarize existing computational models that are relevant to describe phototrophic growth and seeks to outline their interactions and dependencies. Our ultimate aim is to understand cellular functioning and growth as the outcome of a coordinated operation of diverse yet interconnected cellular processes. PMID:28083530

P1 promoter-driven HNF4α isoforms are specifically repressed by β-catenin signaling in colorectal cancer cells.

PubMed

Babeu, Jean-Philippe; Jones, Christine; Geha, Sameh; Carrier, Julie C; Boudreau, François

2018-06-13

HNF4α is a key nuclear receptor for regulating gene expression in the gut. While both P1 and P2 isoform classes of HNF4α are expressed in colonic epithelium, specific inhibition of P1 isoforms is commonly found in colorectal cancer. Previous studies have suggested that P1 and P2 isoforms may regulate different cellular functions. Despite these advances, it remains unclear whether these isoform classes are functionally divergent in the context of human biology. Here, the consequences of specific inhibition of P1 or P2 isoform expression was measured in a human colorectal cancer cell transcriptome. Results indicate that P1 isoforms were specifically associated with the control of cell metabolism while P2 isoforms globally supported aberrant oncogenic signalization, promoting cancer cell survival and progression. P1 promoter-driven isoform expression was found to be repressed by β-catenin, one of the earliest oncogenic pathways to be activated during colon tumorigenesis. These findings identify a novel cascade by which the expression of P1 isoforms are rapidly shut down in the early stages of colon tumorigenesis, allowing a change in HNF4α-dependent transcriptome thereby promoting colorectal cancer progression. © 2018. Published by The Company of Biologists Ltd.

Manipulating the antigen-specific immune response by the hydrophobicity of amphiphilic poly(γ-glutamic acid) nanoparticles.

PubMed

Shima, Fumiaki; Akagi, Takami; Uto, Tomofumi; Akashi, Mitsuru

2013-12-01

The new generation vaccines are safe but poorly immunogenic, and thus they require the use of adjuvants. However, conventional vaccine adjuvants fail to induce potent cellular immunity, and their toxicity and side-effects hinder the clinical use. Therefore, a vaccine adjuvant which is safe and can induce an antigen-specific cellular immunity-biased immune response is urgently required. In the development of nanoparticle-based vaccine adjuvants, the hydrophobicity is one of the most important factors. It could control the interaction between the encapsulated antigens and/or nanoparticles with immune cells. In this study, nanoparticles (NPs) composed of amphiphilic poly(γ-glutamic acid)-graft-L-phenylalanine ethyl ester (γ-PGA-Phe) with various grafting degrees of hydrophobic side chains were prepared to evaluate the effect of hydrophobicity of vaccine carriers on the antigen encapsulation behavior, cellular uptake, activation of dendritic cells (DCs), and induction of antigen-specific cellular immunity-biased immune responses. These NPs could efficiently encapsulate antigens, and the uptake amount of the encapsulated antigen by DCs was dependent on the hydrophobicity of γ-PGA-Phe NPs. Moreover, the activation potential of the DCs and the induction of antigen-specific cellular immunity were correlated with the hydrophobicity of γ-PGA-Phe NPs. By controlling the hydrophobicity of antigen-encapsulated γ-PGA-Phe NPs, the activation potential of DCs was able to manipulate about 5 to 30-hold than the conventional vaccine, and the cellular immunity was about 10 to 40-hold. These results suggest that the hydrophobicity of NPs is a key factor for changing the interaction between NPs and immune cells, and thus the induction of cellular immunity-biased immune response could be achieved by controlling the hydrophobicity of them. Copyright © 2013 Elsevier Ltd. All rights reserved.

Developmental Regulation of Nucleolus Size during Drosophila Eye Differentiation

PubMed Central

Baker, Nicholas E.

2013-01-01

When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals. PMID:23472166

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

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

Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian

In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of themore » intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.« less

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

DOE PAGES

Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; ...

2014-10-13

In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of themore » intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.« less

Developmental regulation of nucleolus size during Drosophila eye differentiation.

PubMed

Baker, Nicholas E

2013-01-01

When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals.

Creeping, drinking, dying: the cinematic portal and the microscopic world of the twentieth-century cell.

PubMed

Landecker, Hannah

2011-09-01

Film scholars have long posed the question of the specificity of the film medium and the apparatus of cinema, asking what is unique to cinema, how it constrains and enables filmmakers and audiences in particular ways that other media do not. This question has rarely been considered in relation to scientific film, and here it is posed within the specific context of cell biology: What does the use oftime-based media such as film coupled with the microscope allow scientists to experience that other visualization practices do not? Examining three episodes in the twentieth-century study of the cell, this article argues that the apparatus ofmicrocinematography constitutes what might be thought of as a technical portal to another world, a door that determines the experience of the world that lies on the other side of it. In this case, the design of apparatuses to capture time-lapsed images enabled the acceleration of cellular time, bringing it into the realm of human perception and experience. Further, the experience of the cellular temporal world was part of a distinct kind of cell biology, one that was focused on behavior rather than structure, focused on the relation between cells, and between the cell and its milieu rather than on cell-intrinsic features such as chromosomes or organelles. As such, the instruments and technical design of the microcinematographic apparatus may be understood as a kind of materialized epistemology, the history of which can elucidate how cinema was and is used to produce scientific knowledge.

Carcinogenic chemicals in food: evaluating the health risk.

PubMed

Abbott, P J

1992-04-01

The presence of a low level of potentially harmful chemicals in food continues to be a concern to many individuals. A major concern is that these chemicals, which can be synthetic or naturally occurring, may be a causative factor in human cancer. Synthetic chemicals in food may be present either as specific additives or as contaminants derived from environmental or agricultural chemicals. Food also contains a variety of naturally occurring chemicals derived from vegetables or other plants. These may in some cases be considered as contaminants, and are occasionally used as specific additives. New chemicals can also be formed during the cooking or preserving processes. The capacity of any of these chemicals to induce cellular damage and mutation is minimized by natural defence systems such as an efficient cellular detoxification system and DNA repair. The factors influencing tumour formation in humans are numerous and interrelated and exposure to minor dietary chemicals needs to be considered in this context. Thus, the results of animal carcinogenicity assays on individual chemicals need to be interpreted with care, taking into account the mechanisms by which mutagenic and other chemicals initiate cancer, as well as the level of human exposure to these chemicals. Further research is necessary to determine the role, if any, of minor dietary components in tumour formation. Meanwhile, there needs to be a more holistic approach to the multitude of factors, including total diet, that may influence human cancer incidence. In this way, the relative risk of dietary chemicals may be given a more meaningful perspective for health professionals and consumers alike.

The FDA approved PI3K inhibitor GDC-0941 enhances in vitro the anti-neoplastic efficacy of Axitinib against c-myc-amplified high-risk medulloblastoma.

PubMed

Ehrhardt, Michael; Craveiro, Rogerio B; Velz, Julia; Olschewski, Martin; Casati, Anna; Schönberger, Stefan; Pietsch, Torsten; Dilloo, Dagmar

2018-04-01

Aberrant receptor kinase signalling and tumour neovascularization are hallmarks of medulloblastoma development and are both considered valuable therapeutic targets. In addition to VEGFR1/2, expression of PDGFR α/β in particular has been documented as characteristic of metastatic disease correlating with poor prognosis. Therefore, we have been suggested that the clinically approved multi-kinase angiogenesis inhibitor Axitinib, which specifically targets these kinases, might constitute a promising option for medulloblastoma treatment. Indeed, our results delineate anti-neoplastic activity of Axitinib in medulloblastoma cell lines modelling the most aggressive c-myc-amplified Non-WNT/Non-SHH and SHH-TP53-mutated tumours. Exposure of medulloblastoma cell lines to Axitinib results in marked inhibition of proliferation and profound induction of cell death. The differential efficacy of Axitinib is in line with target expression of medulloblastoma cells identifying VEGFR 1/2, PDGFR α/β and c-kit as potential markers for drug application. The high specificity of Axitinib and the consequential low impact on the haematopoietic and immune system render this drug ideal multi-modal treatment approaches. In this context, we demonstrate that the clinically available PI3K inhibitor GDC-0941 enhances the anti-neoplastic efficacy of Axitinib against c-myc-amplified medulloblastoma. Our findings provide a rational to further evaluate Axitinib alone and in combination with other therapeutic agents for the treatment of most aggressive medulloblastoma subtypes. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Intermediate Filaments and the Regulation of Cell Motility during Regeneration and Wound Healing.

PubMed

Cheng, Fang; Eriksson, John E

2017-09-01

SUMMARYIntermediate filaments (IFs) comprise a diverse group of flexible cytoskeletal structures, the assembly, dynamics, and functions of which are regulated by posttranslational modifications. Characteristically, the expression of IF proteins is specific for tissues, differentiation stages, cell types, and functional contexts. Recent research has rapidly expanded the knowledge of IF protein functions. From being regarded as primarily structural proteins, it is now well established that IFs act as powerful modulators of cell motility and migration, playing crucial roles in wound healing and tissue regeneration, as well as inflammatory and immune responses. Although many of these IF-associated functions are essential for tissue repair, the involvement of IF proteins has been established in many additional facets of tissue healing and regeneration. Here, we review the recent progress in understanding the multiple functions of cytoplasmic IFs that relate to cell motility in the context of wound healing, taking examples from studies on keratin, vimentin, and nestin. Wound healing and regeneration include orchestration of a broad range of cellular processes, including regulation of cell attachment and migration, proliferation, differentiation, immune responses, angiogenesis, and remodeling of the extracellular matrix. In this respect, IF proteins now emerge as multifactorial and tissue-specific integrators of tissue regeneration, thereby acting as essential guardian biopolymers at the interface between health and disease, the failing of which contributes to a diverse range of pathologies. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

Visualizing Viral Protein Structures in Cells Using Genetic Probes for Correlated Light and Electron Microscopy

PubMed Central

Ou, Horng D.; Deerinck, Thomas J.; Bushong, Eric; Ellisman, Mark H.; O’Shea, Clodagh C.

2015-01-01

Structural studies of viral proteins most often use high-resolution techniques such as X-ray crystallography, nuclear magnetic resonance, single particle negative stain, or cryo-electron microscopy (EM) to reveal atomic interactions of soluble, homogeneous viral proteins or viral protein complexes. Once viral proteins or complexes are separated from their host’s cellular environment, their natural in-situ structure and details of how they interact with other cellular components may be lost. EM has been an invaluable tool in virology since its introduction in the late 1940’s and subsequent application to cells in the 1950’s. EM studies have expanded our knowledge of viral entry, viral replication, alteration of cellular components, and viral lysis. Most of these early studies were focused on conspicuous morphological cellular changes, because classic EM metal stains were designed to highlight classes of cellular structures rather than specific molecular structures. Much later, to identify viral proteins inducing specific structural configurations at the cellular level, immunostaining with a primary antibody followed by colloidal gold secondary antibody was employed to mark the location of specific viral proteins. This technique can suffer from artifacts in cellular ultrastructure due to compromises required to provide access to the immuno-reagents. Immunolocalization methods also require the generation of highly specific antibodies, which may not be available for every viral protein. Here we discuss new methods to visualize viral proteins and structures at high resolutions in-situ using correlated light and electron microscopy (CLEM). We discuss the use of genetically encoded protein fusions that oxidize diaminobenzidine (DAB) into an osmiophilic polymer that can be visualized by EM. Detailed protocols for applying the genetically encoded photo-oxidizing protein MiniSOG to a viral protein, photo-oxidation of the fusion protein to yield DAB polymer staining, and preparation of photo-oxidized samples for TEM and serial block-face scanning EM (SBEM) for large-scale volume EM data acquisition are also presented. As an example, we discuss the recent multi-scale analysis of Adenoviral protein E4-ORF3 that reveals a new type of multi-functional polymer that disrupts multiple cellular proteins. This new capability to visualize unambiguously specific viral protein structures at high resolutions in the native cellular environment is revealing new insights into how they usurp host proteins and functions to drive pathological viral replication. PMID:26066760

Visualizing viral protein structures in cells using genetic probes for correlated light and electron microscopy.

PubMed

Ou, Horng D; Deerinck, Thomas J; Bushong, Eric; Ellisman, Mark H; O'Shea, Clodagh C

2015-11-15

Structural studies of viral proteins most often use high-resolution techniques such as X-ray crystallography, nuclear magnetic resonance, single particle negative stain, or cryo-electron microscopy (EM) to reveal atomic interactions of soluble, homogeneous viral proteins or viral protein complexes. Once viral proteins or complexes are separated from their host's cellular environment, their natural in situ structure and details of how they interact with other cellular components may be lost. EM has been an invaluable tool in virology since its introduction in the late 1940's and subsequent application to cells in the 1950's. EM studies have expanded our knowledge of viral entry, viral replication, alteration of cellular components, and viral lysis. Most of these early studies were focused on conspicuous morphological cellular changes, because classic EM metal stains were designed to highlight classes of cellular structures rather than specific molecular structures. Much later, to identify viral proteins inducing specific structural configurations at the cellular level, immunostaining with a primary antibody followed by colloidal gold secondary antibody was employed to mark the location of specific viral proteins. This technique can suffer from artifacts in cellular ultrastructure due to compromises required to provide access to the immuno-reagents. Immunolocalization methods also require the generation of highly specific antibodies, which may not be available for every viral protein. Here we discuss new methods to visualize viral proteins and structures at high resolutions in situ using correlated light and electron microscopy (CLEM). We discuss the use of genetically encoded protein fusions that oxidize diaminobenzidine (DAB) into an osmiophilic polymer that can be visualized by EM. Detailed protocols for applying the genetically encoded photo-oxidizing protein MiniSOG to a viral protein, photo-oxidation of the fusion protein to yield DAB polymer staining, and preparation of photo-oxidized samples for TEM and serial block-face scanning EM (SBEM) for large-scale volume EM data acquisition are also presented. As an example, we discuss the recent multi-scale analysis of Adenoviral protein E4-ORF3 that reveals a new type of multi-functional polymer that disrupts multiple cellular proteins. This new capability to visualize unambiguously specific viral protein structures at high resolutions in the native cellular environment is revealing new insights into how they usurp host proteins and functions to drive pathological viral replication. Copyright © 2015 Elsevier Inc. All rights reserved.

Interleukin-6 Reduces β-Cell Oxidative Stress by Linking Autophagy With the Antioxidant Response.

PubMed

Marasco, Michelle R; Conteh, Abass M; Reissaus, Christopher A; Cupit V, John E; Appleman, Evan M; Mirmira, Raghavendra G; Linnemann, Amelia K

2018-05-21

Production of reactive oxygen species (ROS) is a key instigator of β-cell dysfunction in diabetes. The pleiotropic cytokine IL-6 has previously been linked to β-cell autophagy but has not been studied in the context of β-cell antioxidant response. We used a combination of animal models of diabetes and analysis of cultured human islets and rodent β-cells to study how IL-6 influences antioxidant response. We show that IL-6 couples autophagy to antioxidant response to reduce β-cell and human islet ROS. β cell-specific loss of IL-6 signaling in vivo renders mice more susceptible to oxidative damage and cell death by the selective β-cell toxins streptozotocin and alloxan. IL-6-driven ROS reduction is associated with an increase in the master antioxidant factor NRF2, which rapidly translocates to the mitochondria to decrease mitochondrial activity and stimulate mitophagy. IL-6 also initiates a robust transient drop in cellular cAMP, likely contributing to the stimulation of mitophagy for ROS mitigation. Our findings suggest that coupling autophagy to antioxidant response in the β cell leads to stress adaptation that can reduce cellular apoptosis. These findings have implications for β-cell survival under diabetogenic conditions and present novel targets for therapeutic intervention. © 2018 by the American Diabetes Association.

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.

Berberine as a photosensitizing agent for antitumoral photodynamic therapy: Insights into its association to low density lipoproteins.

PubMed

Luiza Andreazza, Nathalia; Vevert-Bizet, Christine; Bourg-Heckly, Geneviève; Sureau, Franck; José Salvador, Marcos; Bonneau, Stephanie

2016-08-20

Recent years have seen a growing interest in Berberine, a phytochemical with multispectrum therapeutic activities, as anti-tumoral agent for photodynamic therapy (PDT). In this context, low density lipoproteins (LDL) play a key role in the delivery of the photosensitizer in tumor cells. We correlate the physicochemical parameters of the berberine association to LDL with the influence of LDL-delivery on its accumulation in a glioma cell line and on its photo-induced activity in view of antitumor PDT. Our results evidence an important binding of 400 berberine molecules per LDL. Changes in berberine and apoprotein fluorescence suggest different fixation types, involving various LDL compartments including the vicinity of the apoprotein. The berberine association to LDL does not affect their recognition by the specific B/E receptors, of which over-expression increases the cellular uptake of LDL-preloaded berberine. Fluorescence microscopy evidences the mitochondrial labeling of the glioma model cells, with no significant modification upon LDL-delivery. Moreover, the cellular delivery of berberine by LDL increases its photocytotoxic effects on such cells. So, this research illustrates the potential of berberine as a photosensitizing agent for PDT, in particular due to their behavior towards LDL as plasma vehicles, and gives insights into its mechanisms of cell uptake. Copyright © 2016. Published by Elsevier B.V.

Diverse activities of viral cis-acting RNA regulatory elements revealed using multicolor, long-term, single-cell imaging

PubMed Central

Pocock, Ginger M.; Zimdars, Laraine L.; Yuan, Ming; Eliceiri, Kevin W.; Ahlquist, Paul; Sherer, Nathan M.

2017-01-01

Cis-acting RNA structural elements govern crucial aspects of viral gene expression. How these structures and other posttranscriptional signals affect RNA trafficking and translation in the context of single cells is poorly understood. Herein we describe a multicolor, long-term (>24 h) imaging strategy for measuring integrated aspects of viral RNA regulatory control in individual cells. We apply this strategy to demonstrate differential mRNA trafficking behaviors governed by RNA elements derived from three retroviruses (HIV-1, murine leukemia virus, and Mason-Pfizer monkey virus), two hepadnaviruses (hepatitis B virus and woodchuck hepatitis virus), and an intron-retaining transcript encoded by the cellular NXF1 gene. Striking behaviors include “burst” RNA nuclear export dynamics regulated by HIV-1’s Rev response element and the viral Rev protein; transient aggregations of RNAs into discrete foci at or near the nuclear membrane triggered by multiple elements; and a novel, pulsiform RNA export activity regulated by the hepadnaviral posttranscriptional regulatory element. We incorporate single-cell tracking and a data-mining algorithm into our approach to obtain RNA element–specific, high-resolution gene expression signatures. Together these imaging assays constitute a tractable, systems-based platform for studying otherwise difficult to access spatiotemporal features of viral and cellular gene regulation. PMID:27903772

The RanGTP Pathway: From Nucleo-Cytoplasmic Transport to Spindle Assembly and Beyond

PubMed Central

Cavazza, Tommaso; Vernos, Isabelle

2016-01-01

The small GTPase Ran regulates the interaction of transport receptors with a number of cellular cargo proteins. The high affinity binding of the GTP-bound form of Ran to import receptors promotes cargo release, whereas its binding to export receptors stabilizes their interaction with the cargo. This basic mechanism linked to the asymmetric distribution of the two nucleotide-bound forms of Ran between the nucleus and the cytoplasm generates a switch like mechanism controlling nucleo-cytoplasmic transport. Since 1999, we have known that after nuclear envelope breakdown (NEBD) Ran and the above transport receptors also provide a local control over the activity of factors driving spindle assembly and regulating other aspects of cell division. The identification and functional characterization of RanGTP mitotic targets is providing novel insights into mechanisms essential for cell division. Here we review our current knowledge on the RanGTP system and its regulation and we focus on the recent advances made through the characterization of its mitotic targets. We then briefly review the novel functions of the pathway that were recently described. Altogether, the RanGTP system has moonlighting functions exerting a spatial control over protein interactions that drive specific functions depending on the cellular context. PMID:26793706

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

Common and distinctive localization patterns of Crumbs polarity complex proteins in the mammalian eye.

PubMed

Kim, Jin Young; Song, Ji Yun; Karnam, Santi; Park, Jun Young; Lee, Jamie J H; Kim, Seonhee; Cho, Seo-Hee

2015-01-01

Crumbs polarity complex proteins are essential for cellular and tissue polarity, and for adhesion of epithelial cells. In epithelial tissues deletion of any of three core proteins disrupts localization of the other proteins, indicating structural and functional interdependence among core components. Despite previous studies of function and co-localization that illustrated the properties that these proteins share, it is not known whether an individual component of the complex plays a distinct role in a unique cellular and developmental context. In order to investigate this question, we primarily used confocal imaging to determine the expression and subcellular localization of the core Crumbs polarity complex proteins during ocular development. Here we show that in developing ocular tissues core Crumbs polarity complex proteins, Crb, Pals1 and Patj, generally appear in an overlapping pattern with some exceptions. All three core complex proteins localize to the apical junction of the retinal and lens epithelia. Pals1 is also localized in the Golgi of the retinal cells and Patj localizes to the nuclei of the apically located subset of progenitor cells. These findings suggest that core Crumbs polarity complex proteins exert common and independent functions depending on cellular context. Copyright © 2015 Elsevier B.V. All rights reserved.

Regulation of the ErbB network by the MIG6 feedback loop in physiology, tumor suppression and responses to oncogene-targeted therapeutics.

PubMed

Anastasi, Sergio; Lamberti, Dante; Alemà, Stefano; Segatto, Oreste

2016-02-01

The ErbB signaling network instructs the execution of key cellular programs, such as cell survival, proliferation and motility, through the generation of robust signals of defined strength and duration. In contrast, unabated ErbB signaling disrupts tissue homeostasis and leads to cell transformation. Cells oppose the threat inherent in excessive ErbB activity through several mechanisms of negative feedback regulation. Inducible feedback inhibitors (IFIs) are expressed in the context of transcriptional responses triggered by ErbB signaling, thus being uniquely suited to regulate ErbB activity during the execution of complex cellular programs. This review focuses on MIG6, an IFI that restrains ErbB signaling by mediating ErbB kinase suppression and receptor down-regulation. We will review key issues in MIG6 function, regulation and tumor suppressor activity. Subsequently, the role for MIG6 loss in the pathogenesis of tumors driven by ErbB oncogenes as well as in the generation of cellular addiction to ErbB signaling will be discussed. We will conclude by analyzing feedback inhibition by MIG6 in the context of therapies directed against ErbB and non-ErbB oncogenes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mechanisms of stress-induced cellular HSP72 release: implications for exercise-induced increases in extracellular HSP72.

PubMed

Lancaster, Graeme I; Febbraio, Mark A

2005-01-01

The heat shock proteins are a family of highly conserved proteins with critical roles in maintaining cellular homeostasis and in protecting the cell from stressful conditions. While the critical intracellular roles of heat shock proteins are undisputed, evidence suggests that the cell possess the necessary machinery to actively secrete specific heat shock proteins in response to cellular stress. In this review, we firstly discuss the evidence that physical exercise induces the release of heat shock protein 72 from specific tissues in humans. Importantly, it appears as though this release is the result of an active secretory process, as opposed to non-specific processes such as cell lysis. Next we discuss recent in vitro evidence that has identified a mechanistic basis for the observation that cellular stress induces the release of a specific subset of heat shock proteins. Importantly, while the classical protein secretory pathway does not seem to be involved in the stress-induced release of HSP72, we discuss the evidence that lipid-rafts and exosomes are important mediators of the stress-induced release of HSP72.

Inhibition of microtubules and dynein rescues human immunodeficiency virus type 1 from owl monkey TRIMCyp-mediated restriction in a cellular context-specific fashion.

PubMed

Pawlica, Paulina; Dufour, Caroline; Berthoux, Lionel

2015-04-01

IFN-induced restriction factors can significantly affect the replicative capacity of retroviruses in mammals. TRIM5α (tripartite motif protein 5, isoform α) is a restriction factor that acts at early stages of the virus life cycle by intercepting and destabilizing incoming retroviral cores. Sensitivity to TRIM5α maps to the N-terminal domain of the retroviral capsid proteins. In several New World and Old World monkey species, independent events of retrotransposon-mediated insertion of the cyclophilin A (CypA)-coding sequence in the trim5 gene have given rise to TRIMCyp (also called TRIM5-CypA), a hybrid protein that is active against some lentiviruses in a species-specific fashion. In particular, TRIMCyp from the owl monkey (omkTRIMCyp) very efficiently inhibits human immunodeficiency virus type 1 (HIV-1). Previously, we showed that disrupting the integrity of microtubules (MTs) and of cytoplasmic dynein complexes partially rescued replication of retroviruses, including HIV-1, from restriction mediated by TRIM5α. Here, we showed that efficient restriction of HIV-1 by omkTRIMCyp was similarly dependent on the MT network and on dynein complexes, but in a context-dependent fashion. When omkTRIMCyp was expressed in human HeLa cells, restriction was partially counteracted by pharmacological agents targeting MTs or by small interfering RNA-mediated inhibition of dynein. The same drugs (nocodazole and paclitaxel) also rescued HIV-1 from restriction in cat CRFK cells, although to a lesser extent. Strikingly, neither nocodazole, paclitaxel nor depletion of the dynein heavy chain had a significant effect on the restriction of HIV-1 in an owl monkey cell line. These results suggested the existence of cell-specific functional interactions between MTs/dynein and TRIMCyp. © 2015 The Authors.

Ultra-Sensitive Droplet Digital PCR for the Assessment of Microchimerism in Cellular Therapies.

PubMed

Kliman, David; Castellano-Gonzalez, Gloria; Withers, Barbara; Street, Janine; Tegg, Elizabeth; Mirochnik, Oksana; Lai, Joey; Clancy, Leighton; Gottlieb, David; Blyth, Emily

2018-05-01

Current techniques to assess chimerism after hematopoietic stem cell transplantation (HSCT) are limited in both sensitivity and precision. These drawbacks are problematic in the context of cellular therapies that frequently result in microchimerism (donor chimerism <1%). We have developed a highly sensitive droplet digital PCR (ddPCR) assay using commercially available regents with good performance throughout the range of clinically relevant chimerism measurements, including microchimerism. We tested the assay using spiked samples of known donor-recipient ratios and in clinical samples from HSCT recipients and patients enrolled on clinical trials of microtransplantation and third-party virus-specific T cells (VSTs). The levels of detection and quantification of the assay were .008% and .023%, with high levels of precision with samples of DNA content ranging from 1 to 300 ng DNA. From the panel of 29 insertion-deletion probes multiple informative markers were found for each of 43 HSCT donor-recipient pairs. In the case of third-party cellular therapies in which there were 3 DNA contributors (recipient, HSCT donor, and T-cell donor), a marker to detect the cellular product in a background of recipient and donor cells was available for 11 of 12 cases (92%). Chimerism by ddPCR was able to quantify chimerism in HSCT recipients and comparison against standard STR analysis in 8 HSCT patients demonstrated similar results, with the advantage of fast turnaround time. Persistence of donor microchimerism in patients undergoing microtransplantation for acute myeloid leukemia was detectable for up to 57 days in peripheral blood and bone marrow. The presence of microtransplant product DNA in bone marrow T cells after cell sorting was seen in the 1 patient tested. In patients receiving third-party VSTs for treatment of refractory viral infections, VST donor DNA was detected at low levels in 7 of 9 cases. ddPCR offers advantages over currently available methods for assessment of chimerism in standard HSCT and cellular therapies. Copyright © 2018 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Cellular reprogramming through mitogen-activated protein kinases.

PubMed

Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

2015-01-01

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression-including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

Crk1/2 and CrkL form a hetero-oligomer and functionally complement each other during podocyte morphogenesis

PubMed Central

Zhang, Jidong; Verma, Rakesh; Park, Tae-Ju; Wong, Hetty; Curran, Tom; Nihalani, Deepak; Holzman, Lawrence B.

2014-01-01

Activation of the slit diaphragm protein Nephrin induces actin cytoskeletal remodeling resulting in lamellipodia formation in podocytes in vitro in a phosphatidylinositol-3 kinase, focal adhesion kinase, Cas, and Crk1/2-dependent fashion. In mice, podocyte-specific deletion of Crk1/2 prevents or attenuates foot process effacement in two models of podocyte injury. This suggests that cellular mechanisms governing lamellipodial protrusion in vitro are similar to those in vivo during foot process effacement. Since Crk1/2 null mice develop and aged normally, we tested whether the Crk1/2 paralog, CrkL, functionally complements Crk1/2 in a podocyte-specific context. Podocyte-specific CrkL null mice, like podocyte-specific Crk1/2 null mice, developed and aged normally but were protected from protamine sulfate-induced foot process effacement. Simultaneous podocyte-specific deletion of Crk1/2 and CrkL resulted in albuminuria detected by six weeks post-partum and associated with altered podocyte process architecture. Nephrin-induced lamellipodia formation in podocytes in vitro was CrkL-dependent. CrkL formed a heterooligomer with Crk2 and, like Crk2, was recruited to tyrosine phosphorylated Nephrin. Thus, Crk1/2 and CrkL are physically-linked, functionally complement each other during podocyte foot process spreading, and together are required for developing typical foot process architecture. PMID:24499776

ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

PubMed

Karayazi Atici, Ödül; Urbanska, Anna; Gopinathan, Sesha Gopal; Boutillon, Florence; Goffin, Vincent; Shemanko, Carrie S

2018-02-01

Prolactin (PRL) acts as a survival factor for breast cancer cells, but the PRL signaling pathway and the mechanism are unknown. Previously, we identified the master chaperone, heat shock protein 90 (HSP90) α, as a prolactin-Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) target gene involved in survival, and here we investigated the role of HSP90 in the mechanism of PRL-induced viability in response to DNA damage. The ataxia-telangiectasia mutated kinase (ATM) protein plays a critical role in the cellular response to double-strand DNA damage. We observed that PRL increased viability of breast cancer cells treated with doxorubicin or etoposide. The increase in cellular resistance is specific to the PRL receptor, because the PRL receptor antagonist, Δ1-9-G129R-hPRL, prevented the increase in viability. Two different HSP90 inhibitors, 17-allylamino-17-demethoxygeldanamycin and BIIB021, reduced the PRL-mediated increase in cell viability of doxorubicin-treated cells and led to a decrease in JAK2, ATM, and phosphorylated ATM protein levels. Inhibitors of JAK2 (G6) and ATM (KU55933) abolished the PRL-mediated increase in cell viability of DNA-damaged cells, supporting the involvement of each, as well as the crosstalk of ATM with the PRL pathway in the context of DNA damage. Drug synergism was detected between the ATM inhibitor (KU55933) and doxorubicin and between the HSP90 inhibitor (BIIB021) and doxorubicin. Short interfering RNA directed against ATM prevented the PRL-mediated increase in cell survival in two-dimensional cell culture, three-dimensional collagen gel cultures, and clonogenic cell survival, after doxorubicin treatment. Our results indicate that ATM contributes to the PRL-JAK2-STAT5-HSP90 pathway in mediating cellular resistance to DNA-damaging agents. Copyright © 2018 Endocrine Society.

Resource Allocation Algorithms for the Next Generation Cellular Networks

NASA Astrophysics Data System (ADS)

Amzallag, David; Raz, Danny

This chapter describes recent results addressing resource allocation problems in the context of current and future cellular technologies. We present models that capture several fundamental aspects of planning and operating these networks, and develop new approximation algorithms providing provable good solutions for the corresponding optimization problems. We mainly focus on two families of problems: cell planning and cell selection. Cell planning deals with choosing a network of base stations that can provide the required coverage of the service area with respect to the traffic requirements, available capacities, interference, and the desired QoS. Cell selection is the process of determining the cell(s) that provide service to each mobile station. Optimizing these processes is an important step towards maximizing the utilization of current and future cellular networks.

An ontology for major histocompatibility restriction.

PubMed

Vita, Randi; Overton, James A; Seymour, Emily; Sidney, John; Kaufman, Jim; Tallmadge, Rebecca L; Ellis, Shirley; Hammond, John; Butcher, Geoff W; Sette, Alessandro; Peters, Bjoern

2016-01-01

MHC molecules are a highly diverse family of proteins that play a key role in cellular immune recognition. Over time, different techniques and terminologies have been developed to identify the specific type(s) of MHC molecule involved in a specific immune recognition context. No consistent nomenclature exists across different vertebrate species. To correctly represent MHC related data in The Immune Epitope Database (IEDB), we built upon a previously established MHC ontology and created an ontology to represent MHC molecules as they relate to immunological experiments. This ontology models MHC protein chains from 16 species, deals with different approaches used to identify MHC, such as direct sequencing verses serotyping, relates engineered MHC molecules to naturally occurring ones, connects genetic loci, alleles, protein chains and multi-chain proteins, and establishes evidence codes for MHC restriction. Where available, this work is based on existing ontologies from the OBO foundry. Overall, representing MHC molecules provides a challenging and practically important test case for ontology building, and could serve as an example of how to integrate other ontology building efforts into web resources.

MicroRNA Intercellular Transfer and Bioelectrical Regulation of Model Multicellular Ensembles by the Gap Junction Connectivity.

PubMed

Cervera, Javier; Meseguer, Salvador; Mafe, Salvador

2017-08-17

We have studied theoretically the microRNA (miRNA) intercellular transfer through voltage-gated gap junctions in terms of a biophysically grounded system of coupled differential equations. Instead of modeling a specific system, we use a general approach describing the interplay between the genetic mechanisms and the single-cell electric potentials. The dynamics of the multicellular ensemble are simulated under different conditions including spatially inhomogeneous transcription rates and local intercellular transfer of miRNAs. These processes result in spatiotemporal changes of miRNA, mRNA, and ion channel protein concentrations that eventually modify the bioelectrical states of small multicellular domains because of the ensemble average nature of the electrical potential. The simulations allow a qualitative understanding of the context-dependent nature of the effects observed when specific signaling molecules are transferred through gap junctions. The results suggest that an efficient miRNA intercellular transfer could permit the spatiotemporal control of small cellular domains by the conversion of single-cell genetic and bioelectric states into multicellular states regulated by the gap junction interconnectivity.

Physiological and Transcriptional Responses of Saccharomyces cerevisiae to Zinc Limitation in Chemostat Cultures †

PubMed Central

De Nicola, Raffaele; Hazelwood, Lucie A.; De Hulster, Erik A. F.; Walsh, Michael C.; Knijnenburg, Theo A.; Reinders, Marcel J. T.; Walker, Graeme M.; Pronk, Jack T.; Daran, Jean-Marc; Daran-Lapujade, Pascale

2007-01-01

Transcriptional responses of the yeast Saccharomyces cerevisiae to Zn availability were investigated at a fixed specific growth rate under limiting and abundant Zn concentrations in chemostat culture. To investigate the context dependency of this transcriptional response and eliminate growth rate-dependent variations in transcription, yeast was grown under several chemostat regimens, resulting in various carbon (glucose), nitrogen (ammonium), zinc, and oxygen supplies. A robust set of genes that responded consistently to Zn limitation was identified, and the set enabled the definition of the Zn-specific Zap1p regulon, comprised of 26 genes and characterized by a broader zinc-responsive element consensus (MHHAACCBYNMRGGT) than so far described. Most surprising was the Zn-dependent regulation of genes involved in storage carbohydrate metabolism. Their concerted down-regulation was physiologically relevant as revealed by a substantial decrease in glycogen and trehalose cellular content under Zn limitation. An unexpectedly large number of genes were synergistically or antagonistically regulated by oxygen and Zn availability. This combinatorial regulation suggested a more prominent involvement of Zn in mitochondrial biogenesis and function than hitherto identified. PMID:17933919

Modulation of PPAR activity via phosphorylation

PubMed Central

Burns, Katherine A.; Vanden Heuvel, John P.

2009-01-01

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily of transcription factors that respond to specific ligands by altering gene expression in a cell-, developmental- and sex-specific manner. Three subtypes of this receptor have been discovered (PPARα, β and γ), each apparently evolving to fulfill different biological niches. PPARs control a variety of target genes involved in lipid homeostasis, diabetes and cancer. Similar to other nuclear receptors, the PPARs are phosphoproteins and their transcriptional activity is affected by cross-talk with kinases and phosphatases. Phosphorylation by the mitogen-activated protein kinases (ERK- and p38-MAPK), Protein Kinase A and C (PKA, PKC), AMP Kinase (AMPK) and glycogen synthase kinase-3 (GSK3) affect their activity in a ligand-dependent or -independent manner. The effects of phosphorylation depend on the cellular context, receptor subtype and residue metabolized which can be manifested at several steps in the PPAR activation sequence including ligand affinity, DNA binding, coactivator recruitment and proteasomal degradation. The review will summarize the known PPAR kinases that directly act on these receptors, the sites affected and the result of this modification on receptor activity. PMID:17560826

Epidermal growth factor receptor and variant III targeted immunotherapy.

PubMed

Congdon, Kendra L; Gedeon, Patrick C; Suryadevara, Carter M; Caruso, Hillary G; Cooper, Laurence J N; Heimberger, Amy B; Sampson, John H

2014-10-01

Immunotherapeutic approaches to cancer have shown remarkable promise. A critical barrier to successfully executing such immune-mediated interventions is the selection of safe yet immunogenic targets. As patient deaths have occurred when tumor-associated antigens shared by normal tissue have been targeted by strong cellular immunotherapeutic platforms, route of delivery, target selection and the immune-mediated approach undertaken must work together to maximize efficacy with safety. Selected tumor-specific targets can spare potential toxicity to normal tissue; however, they are far less common than tumor-associated antigens and may not be present on all patients. In the context of immunotherapy for high-grade glioma, 2 of the most prominently studied antigens are the tumor-associated epidermal growth factor receptor and its tumor-specific genetic deletion variant III. In this review, we will summarize the immune-mediated strategies employed against these targets as well as the caveats particular to these approaches. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Systems Biology Approach to the Dissection of the Complexity of Regulatory Networks in the S. scrofa Cardiocirculatory System

PubMed Central

Martini, Paolo; Sales, Gabriele; Calura, Enrica; Brugiolo, Mattia; Lanfranchi, Gerolamo; Romualdi, Chiara; Cagnin, Stefano

2013-01-01

Genome-wide experiments are routinely used to increase the understanding of the biological processes involved in the development and maintenance of a variety of pathologies. Although the technical feasibility of this type of experiment has improved in recent years, data analysis remains challenging. In this context, gene set analysis has emerged as a fundamental tool for the interpretation of the results. Here, we review strategies used in the gene set approach, and using datasets for the pig cardiocirculatory system as a case study, we demonstrate how the use of a combination of these strategies can enhance the interpretation of results. Gene set analyses are able to distinguish vessels from the heart and arteries from veins in a manner that is consistent with the different cellular composition of smooth muscle cells. By integrating microRNA elements in the regulatory circuits identified, we find that vessel specificity is maintained through specific miRNAs, such as miR-133a and miR-143, which show anti-correlated expression with their mRNA targets. PMID:24284405

FRET-based genetically-encoded sensors for quantitative monitoring of metabolites.

PubMed

Mohsin, Mohd; Ahmad, Altaf; Iqbal, Muhammad

2015-10-01

Neighboring cells in the same tissue can exist in different states of dynamic activities. After genomics, proteomics and metabolomics, fluxomics is now equally important for generating accurate quantitative information on the cellular and sub-cellular dynamics of ions and metabolite, which is critical for functional understanding of organisms. Various spectrometry techniques are used for monitoring ions and metabolites, although their temporal and spatial resolutions are limited. Discovery of the fluorescent proteins and their variants has revolutionized cell biology. Therefore, novel tools and methods targeting sub-cellular compartments need to be deployed in specific cells and targeted to sub-cellular compartments in order to quantify the target-molecule dynamics directly. We require tools that can measure cellular activities and protein dynamics with sub-cellular resolution. Biosensors based on fluorescence resonance energy transfer (FRET) are genetically encoded and hence can specifically target sub-cellular organelles by fusion to proteins or targetted sequences. Since last decade, FRET-based genetically encoded sensors for molecules involved in energy production, reactive oxygen species and secondary messengers have helped to unravel key aspects of cellular physiology. This review, describing the design and principles of sensors, presents a database of sensors for different analytes/processes, and illustrate examples of application in quantitative live cell imaging.

Chimeric antigen receptor engineering: a right step in the evolution of adoptive cellular immunotherapy.

PubMed

Figueroa, Jose A; Reidy, Adair; Mirandola, Leonardo; Trotter, Kayley; Suvorava, Natallia; Figueroa, Alejandro; Konala, Venu; Aulakh, Amardeep; Littlefield, Lauren; Grizzi, Fabio; Rahman, Rakhshanda Layeequr; Jenkins, Marjorie R; Musgrove, Breeanna; Radhi, Saba; D'Cunha, Nicholas; D'Cunha, Luke N; Hermonat, Paul L; Cobos, Everardo; Chiriva-Internati, Maurizio

2015-03-01

Cancer immunotherapy comprises different therapeutic strategies that exploit the use of distinct components of the immune system, with the common goal of specifically targeting and eradicating neoplastic cells. These varied approaches include the use of specific monoclonal antibodies, checkpoint inhibitors, cytokines, therapeutic cancer vaccines and cellular anticancer strategies such as activated dendritic cell (DC) vaccines, tumor-infiltrating lymphocytes (TILs) and, more recently, genetically engineered T cells. Each one of these approaches has demonstrated promise, but their generalized success has been hindered by the paucity of specific tumor targets resulting in suboptimal tumor responses and unpredictable toxicities. This review will concentrate on recent advances on the use of engineered T cells for adoptive cellular immunotherapy (ACI) in cancer.

A grid matrix-based Raman spectroscopic method to characterize different cell milieu in biopsied axillary sentinel lymph nodes of breast cancer patients.

PubMed

Som, Dipasree; Tak, Megha; Setia, Mohit; Patil, Asawari; Sengupta, Amit; Chilakapati, C Murali Krishna; Srivastava, Anurag; Parmar, Vani; Nair, Nita; Sarin, Rajiv; Badwe, R

2016-01-01

Raman spectroscopy which is based upon inelastic scattering of photons has a potential to emerge as a noninvasive bedside in vivo or ex vivo molecular diagnostic tool. There is a need to improve the sensitivity and predictability of Raman spectroscopy. We developed a grid matrix-based tissue mapping protocol to acquire cellular-specific spectra that also involved digital microscopy for localizing malignant and lymphocytic cells in sentinel lymph node biopsy sample. Biosignals acquired from specific cellular milieu were subjected to an advanced supervised analytical method, i.e., cross-correlation and peak-to-peak ratio in addition to PCA and PC-LDA. We observed decreased spectral intensity as well as shift in the spectral peaks of amides and lipid bands in the completely metastatic (cancer cells) lymph nodes with high cellular density. Spectral library of normal lymphocytes and metastatic cancer cells created using the cellular specific mapping technique can be utilized to create an automated smart diagnostic tool for bench side screening of sampled lymph nodes. Spectral library of normal lymphocytes and metastatic cancer cells created using the cellular specific mapping technique can be utilized to develop an automated smart diagnostic tool for bench side screening of sampled lymph nodes supported by ongoing global research in developing better technology and signal and big data processing algorithms.

Mitochondrion

ERIC Educational Resources Information Center

Goodsell, David S.

2010-01-01

Diverse biological data may be used to create illustrations of molecules in their cellular context. I describe the scientific results that support a recent textbook illustration of a mitochondrion. The image magnifies a portion of the mitochondrion by one million times, showing the location and form of membranes and individual macromolecules,…

RNA Imaging with Multiplexed Error Robust Fluorescence in situ Hybridization

PubMed Central

Moffitt, Jeffrey R.; Zhuang, Xiaowei

2016-01-01

Quantitative measurements of both the copy number and spatial distribution of large fractions of the transcriptome in single-cells could revolutionize our understanding of a variety of cellular and tissue behaviors in both healthy and diseased states. Single-molecule Fluorescence In Situ Hybridization (smFISH)—an approach where individual RNAs are labeled with fluorescent probes and imaged in their native cellular and tissue context—provides both the copy number and spatial context of RNAs but has been limited in the number of RNA species that can be measured simultaneously. Here we describe Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH), a massively parallelized form of smFISH that can image and identify hundreds to thousands of different RNA species simultaneously with high accuracy in individual cells in their native spatial context. We provide detailed protocols on all aspects of MERFISH, including probe design, data collection, and data analysis to allow interested laboratories to perform MERFISH measurements themselves. PMID:27241748

E3Net: a system for exploring E3-mediated regulatory networks of cellular functions.

PubMed

Han, Youngwoong; Lee, Hodong; Park, Jong C; Yi, Gwan-Su

2012-04-01

Ubiquitin-protein ligase (E3) is a key enzyme targeting specific substrates in diverse cellular processes for ubiquitination and degradation. The existing findings of substrate specificity of E3 are, however, scattered over a number of resources, making it difficult to study them together with an integrative view. Here we present E3Net, a web-based system that provides a comprehensive collection of available E3-substrate specificities and a systematic framework for the analysis of E3-mediated regulatory networks of diverse cellular functions. Currently, E3Net contains 2201 E3s and 4896 substrates in 427 organisms and 1671 E3-substrate specific relations between 493 E3s and 1277 substrates in 42 organisms, extracted mainly from MEDLINE abstracts and UniProt comments with an automatic text mining method and additional manual inspection and partly from high throughput experiment data and public ubiquitination databases. The significant functions and pathways of the extracted E3-specific substrate groups were identified from a functional enrichment analysis with 12 functional category resources for molecular functions, protein families, protein complexes, pathways, cellular processes, cellular localization, and diseases. E3Net includes interactive analysis and navigation tools that make it possible to build an integrative view of E3-substrate networks and their correlated functions with graphical illustrations and summarized descriptions. As a result, E3Net provides a comprehensive resource of E3s, substrates, and their functional implications summarized from the regulatory network structures of E3-specific substrate groups and their correlated functions. This resource will facilitate further in-depth investigation of ubiquitination-dependent regulatory mechanisms. E3Net is freely available online at http://pnet.kaist.ac.kr/e3net.

Techniques for the Cellular and Subcellular Localization of Endocannabinoid Receptors and Enzymes in the Mammalian Brain.

PubMed

Cristino, Luigia; Imperatore, Roberta; Di Marzo, Vincenzo

2017-01-01

This chapter attempts to piece together knowledge about new advanced microscopy techniques to study the neuroanatomical distribution of endocannabinoid receptors and enzymes at the level of cellular and subcellular structures and organelles in the brain. Techniques ranging from light to electron microscopy up to the new advanced LBM, PALM, and STORM super-resolution microscopy will be discussed in the context of their contribution to define the spatial distribution and organization of receptors and enzymes of the endocannabinoid system (ECS), and to better understand ECS brain functions. © 2017 Elsevier Inc. All rights reserved.

Molecular deconstruction, detection, and computational prediction of microenvironment-modulated cellular responses to cancer therapeutics.

PubMed

Labarge, Mark A; Parvin, Bahram; Lorens, James B

2014-04-01

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 have revealed a rich interplay between the genome and extracellular effectors in determining cellular phenotypes and in a number of cases have 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. Copyright © 2014 Elsevier B.V. All rights reserved.

The protein expression landscape of the Arabidopsis root

PubMed Central

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

2012-01-01

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

Social impact evaluation : Some implications of the specific decisional context approach for anticipatory project assessment with special reference to available alternatives and to techniques of evaluating the social impacts of the anticipated effects of such alternatives

NASA Technical Reports Server (NTRS)

Mayo, L. H.

1972-01-01

The implications are explored of the specific decision context approach to anticipatory project assessment. More specifically, it is hypothesized that with respect to any given effect of a proposed project or action (mobility, job opportunities, air pollution, population distribution, etc.) such effect will likely differ in probability and/or magnitude from one decisional context to another; that the social desirability or undesirability of a given effect is a function (will differ with) each specific decisional context; that therefore the social impact of such effect will in all likelihood differ with each specific decisional context; and that the social significance of even the dame social impact of a given effect will vary from one decisional context to another when such social impact interacts with (competes with or reinforces) the social impacts of other effects. It also follows from this analysis that the respective roles of scientific method (demonstrable data) and adversarial system will not only differ with each specific decisional context but with each alternative course of action available to the decisional entity in each specific context.

The fate of chemoresistance in triple negative breast cancer (TNBC)

PubMed Central

O’Reilly, Elma A.; Gubbins, Luke; Sharma, Shiva; Tully, Riona; Guang, Matthew Ho Zhing; Weiner-Gorzel, Karolina; McCaffrey, John; Harrison, Michele; Furlong, Fiona; Kell, Malcolm; McCann, Amanda

2015-01-01

Background Treatment options for women presenting with triple negative breast cancer (TNBC) are limited due to the lack of a therapeutic target and as a result, are managed with standard chemotherapy such as paclitaxel (Taxol®). Following chemotherapy, the ideal tumour response is apoptotic cell death. Post-chemotherapy, cells can maintain viability by undergoing viable cellular responses such as cellular senescence, generating secretomes which can directly enhance the malignant phenotype. Scope of Review How tumour cells retain viability in response to chemotherapeutic engagement is discussed. In addition we discuss the implications of this retained tumour cell viability in the context of the development of recurrent and metastatic TNBC disease. Current adjuvant and neo-adjuvant treatments available and the novel potential therapies that are being researched are also reviewed. Major conclusions Cellular senescence and cytoprotective autophagy are potential mechanisms of chemoresistance in TNBC. These two non-apoptotic outcomes in response to chemotherapy are inextricably linked and are neglected outcomes of investigation in the chemotherapeutic arena. Cellular fate assessments may therefore have the potential to predict TNBC patient outcome. General Significance Focusing on the fact that cancer cells can bypass the desired cellular apoptotic response to chemotherapy through cellular senescence and cytoprotective autophagy will highlight the importance of targeting non-apoptotic survival pathways to enhance chemotherapeutic efficacy. PMID:26676166

The development and application of a quantitative peptide microarray platform to SH2 domain specificity space

NASA Astrophysics Data System (ADS)

Engelmann, Brett Warren

The Src homology 2 (SH2) domains evolved alongside protein tyrosine kinases (PTKs) and phosphatases (PTPs) in metazoans to recognize the phosphotyrosine (pY) post-translational modification. The human genome encodes 121 SH2 domains within 111 SH2 domain containing proteins that represent the primary mechanism for cellular signal transduction immediately downstream of PTKs. Despite pY recognition contributing to roughly half of the binding energy, SH2 domains possess substantial binding specificity, or affinity discrimination between phosphopeptide ligands. This specificity is largely imparted by amino acids (AAs) adjacent to the pY, typically from positions +1 to +4 C-terminal to the pY. Much experimental effort has been undertaken to construct preferred binding motifs for many SH2 domains. However, due to limitations in previous experimental methodologies these motifs do not account for the interplay between AAs. It was therefore not known how AAs within the context of individual peptides function to impart SH2 domain specificity. In this work we identified the critical role context plays in defining SH2 domain specificity for physiological ligands. We also constructed a high quality interactome using 50 SH2 domains and 192 physiological ligands. We next developed a quantitative high-throughput (Q-HTP) peptide microarray platform to assess the affinities four SH2 domains have for 124 physiological ligands. We demonstrated the superior characteristics of our platform relative to preceding approaches and validated our results using established biophysical techniques, literature corroboration, and predictive algorithms. The quantitative information provided by the arrays was leveraged to investigate SH2 domain binding distributions and identify points of binding overlap. Our microarray derived affinity estimates were integrated to produce quantitative interaction motifs capable of predicting interactions. Furthermore, our microarrays proved capable of resolving subtle contextual differences within motifs that modulate interaction affinities. We conclude that contextually informed specificity profiling of protein interaction domains using the methodologies developed in this study can inform efforts to understand the interconnectivity of signaling networks in normal and aberrant states. Three supplementary tables containing detailed lists of peptides, interactions, and sources of corroborative information are provided.

Crosstalk and the evolvability of intracellular communication.

PubMed

Rowland, Michael A; Greenbaum, Joseph M; Deeds, Eric J

2017-07-10

Metazoan signalling networks are complex, with extensive crosstalk between pathways. It is unclear what pressures drove the evolution of this architecture. We explore the hypothesis that crosstalk allows different cell types, each expressing a specific subset of signalling proteins, to activate different outputs when faced with the same inputs, responding differently to the same environment. We find that the pressure to generate diversity leads to the evolution of networks with extensive crosstalk. Using available data, we find that human tissues exhibit higher levels of diversity between cell types than networks with random expression patterns or networks with no crosstalk. We also find that crosstalk and differential expression can influence drug activity: no protein has the same impact on two tissues when inhibited. In addition to providing a possible explanation for the evolution of crosstalk, our work indicates that consideration of cellular context will likely be crucial for targeting signalling networks.

Tie2 and Eph Receptor Tyrosine Kinase Activation and Signaling

PubMed Central

Barton, William A.; Dalton, Annamarie C.; Seegar, Tom C.M.; Himanen, Juha P.

2014-01-01

The Eph and Tie cell surface receptors mediate a variety of signaling events during development and in the adult organism. As other receptor tyrosine kinases, they are activated on binding of extracellular ligands and their catalytic activity is tightly regulated on multiple levels. The Eph and Tie receptors display some unique characteristics, including the requirement of ligand-induced receptor clustering for efficient signaling. Interestingly, both Ephs and Ties can mediate different, even opposite, biological effects depending on the specific ligand eliciting the response and on the cellular context. Here we discuss the structural features of these receptors, their interactions with various ligands, as well as functional implications for downstream signaling initiation. The Eph/ephrin structures are already well reviewed and we only provide a brief overview on the initial binding events. We go into more detail discussing the Tie-angiopoietin structures and recognition. PMID:24478383

Endo-β-Glucosidase Tag Allows Dual Detection of Fusion Proteins by Fluorescent Mechanism-Based Probes and Activity Measurement.

PubMed

Kallemeijn, Wouter W; Scheij, Saskia; Voorn-Brouwer, Tineke M; Witte, Martin D; Verhoek, Marri; Overkleeft, Hermen S; Boot, Rolf G; Aerts, Johannes M F G

2016-09-15

β-Glucoside-configured cyclophellitols are activity-based probes (ABPs) that allow sensitive detection of β-glucosidases. Their applicability to detect proteins fused with β-glucosidase was investigated in the cellular context. The tag was Rhodococcus sp. M-777 endoglycoceramidase II (EGCaseII), based on its lack of glycans and ability to hydrolyze fluorogenic 4-methylumbelliferyl β-d-lactoside (an activity absent in mammalian cells). Specific dual detection of fusion proteins was possible in vitro and in situ by using fluorescent ABPs and a fluorogenic substrate. Pre-blocking with conduritol β-epoxide (a poor inhibitor of EGCaseII) eliminated ABP labeling of endogenous β-glucosidases. ABPs equipped with biotin allowed convenient purification of the fusion proteins. Diversification of ABPs (distinct fluorophores, fluorogenic high-resolution detection moieties) should assist further research in living cells and organisms. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chimeric Antigen Receptors T Cell Therapy in Solid Tumor: Challenges and Clinical Applications.

PubMed

Mirzaei, Hamid R; Rodriguez, Analiz; Shepphird, Jennifer; Brown, Christine E; Badie, Behnam

2017-01-01

Adoptive cellular immunotherapy (ACT) employing engineered T lymphocytes expressing chimeric antigen receptors (CARs) has demonstrated promising antitumor effects in advanced hematologic cancers, such as relapsed or refractory acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma, supporting the translation of ACT to non-hematological malignancies. Although CAR T cell therapy has made remarkable strides in the treatment of patients with certain hematological cancers, in solid tumors success has been limited likely due to heterogeneous antigen expression, immunosuppressive networks in the tumor microenvironment limiting CAR T cell function and persistence, and suboptimal trafficking to solid tumors. Here, we outline specific approaches to overcome barriers to CAR T cell effectiveness in the context of the tumor microenvironment and offer our perspective on how expanding the use of CAR T cells in solid tumors may require modifications in CAR T cell design. We anticipate these modifications will further expand CAR T cell therapy in clinical practice.

[Role of long-term potentiation in mechanism of the conditioned learning].

PubMed

Tsvetkov, E A; Suderevskaia, E I; Veselkin, N P

2011-01-01

The review analyzes the fundamental problem of study of the neuronal mechanisms underlying processes of learning and memory. As a neuronal models of these phenomena there was considered one of the cellular phenomena that has characteristics similar with those in the process of "remembering"--such as the long-term potentiation (LTP). LTP is easily reproduced in certain synapses of the central nervous system, specifically in synapses of hippocampus and amygdala. As to the behavioral model of learning, there was used the conditioned learning, in frames of which production of the context-dependent/independent conditioned reaction was considered. Analysis of literature data has allowed showing that various stages of LTP produced on synapses of hippocampus or amygdala can be comparable with certain phase of the process of learning. Based on the exposed material the authors conclude that plastic changes of synapses of hippocampus and amygdala can represent the morphological substrate of some kinds of learning and memory.

The macro- and microcirculation of the kidney.

PubMed

Guerci, Philippe; Ergin, Bulent; Ince, Can

2017-09-01

Acute kidney injury (AKI) remains one of the main causes of morbidity and mortality in the intensive care medicine today. Its pathophysiology and progress to chronic kidney disease is still under investigation. In addition, the lack of techniques to adequately monitor renal function and microcirculation at the bedside makes its therapeutic resolution challenging. In this article, we review current concepts related to renal hemodynamics compromise as being the event underlying AKI. In doing so, we discuss the physiology of the renal circulation and the effects of alterations in systemic hemodynamics that lead to renal injury specifically in the context of reperfusion injury and sepsis. The ultimate key culprit of AKI leading to failure is the dysfunction of the renal microcirculation. The cellular and subcellular components of the renal microcirculation are discussed and how their injury contributes to AKI is described. Copyright © 2017. Published by Elsevier Ltd.

The Brainstem and Serotonin in the Sudden Infant Death Syndrome

PubMed Central

Kinney, Hannah C.; Richerson, George B.; Dymecki, Susan M.; Darnall, Robert A.; Nattie, Eugene E.

2012-01-01

The sudden infant death syndrome (SIDS) is the sudden death of an infant under one year of age that is typically associated with sleep and that remains unexplained after a complete autopsy and death scene investigation. A leading hypothesis about its pathogenesis is that many cases result from defects in brainstem-mediated protective responses to homeostatic stressors occurring during sleep in a critical developmental period. Here we review the evidence for the brainstem hypothesis in SIDS with a focus upon abnormalities related to the neurotransmitter serotonin in the medulla oblongata, as these are the most robust pathologic findings to date. In this context, we synthesize the human autopsy data with genetic, whole-animal, and cellular data concerning the function and development of the medullary serotonergic system. These emerging data suggest an important underlying mechanism in SIDS that may help lead to identification of infants at risk and specific interventions to prevent death. PMID:19400695

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

Systems Genetics: A Novel Approach to Dissect the Genetic Basis of Osteoporosis

PubMed Central

Farber, Charles R.

2012-01-01

From the early 1990s to the middle of the last decade, the search for genes influencing osteoporosis proved difficult with few successes. However, over the last 5 years this has begun to change with the introduction of genome-wide association (GWA) studies. In this short period of time, GWA studies have significantly accelerated the pace of gene discovery, leading to the identification of nearly 100 independent associations for osteoporosis-related traits. However, GWA does not specifically pinpoint causal genes or provide functional context for associations. Thus, there is a need for approaches that provide systems-level insight on how associated variants influence cellular function, downstream gene networks, and ultimately disease. In this review we discuss the emerging field of “systems genetics” and how it is being used in combination with and independent of GWA to improve our understanding of the molecular mechanisms involved in bone fragility. PMID:22802146

Rapid control of male typical behaviors by brain-derived estrogens

PubMed Central

Cornil, Charlotte A.; Ball, Gregory F.; Balthazart, Jacques

2012-01-01

Beside their genomic mode of action, estrogens also activate a variety of cellular signaling pathways through non-genomic mechanisms. Until recently, little was known regarding the functional significance of such actions in males and the mechanism that control local estrogen concentration with a spatial and time resolution compatible with these non-genomic actions had rarely been examined. Here, we review evidence that estrogens rapidly modulate a variety of behaviors in male vertebrates. Then, we present in vitro work supporting the existence of a control mechanism of local brain estrogen synthesis by aromatase along with in vivo evidence that rapid changes in aromatase activity also occur in a region-specific manner in response to changes in the social or environmental context. Finally, we suggest that the brain estrogen provision may also play a significant role in females. Together these data bolster the hypothesis that brain-derived estrogens should be considered as neuromodulators. PMID:22983088

Mechanisms of viral mutation.

PubMed

Sanjuán, Rafael; Domingo-Calap, Pilar

2016-12-01

The remarkable capacity of some viruses to adapt to new hosts and environments is highly dependent on their ability to generate de novo diversity in a short period of time. Rates of spontaneous mutation vary amply among viruses. RNA viruses mutate faster than DNA viruses, single-stranded viruses mutate faster than double-strand virus, and genome size appears to correlate negatively with mutation rate. Viral mutation rates are modulated at different levels, including polymerase fidelity, sequence context, template secondary structure, cellular microenvironment, replication mechanisms, proofreading, and access to post-replicative repair. Additionally, massive numbers of mutations can be introduced by some virus-encoded diversity-generating elements, as well as by host-encoded cytidine/adenine deaminases. Our current knowledge of viral mutation rates indicates that viral genetic diversity is determined by multiple virus- and host-dependent processes, and that viral mutation rates can evolve in response to specific selective pressures.

A modular approach for multifunctional polymersomes with controlled adhesive properties.

PubMed

Petit, Julien; Thomi, Laura; Schultze, Jennifer; Makowski, Marcin; Negwer, Inka; Koynov, Kaloian; Herminghaus, Stephan; Wurm, Frederik R; Bäumchen, Oliver; Landfester, Katharina

2018-02-14

The bottom-up approach in synthetic biology involves the engineering of synthetic cells by designing biological and chemical building blocks, which can be combined in order to mimic cellular functions. The first step for mimicking a living cell is the design of an appropriate compartment featuring a multifunctional membrane. This is of particular interest since it allows for the selective attachment of different groups or molecules to the membrane. In this context, we report on a modular approach for polymeric vesicles, so-called polymersomes, with a multifunctional surface, namely hydroxyl, alkyne and acrylate groups. We demonstrate that the surface of the polymersome can be functionalized to facilitate imaging, via fluorescent dyes, or to improve the specific adhesion to surfaces by using a biotin functionalization. This generally applicable multifunctionality allows for the covalent integration of various molecules in the membrane of a synthetic cell.

Carboxysomes: metabolic modules for CO 2 fixation

DOE PAGES

Turmo, Aiko; Gonzalez-Esquer, Cesar Raul; Kerfeld, Cheryl A.

2017-08-14

The carboxysome is a bacterial microcompartment encapsulating the enzymes carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase. As the site of CO 2 fixation, it serves an essential role in the carbon dioxide concentrating mechanism of many chemoautotrophs and all cyanobacteria. Carboxysomes and other bacterial microcompartments self-assemble through specific protein–protein interactions that are typically mediated by conserved protein domains. In this review, we frame our current understanding of carboxysomes in the context of their component protein domains with their inherent function as the ‘building blocks’ of carboxysomes. These building blocks are organized in genetic modules (conserved chromosomal loci) that encode for carboxysomes andmore » ancillary proteins essential for the integration of the organelle with the rest of cellular metabolism. This conceptual framework provides the foundation for ‘plug-and-play’ engineering of carboxysomes as CO 2 fixation modules in a variety of biotechnological applications.« less

Properties of an unusual DNA primase from an archaeal plasmid

PubMed Central

Beck, Kirsten; Lipps, Georg

2007-01-01

Primases are specialized DNA-dependent RNA polymerases that synthesize a short oligoribonucleotide complementary to single-stranded template DNA. In the context of cellular DNA replication, primases are indispensable since DNA polymerases are not able to start DNA polymerization de novo. The primase activity of the replication protein from the archaeal plasmid pRN1 synthesizes a rather unusual mixed primer consisting of a single ribonucleotide at the 5′ end followed by seven deoxynucleotides. Ribonucleotides and deoxynucleotides are strictly required at the respective positions within the primer. Furthermore, in contrast to other archaeo-eukaryotic primases, the primase activity is highly sequence-specific and requires the trinucleotide motif GTG in the template. Primer synthesis starts outside of the recognition motif, immediately 5′ to the recognition motif. The fidelity of the primase synthesis is high, as non-complementary bases are not incorporated into the primer. PMID:17709343

Isolation and characterization of high affinity aptamers against DNA polymerase iota.

PubMed

Lakhin, Andrei V; Kazakov, Andrei A; Makarova, Alena V; Pavlov, Yuri I; Efremova, Anna S; Shram, Stanislav I; Tarantul, Viacheslav Z; Gening, Leonid V

2012-02-01

Human DNA-polymerase iota (Pol ι) is an extremely error-prone enzyme and the fidelity depends on the sequence context of the template. Using the in vitro systematic evolution of ligands by exponential enrichment (SELEX) procedure, we obtained an oligoribonucleotide with a high affinity to human Pol ι, named aptamer IKL5. We determined its dissociation constant with homogenous preparation of Pol ι and predicted its putative secondary structure. The aptamer IKL5 specifically inhibits DNA-polymerase activity of the purified enzyme Pol ι, but did not inhibit the DNA-polymerase activities of human DNA polymerases beta and kappa. IKL5 suppressed the error-prone DNA-polymerase activity of Pol ι also in cellular extracts of the tumor cell line SKOV-3. The aptamer IKL5 is useful for studies of the biological role of Pol ι and as a potential drug to suppress the increase of the activity of this enzyme in malignant cells.

A cellular and regulatory map of the cholinergic nervous system of C. elegans

PubMed Central

Pereira, Laura; Kratsios, Paschalis; Serrano-Saiz, Esther; Sheftel, Hila; Mayo, Avi E; Hall, David H; White, John G; LeBoeuf, Brigitte; Garcia, L Rene; Alon, Uri; Hobert, Oliver

2015-01-01

Nervous system maps are of critical importance for understanding how nervous systems develop and function. We systematically map here all cholinergic neuron types in the male and hermaphrodite C. elegans nervous system. We find that acetylcholine (ACh) is the most broadly used neurotransmitter and we analyze its usage relative to other neurotransmitters within the context of the entire connectome and within specific network motifs embedded in the connectome. We reveal several dynamic aspects of cholinergic neurotransmitter identity, including a sexually dimorphic glutamatergic to cholinergic neurotransmitter switch in a sex-shared interneuron. An expression pattern analysis of ACh-gated anion channels furthermore suggests that ACh may also operate very broadly as an inhibitory neurotransmitter. As a first application of this comprehensive neurotransmitter map, we identify transcriptional regulatory mechanisms that control cholinergic neurotransmitter identity and cholinergic circuit assembly. DOI: http://dx.doi.org/10.7554/eLife.12432.001 PMID:26705699

Retinoids and Retinal Diseases

PubMed Central

Kiser, Philip D.; Palczewski, Krzysztof

2016-01-01

Recent progress in molecular understanding of the retinoid cycle in mammalian retina stems from painstaking biochemical reconstitution studies supported by natural or engineered animal models with known genetic lesions and studies of humans with specific genetic blinding diseases. Structural and membrane biology have been used to detect critical retinal enzymes and proteins and their substrates and ligands, placing them in a cellular context. These studies have been supplemented by analytical chemistry methods that have identified small molecules by their spectral characteristics, often in conjunction with the evaluation of models of animal retinal disease. It is from this background that rational therapeutic interventions to correct genetic defects or environmental insults are identified. Thus, most presently accepted modulators of the retinoid cycle already have demonstrated promising results in animal models of retinal degeneration. These encouraging signs indicate that some human blinding diseases can be alleviated by pharmacological interventions. PMID:27917399

T-cell epitope prediction and immune complex simulation using molecular dynamics: state of the art and persisting challenges

PubMed Central

2010-01-01

Atomistic Molecular Dynamics provides powerful and flexible tools for the prediction and analysis of molecular and macromolecular systems. Specifically, it provides a means by which we can measure theoretically that which cannot be measured experimentally: the dynamic time-evolution of complex systems comprising atoms and molecules. It is particularly suitable for the simulation and analysis of the otherwise inaccessible details of MHC-peptide interaction and, on a larger scale, the simulation of the immune synapse. Progress has been relatively tentative yet the emergence of truly high-performance computing and the development of coarse-grained simulation now offers us the hope of accurately predicting thermodynamic parameters and of simulating not merely a handful of proteins but larger, longer simulations comprising thousands of protein molecules and the cellular scale structures they form. We exemplify this within the context of immunoinformatics. PMID:21067546

Preferential tumor cellular uptake and retention of indocyanine green for in vivo tumor imaging.

PubMed

Onda, Nobuhiko; Kimura, Masayuki; Yoshida, Toshinori; Shibutani, Makoto

2016-08-01

Indocyanine green (ICG) is a fluorescent agent approved for clinical applications by the Food and Drug Administration and European Medicines Agency. This study examined the mechanism of tumor imaging using intravenously administered ICG. The in vivo kinetics of intravenously administered ICG were determined in tumor xenografts using microscopic approaches that enabled both spatio-temporal and high-magnification analyses. The mechanism of ICG-based tumor imaging was examined at the cellular level in six phenotypically different human colon cancer cell lines exhibiting different grades of epithelioid organization. ICG fluorescence imaging detected xenograft tumors, even those < 1 mm in size, based on their preferential cellular uptake and retention of the dye following its rapid tissue-non-specific delivery, in contrast to its rapid clearance by normal tissue. Live-cell imaging revealed that cellular ICG uptake is temperature-dependent and occurs after ICG binding to the cellular membrane, a pattern suggesting endocytic uptake as the mechanism. Cellular ICG uptake correlated inversely with the formation of tight junctions. Intracellular ICG was entrapped in the membrane traffic system, resulting in its slow turnover and prolonged retention by tumor cells. Our results suggest that tumor-specific imaging by ICG involves non-specific delivery of the dye to tissues followed by preferential tumor cellular uptake and retention. The tumor cell-preference of ICG is driven by passive tumor cell-targeting, the inherent ability of ICG to bind to cell membranes, and the high endocytic activity of tumor cells in association with the disruption of their tight junctions. © 2016 UICC.

Escherichia Coli

ERIC Educational Resources Information Center

Goodsell, David S.

2009-01-01

Diverse biological data may be used to create illustrations of molecules in their cellular context. I describe the scientific results that support a recent textbook illustration of an "Escherichia coli cell". The image magnifies a portion of the bacterium at one million times, showing the location and form of individual macromolecules. Results…

Eukaryotic Cell Panorama

ERIC Educational Resources Information Center

Goodsell, David S.

2011-01-01

Diverse biological data may be used to create illustrations of molecules in their cellular context. This report describes the scientific results that support an illustration of a eukaryotic cell, enlarged by one million times to show the distribution and arrangement of macromolecules. The panoramic cross section includes eight panels that extend…

The Role of Auxiliary Subunits for the Functional Diversity of Voltage-Gated Calcium Channels

PubMed Central

Campiglio, Marta; Flucher, Bernhard E

2015-01-01

Voltage-gated calcium channels (VGCCs) represent the sole mechanism to convert membrane depolarization into cellular functions like secretion, contraction, or gene regulation. VGCCs consist of a pore-forming α1 subunit and several auxiliary channel subunits. These subunits come in multiple isoforms and splice-variants giving rise to a stunning molecular diversity of possible subunit combinations. It is generally believed that specific auxiliary subunits differentially regulate the channels and thereby contribute to the great functional diversity of VGCCs. If auxiliary subunits can associate and dissociate from pre-existing channel complexes, this would allow dynamic regulation of channel properties. However, most auxiliary subunits modulate current properties very similarly, and proof that any cellular calcium channel function is indeed modulated by the physiological exchange of auxiliary subunits is still lacking. In this review we summarize available information supporting a differential modulation of calcium channel functions by exchange of auxiliary subunits, as well as experimental evidence in support of alternative functions of the auxiliary subunits. At the heart of the discussion is the concept that, in their native environment, VGCCs function in the context of macromolecular signaling complexes and that the auxiliary subunits help to orchestrate the diverse protein–protein interactions found in these calcium channel signalosomes. Thus, in addition to a putative differential modulation of current properties, differential subcellular targeting properties and differential protein–protein interactions of the auxiliary subunits may explain the need for their vast molecular diversity. J. Cell. Physiol. 999: 00–00, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. J. Cell. Physiol. 230: 2019–2031, 2015. © 2015 Wiley Periodicals, Inc. PMID:25820299

Protective effects of sirtuins in cardiovascular diseases: from bench to bedside

PubMed Central

Winnik, Stephan; Auwerx, Johan; Sinclair, David A.; Matter, Christian M.

2015-01-01

Sirtuins (Sirt1–Sirt7) comprise a family of nicotinamide adenine dinucleotide (NAD+)-dependent enzymes. While deacetylation reflects their main task, some of them have deacylase, adenosine diphosphate-ribosylase, demalonylase, glutarylase, and desuccinylase properties. Activated upon caloric restriction and exercise, they control critical cellular processes in the nucleus, cytoplasm, and mitochondria to maintain metabolic homeostasis, reduce cellular damage and dampen inflammation—all of which serve to protect against a variety of age-related diseases, including cardiovascular pathologies. This review focuses on the cardiovascular effects of Sirt1, Sirt3, Sirt6, and Sirt7. Most is known about Sirt1. This deacetylase protects from endothelial dysfunction, atherothrombosis, diet-induced obesity, type 2 diabetes, liver steatosis, and myocardial infarction. Sirt3 provides beneficial effects in the context of left ventricular hypertrophy, cardiomyopathy, oxidative stress, metabolic homeostasis, and dyslipidaemia. Sirt6 is implicated in ameliorating dyslipidaemia, cellular senescence, and left ventricular hypertrophy. Sirt7 plays a role in lipid metabolism and cardiomyopathies. Most of these data were derived from experimental findings in genetically modified mice, where NFκB, Pcsk9, low-density lipoprotein-receptor, PPARγ, superoxide dismutase 2, poly[adenosine diphosphate-ribose] polymerase 1, and endothelial nitric oxide synthase were identified among others as crucial molecular targets and/or partners of sirtuins. Of note, there is translational evidence for a role of sirtuins in patients with endothelial dysfunction, type 1 or type 2 diabetes and longevity. Given the availability of specific Sirt1 activators or pan-sirtuin activators that boost levels of the sirtuin cofactor NAD+, we anticipate that this field will move quickly from bench to bedside. PMID:26112889

Modulation of Estrogen Response Element-Driven Gene Expressions and Cellular Proliferation with Polar Directions by Designer Transcription Regulators

PubMed Central

Muyan, Mesut; Güpür, Gizem; Yaşar, Pelin; Ayaz, Gamze; User, Sırma Damla; Kazan, Hasan Hüseyin; Huang, Yanfang

2015-01-01

Estrogen receptor α (ERα), as a ligand-dependent transcription factor, mediates 17β-estradiol (E2) effects. ERα is a modular protein containing a DNA binding domain (DBD) and transcription activation domains (AD) located at the amino- and carboxyl-termini. The interaction of the E2-activated ERα dimer with estrogen response elements (EREs) of genes constitutes the initial step in the ERE-dependent signaling pathway necessary for alterations of cellular features. We previously constructed monomeric transcription activators, or monotransactivators, assembled from an engineered ERE-binding module (EBM) using the ERα-DBD and constitutively active ADs from other transcription factors. Monotransactivators modulated cell proliferation by activating and repressing ERE-driven gene expressions that simulate responses observed with E2-ERα. We reasoned here that integration of potent heterologous repression domains (RDs) into EBM could generate monotransrepressors that alter ERE-bearing gene expressions and cellular proliferation in directions opposite to those observed with E2-ERα or monotransactivators. Consistent with this, monotransrepressors suppressed reporter gene expressions that emulate the ERE-dependent signaling pathway. Moreover, a model monotransrepressor regulated DNA synthesis, cell cycle progression and proliferation of recombinant adenovirus infected ER-negative cells through decreasing as well as increasing gene expressions with polar directions compared with E2-ERα or monotransactivator. Our results indicate that an ‘activator’ or a ‘repressor’ possesses both transcription activating/enhancing and repressing/decreasing abilities within a chromatin context. Offering a protein engineering platform to alter signal pathway-specific gene expressions and cell growth, our approach could also be used for the development of tools for epigenetic modifications and for clinical interventions wherein multigenic de-regulations are an issue. PMID:26295471

HSV-I and the cellular DNA damage response.

PubMed

Smith, Samantha; Weller, Sandra K

2015-04-01

Peter Wildy first observed genetic recombination between strains of HSV in 1955. At the time, knowledge of DNA repair mechanisms was limited, and it has only been in the last decade that particular DNA damage response (DDR) pathways have been examined in the context of viral infections. One of the first reports addressing the interaction between a cellular DDR protein and HSV-1 was the observation by Lees-Miller et al . that DNA-dependent protein kinase catalytic subunit levels were depleted in an ICP0-dependent manner during Herpes simplex virus 1 infection. Since then, there have been numerous reports describing the interactions between HSV infection and cellular DDR pathways. Due to space limitations, this review will focus predominantly on the most recent observations regarding how HSV navigates a potentially hostile environment to replicate its genome.

Label-Free Analysis of Cellular Lipid Droplet Formation by Non-Linear Microscopy

NASA Astrophysics Data System (ADS)

Schie, Iwan W.

Cellular lipid droplets (LD) are cellular organelles that can be found in every cell type. Recent research indicates that cellular LD are involved in a large number of cellular metabolic functions, such as lipid metabolism, protection from lipotoxicity, protein storage and degradation, and many more. LD formation is frequently associated with adverse health effects, i.e. alcoholic and non-alcoholic fatty liver disease, diabetes type-2, as well as many cardiovascular disorders. Despite their wide presence, LDs are the least studied and most poorly understood cellular organelles. Typically, LDs are investigated using fluorescence-based techniques that require staining with exogenous fluorophores. Other techniques, e.g. biochemical assays, require the destruction of cells that prohibit the analysis of living cells. Therefore, in my thesis research I developed a novel compound fast-scanning nonlinear optical microscope equipped with the ability to also acquire Raman spectra at specific image locations. This system allows us to image label-free cellular LD formation in living cells and analyze the composition of single cellular LDs. Images can be acquired at near video-rate (˜16 frames/s). Furthermore, the system has the ability to acquire very large images of tissue of up to 7.5x15 cm2 total area by stitching together scans with dimensions of 1x1 mm2 in less than 1 minute. The system also enables the user to acquire Raman spectra from points of interest in the multiphoton images and provides chemically-specific data from sample volumes as small as 1 femtoliter. In my thesis I used this setup to determine the effects of VLDL lipolysis products on primary rat hepatocytes. By analyzing the Raman spectra and comparing the peak ratios for saturated and unsaturated fatty acid it was determined that the small cellular LD are highly saturated, while large cellular LDs contain mostly unsaturated lipids. Furthermore, I established a method to determine the specific contribution of each individual fatty acids to a single cellular LD based on non-negative least squares analysis. The calculated quantities for oleic and palmitic acid from 10 individual cellular LDs were compared to results of a gas chromatography (GC) analysis of 2x10 6 cells. The analysis found that the data obtained by Raman spectroscopy of individual LDs closely resemble GC data of a significantly larger number of LDs.

A pharma perspective on the systems medicine and pharmacology of inflammation.

PubMed

Lahoz-Beneytez, Julio; Schnizler, Katrin; Eissing, Thomas

2015-02-01

Biological systems are complex and comprehend multiple scales of organisation. Hence, holistic approaches are necessary to capture the behaviour of these entities from the molecular and cellular to the whole organism level. This also applies to the understanding and treatment of different diseases. Traditional systems biology has been successful in describing different biological phenomena at the cellular level, but it still lacks of a holistic description of the multi-scale interactions within the body. The importance of the physiological context is of particular interest in inflammation. Regulatory agencies have urged the scientific community to increase the translational power of bio-medical research and it has been recognised that modelling and simulation could be a path to follow. Interestingly, in pharma R&D, modelling and simulation has been employed since a long time ago. Systems pharmacology, and particularly physiologically based pharmacokinetic/pharmacodynamic models, serve as a suitable framework to integrate the available and emerging knowledge at different levels of the drug development process. Systems medicine and pharmacology of inflammation will potentially benefit from this framework in order to better understand inflammatory diseases and to help to transfer the vast knowledge on the molecular and cellular level into a more physiological context. Ultimately, this may lead to reliable predictions of clinical outcomes such as disease progression or treatment efficacy, contributing thereby to a better care of patients. Copyright © 2014 Elsevier Inc. All rights reserved.

The evolution of early cellular systems viewed through the lens of biological interactions.

PubMed

Poole, Anthony M; Lundin, Daniel; Rytkönen, Kalle T

2015-01-01

The minimal cell concept represents a pragmatic approach to the question of how few genes are required to run a cell. This is a helpful way to build a parts-list, and has been more successful than attempts to deduce a minimal gene set for life by inferring the gene repertoire of the last universal common ancestor, as few genes trace back to this hypothetical ancestral state. However, the study of minimal cellular systems is the study of biological outliers where, by practical necessity, coevolutionary interactions are minimized or ignored. In this paper, we consider the biological context from which minimal genomes have been removed. For instance, some of the most reduced genomes are from endosymbionts and are the result of coevolutionary interactions with a host; few such organisms are "free-living." As few, if any, biological systems exist in complete isolation, we expect that, as with modern life, early biological systems were part of an ecosystem, replete with organismal interactions. We favor refocusing discussions of the evolution of cellular systems on processes rather than gene counts. We therefore draw a distinction between a pragmatic minimal cell (an interesting engineering problem), a distributed genome (a system resulting from an evolutionary transition involving more than one cell) and the looser coevolutionary interactions that are ubiquitous in ecosystems. Finally, we consider the distributed genome and coevolutionary interactions between genomic entities in the context of early evolution.

Domain Specificity of MAP3K Family Members, MLK and Tak1, for JNK Signaling in Drosophila

PubMed Central

Stronach, Beth; Lennox, Ashley L.; Garlena, Rebecca A.

2014-01-01

A highly diverse set of protein kinases functions as early responders in the mitogen- and stress-activated protein kinase (MAPK/SAPK) signaling pathways. For instance, humans possess 14 MAPK kinase kinases (MAP3Ks) that activate Jun kinase (JNK) signaling downstream. A major challenge is to decipher the selective and redundant functions of these upstream MAP3Ks. Taking advantage of the relative simplicity of Drosophila melanogaster as a model system, we assessed MAP3K signaling specificity in several JNK-dependent processes during development and stress response. Our approach was to generate molecular chimeras between two MAP3K family members, the mixed lineage kinase, Slpr, and the TGF-β activated kinase, Tak1, which share 32% amino acid identity across the kinase domain but otherwise differ in sequence and domain structure, and then test the contributions of various domains for protein localization, complementation of mutants, and activation of signaling. We found that overexpression of the wild-type kinases stimulated JNK signaling in alternate contexts, so cells were capable of responding to both MAP3Ks, but with distinct outcomes. Relative to wild-type, the catalytic domain swaps compensated weakly or not at all, despite having a shared substrate, the JNK kinase Hep. Tak1 C-terminal domain-containing constructs were inhibitory in Tak1 signaling contexts, including tumor necrosis factor-dependent cell death and innate immune signaling; however, depressing antimicrobial gene expression did not necessarily cause phenotypic susceptibility to infection. These same constructs were neutral in the context of Slpr-dependent developmental signaling, reflecting differential subcellular protein localization and by inference, point of activation. Altogether, our findings suggest that the selective deployment of a particular MAP3K can be attributed in part to its inherent sequence differences, cellular localization, and binding partner availability. PMID:24429281

Landauer in the Age of Synthetic Biology: Energy Consumption and Information Processing in Biochemical Networks

NASA Astrophysics Data System (ADS)

Mehta, Pankaj; Lang, Alex H.; Schwab, David J.

2016-03-01

A central goal of synthetic biology is to design sophisticated synthetic cellular circuits that can perform complex computations and information processing tasks in response to specific inputs. The tremendous advances in our ability to understand and manipulate cellular information processing networks raises several fundamental physics questions: How do the molecular components of cellular circuits exploit energy consumption to improve information processing? Can one utilize ideas from thermodynamics to improve the design of synthetic cellular circuits and modules? Here, we summarize recent theoretical work addressing these questions. Energy consumption in cellular circuits serves five basic purposes: (1) increasing specificity, (2) manipulating dynamics, (3) reducing variability, (4) amplifying signal, and (5) erasing memory. We demonstrate these ideas using several simple examples and discuss the implications of these theoretical ideas for the emerging field of synthetic biology. We conclude by discussing how it may be possible to overcome these limitations using "post-translational" synthetic biology that exploits reversible protein modification.

The effect of redox-related species of nitrogen monoxide on transferrin and iron uptake and cellular proliferation of erythroleukemia (K562) cells.

PubMed

Richardson, D R; Neumannova, V; Nagy, E; Ponka, P

1995-10-15

The iron-responsive element-binding protein (IRE-BP) modulates both ferritin mRNA translation and transferrin receptor (TfR) mRNA stability by binding to specific mRNA sequences called iron-responsive elements (IREs). The regulation of IRE-BP in situ could possibly occur either through its Fe-S cluster and/or via free cysteine sulphydryl groups such as cysteine 437 (Philpott et al, J Biol Chem 268:17655, 1993; and Hirling et al, EMBO J 13:453, 1994). Recently, nitrogen monoxide (NO) has been shown to have markedly different biologic effects depending on its redox state (Lipton et al, Nature 364:626, 1993). Considering this fact, it is conceivable that the NO group, as either the nitrosonium ion (NO+) or nitric oxide (NO+), may regulate IRE-BP activity by S-nitrosylation of key sulphydryl groups or via ligation of NO. to the Fe-S cluster, respectively. This hypothesis has been examined using the NO+ generator, sodium nitroprusside (SNP); the NO. generator, S-nitroso-N-acetylpenicillamine (SNAP); and the NO./peroxynitrite (ONOO-) generator, 3-morpholinosydnonimine hydrochloride (SIN-1). Treatment of K562 cells for 18 hours with SNP (1 mmol/L) resulted in a pronounced decrease in both the RNA-binding activity of IRE-BP and the level of TfR mRNA. In addition, Scatchard analysis showed a marked decrease in the number of specific Tf-binding sites, from 590,000/cell (control) to 170,000/cell (test), and there was also a distinct decrease in Fe uptake. Furthermore, SNP did not decrease cellular viability or proliferation. In contrast, the NO. generator, SNAP (1 mmol/L), increased RNA-binding activity of IRE-BP, the level of TfR mRNA, and the number of TfRs in K562 cells. Moreover, both SNAP (1 mmol/L) and SIN-1 (0.5 mmol/L) reduced cellular proliferation. The results are discussed in context of the possible physiologic role of redox-related species of NO in regulating iron metabolism.

Tandem E2F Binding Sites in the Promoter of the p107 Cell Cycle Regulator Control p107 Expression and Its Cellular Functions

PubMed Central

Burkhart, Deborah L.; Wirt, Stacey E.; Zmoos, Anne-Flore; Kareta, Michael S.; Sage, Julien

2010-01-01

The retinoblastoma tumor suppressor (Rb) is a potent and ubiquitously expressed cell cycle regulator, but patients with a germline Rb mutation develop a very specific tumor spectrum. This surprising observation raises the possibility that mechanisms that compensate for loss of Rb function are present or activated in many cell types. In particular, p107, a protein related to Rb, has been shown to functionally overlap for loss of Rb in several cellular contexts. To investigate the mechanisms underlying this functional redundancy between Rb and p107 in vivo, we used gene targeting in embryonic stem cells to engineer point mutations in two consensus E2F binding sites in the endogenous p107 promoter. Analysis of normal and mutant cells by gene expression and chromatin immunoprecipitation assays showed that members of the Rb and E2F families directly bound these two sites. Furthermore, we found that these two E2F sites controlled both the repression of p107 in quiescent cells and also its activation in cycling cells, as well as in Rb mutant cells. Cell cycle assays further indicated that activation of p107 transcription during S phase through the two E2F binding sites was critical for controlled cell cycle progression, uncovering a specific role for p107 to slow proliferation in mammalian cells. Direct transcriptional repression of p107 by Rb and E2F family members provides a molecular mechanism for a critical negative feedback loop during cell cycle progression and tumorigenesis. These experiments also suggest novel therapeutic strategies to increase the p107 levels in tumor cells. PMID:20585628

New target genes of MITF-induced microRNA-211 contribute to melanoma cell invasion.

PubMed

Margue, Christiane; Philippidou, Demetra; Reinsbach, Susanne E; Schmitt, Martina; Behrmann, Iris; Kreis, Stephanie

2013-01-01

The non-coding microRNAs (miRNA) have tissue- and disease-specific expression patterns. They down-regulate target mRNAs, which likely impacts on most fundamental cellular processes. Differential expression patterns of miRNAs are currently being exploited for identification of biomarkers for early disease diagnosis, prediction of progression for melanoma and other cancers and as promising drug targets, since they can easily be inhibited or replaced in a given cellular context. Before successfully manipulating miRNAs in clinical settings, their precise expression levels, endogenous functions and thus their target genes have to be determined. MiR-211, a melanocyte lineage-specific small non-coding miRNA, is located in an intron of TRPM1, a target gene of the microphtalmia-associated transcription factor (MITF). By transcriptionally up-regulating TRPM1, MITF, which is critical for both melanocyte differentiation and survival and for melanoma progression, indirectly drives the expression of miR-211. Expression of this miRNA is often reduced in melanoma samples. Here, we investigated functional roles of miR-211 by identifying and studying new target genes. We show that MITF-correlated miR-211 expression levels are mostly but not always reduced in a panel of 11 melanoma cell lines and in primary and metastatic melanoma compared to normal melanocytes and nevi, respectively. MiR-211 itself only marginally impacted on cell invasion and migration, while perturbation of some new miR-211 target genes, such as AP1S2, SOX11, IGFBP5, and SERINC3 significantly increased invasion. These results and the variable expression levels of miR-211 raise serious doubts on the value of miR-211 as a melanoma tumor-suppressing miRNA and/or as a biomarker for melanoma.

Postnatal odorant exposure induces peripheral olfactory plasticity at the cellular level.

PubMed

Cadiou, Hervé; Aoudé, Imad; Tazir, Bassim; Molinas, Adrien; Fenech, Claire; Meunier, Nicolas; Grosmaitre, Xavier

2014-04-02

Mammalian olfactory sensory neurons (OSNs) form the primary elements of the olfactory system. Inserted in the olfactory mucosa lining of the nasal cavity, they are exposed to the environment and their lifespan is brief. Several reports say that OSNs are regularly regenerated during the entire life and that odorant environment affects the olfactory epithelium. However, little is known about the impact of the odorant environment on OSNs at the cellular level and more precisely in the context of early postnatal olfactory exposure. Here we exposed MOR23-green fluorescent protein (GFP) and M71-GFP mice to lyral or acetophenone, ligands for MOR23 or M71, respectively. Daily postnatal exposure to lyral induces plasticity in the population of OSNs expressing MOR23. Their density decreases after odorant exposure, whereas the amount of MOR23 mRNA and protein remain stable in the whole epithelium. Meanwhile, quantitative PCR indicates that each MOR23 neuron has higher levels of olfactory receptor transcripts and also expresses more CNGA2 and phosphodiesterase 1C, fundamental olfactory transduction pathway proteins. Transcript levels return to baseline after 4 weeks recovery. Patch-clamp recordings reveal that exposed MOR23 neurons respond to lyral with higher sensitivity and broader dynamic range while the responses' kinetics were faster. These effects are specific to the odorant-receptor pair lyral-MOR23: there was no effect of acetophenone on MOR23 neurons and no effect of acetophenone and lyral on the M71 population. Together, our results clearly demonstrate that OSNs undergo specific anatomical, molecular, and functional adaptation when chronically exposed to odorants in the early stage of life.

"Illustrating the Machinery of Life": Viruses

ERIC Educational Resources Information Center

Goodsell, David S.

2012-01-01

Data from electron microscopy, X-ray crystallography, and biophysical analysis are used to create illustrations of viruses in their cellular context. This report describes the scientific data and artistic methods used to create three illustrations: a depiction of the poliovirus lifecycle, budding of influenza virus from a cell surface, and a…

PhosphoregDB: The tissue and sub-cellular distribution of mammalian protein kinases and phosphatases

PubMed Central

Forrest, Alistair RR; Taylor, Darrin F; Fink, J Lynn; Gongora, M Milena; Flegg, Cameron; Teasdale, Rohan D; Suzuki, Harukazu; Kanamori, Mutsumi; Kai, Chikatoshi; Hayashizaki, Yoshihide; Grimmond, Sean M

2006-01-01

Background Protein kinases and protein phosphatases are the fundamental components of phosphorylation dependent protein regulatory systems. We have created a database for the protein kinase-like and phosphatase-like loci of mouse that integrates protein sequence, interaction, classification and pathway information with the results of a systematic screen of their sub-cellular localization and tissue specific expression data mined from the GNF tissue atlas of mouse. Results The database lets users query where a specific kinase or phosphatase is expressed at both the tissue and sub-cellular levels. Similarly the interface allows the user to query by tissue, pathway or sub-cellular localization, to reveal which components are co-expressed or co-localized. A review of their expression reveals 30% of these components are detected in all tissues tested while 70% show some level of tissue restriction. Hierarchical clustering of the expression data reveals that expression of these genes can be used to separate the samples into tissues of related lineage, including 3 larger clusters of nervous tissue, developing embryo and cells of the immune system. By overlaying the expression, sub-cellular localization and classification data we examine correlations between class, specificity and tissue restriction and show that tyrosine kinases are more generally expressed in fewer tissues than serine/threonine kinases. Conclusion Together these data demonstrate that cell type specific systems exist to regulate protein phosphorylation and that for accurate modelling and for determination of enzyme substrate relationships the co-location of components needs to be considered. PMID:16504016

Targeting the relaxin hormonal pathway in prostate cancer.

PubMed

Neschadim, Anton; Summerlee, Alastair J S; Silvertown, Joshua D

2015-11-15

Targeting the androgen signalling pathway has long been the hallmark of anti-hormonal therapy for prostate cancer. However, development of androgen-independent prostate cancer is an inevitable outcome to therapies targeting this pathway, in part, owing to the shift from cancer dependence on androgen signalling for growth in favor of augmentation of other cellular pathways that provide proliferation-, survival- and angiogenesis-promoting signals. This review focuses on the role of the hormone relaxin in the development and progression of prostate cancer, prior to and after the onset of androgen independence, as well as its role in cancers of other reproductive tissues. As the body of literature expands, examining relaxin expression in cancerous tissues and its role in a growing number of in vitro and in vivo cancer models, our understanding of the important involvement of this hormone in cancer biology is becoming clearer. Specifically, the pleiotropic functions of relaxin affecting cell growth, angiogenesis, blood flow, cell migration and extracellular matrix remodeling are examined in the context of cancer progression. The interactions and intercepts of the intracellular signalling pathways of relaxin with the androgen pathway are explored in the context of progression of castration-resistant and androgen-independent prostate cancers. We provide an overview of current anti-hormonal therapeutic treatment options for prostate cancer and delve into therapeutic approaches and development of agents aimed at specifically antagonizing relaxin signalling to curb tumor growth. We also discuss the rationale and challenges utilizing such agents as novel anti-hormonals in the clinic, and their potential to supplement current therapeutic modalities. © 2014 UICC.

From sticky stuff to sweet receptors--achievements, limits and novel approaches to bioadhesion.

PubMed

Lehr, C M

1996-01-01

About 10 years ago, the concept of bioadhesion was introduced into the pharmaceutical literature and has since stimulated much research and development both in academia and in industry. The first generation of bioadhesive drug delivery systems (BBDS) were based on so-called mucoadhesive polymers, i.e. natural or synthetic macromolecules, often already well accepted and used as pharmaceutical excipients for other purposes, which show the remarkable ability to 'stick' to humid or wet mucosal tissue surfaces. While these novel dosage forms were mainly expected to allow for a possible prolongation, better localization or intensified contact to mucosal tissue surfaces, it had to be realized that these goals were often not so easily accomplished, at least not by means of such relatively straightforward technology. However, although not always convincing as a 'pharmaceutical glue', some of the mucoadhesive polymers were found to display other, possibly even more important biological activities, namely to inhibit proteolytic enzymes and/or to modulate the permeability of usually tight epithelial tissue barriers. Such features were found to be particularly useful in the context of peptide and protein drug delivery. But still, the interest in realizing 'true' bioadhesion continues: instead of mucoadhesive polymers, plant or bacterial lectins, i.e. adhesion molecules which specifically bind to sugar moieties of the epithelial cell membrane, are now widely being investigated as drug delivery adjuvants. These second-generation bioadhesives not only provide for cellular binding, but also for subsequent endo- and transcytosis. This makes the novel, specifically bioadhesive molecules particularly interesting for the controlled delivery of DNA/RNA molecules in the context of antisense or gene therapy.

Early spatiotemporal-specific changes in intermediate signals are predictive of cytotoxic sensitivity to TNFα and co-treatments

NASA Astrophysics Data System (ADS)

Loo, Lit-Hsin; Bougen-Zhukov, Nicola Michelle; Tan, Wei-Ling Cecilia

2017-03-01

Signaling pathways can generate different cellular responses to the same cytotoxic agents. Current quantitative models for predicting these differential responses are usually based on large numbers of intracellular gene products or signals at different levels of signaling cascades. Here, we report a study to predict cellular sensitivity to tumor necrosis factor alpha (TNFα) using high-throughput cellular imaging and machine-learning methods. We measured and compared 1170 protein phosphorylation events in a panel of human lung cancer cell lines based on different signals, subcellular regions, and time points within one hour of TNFα treatment. We found that two spatiotemporal-specific changes in an intermediate signaling protein, p90 ribosomal S6 kinase (RSK), are sufficient to predict the TNFα sensitivity of these cell lines. Our models could also predict the combined effects of TNFα and other kinase inhibitors, many of which are not known to target RSK directly. Therefore, early spatiotemporal-specific changes in intermediate signals are sufficient to represent the complex cellular responses to these perturbations. Our study provides a general framework for the development of rapid, signaling-based cytotoxicity screens that may be used to predict cellular sensitivity to a cytotoxic agent, or identify co-treatments that may sensitize or desensitize cells to the agent.

Early spatiotemporal-specific changes in intermediate signals are predictive of cytotoxic sensitivity to TNFα and co-treatments

PubMed Central

Loo, Lit-Hsin; Bougen-Zhukov, Nicola Michelle; Tan, Wei-Ling Cecilia

2017-01-01

Signaling pathways can generate different cellular responses to the same cytotoxic agents. Current quantitative models for predicting these differential responses are usually based on large numbers of intracellular gene products or signals at different levels of signaling cascades. Here, we report a study to predict cellular sensitivity to tumor necrosis factor alpha (TNFα) using high-throughput cellular imaging and machine-learning methods. We measured and compared 1170 protein phosphorylation events in a panel of human lung cancer cell lines based on different signals, subcellular regions, and time points within one hour of TNFα treatment. We found that two spatiotemporal-specific changes in an intermediate signaling protein, p90 ribosomal S6 kinase (RSK), are sufficient to predict the TNFα sensitivity of these cell lines. Our models could also predict the combined effects of TNFα and other kinase inhibitors, many of which are not known to target RSK directly. Therefore, early spatiotemporal-specific changes in intermediate signals are sufficient to represent the complex cellular responses to these perturbations. Our study provides a general framework for the development of rapid, signaling-based cytotoxicity screens that may be used to predict cellular sensitivity to a cytotoxic agent, or identify co-treatments that may sensitize or desensitize cells to the agent. PMID:28272488

Measuring spatial and temporal Ca2+ signals in Arabidopsis plants.

PubMed

Zhu, Xiaohong; Taylor, Aaron; Zhang, Shenyu; Zhang, Dayong; Feng, Ying; Liang, Gaimei; Zhu, Jian-Kang

2014-09-02

Developmental and environmental cues induce Ca(2+) fluctuations in plant cells. Stimulus-specific spatial-temporal Ca(2+) patterns are sensed by cellular Ca(2+) binding proteins that initiate Ca(2+) signaling cascades. However, we still know little about how stimulus specific Ca(2+) signals are generated. The specificity of a Ca(2+) signal may be attributed to the sophisticated regulation of the activities of Ca(2+) channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca(2+) signals at both the tissue and cellular levels. Genetically encoded Ca(2+) indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca(2+) signals. Here we describe instructions for the use of two Ca(2+) detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca(2+) imaging and case12 based live cell confocal fluorescence Ca(2+) imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca(2+) signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca(2+) signals at a high resolution.

Positive-sense RNA viruses reveal the complexity and dynamics of the cellular and viral epitranscriptomes during infection.

PubMed

McIntyre, Will; Netzband, Rachel; Bonenfant, Gaston; Biegel, Jason M; Miller, Clare; Fuchs, Gabriele; Henderson, Eric; Arra, Manoj; Canki, Mario; Fabris, Daniele; Pager, Cara T

2018-06-20

More than 140 post-transcriptional modifications (PTMs) are known to decorate cellular RNAs, but their incidence, identity and significance in viral RNA are still largely unknown. We have developed an agnostic analytical approach to comprehensively survey PTMs on viral and cellular RNAs. Specifically, we used mass spectrometry to analyze PTMs on total RNA isolated from cells infected with Zika virus, Dengue virus, hepatitis C virus (HCV), poliovirus and human immunodeficiency virus type 1. All five RNA viruses significantly altered global PTM landscapes. Examination of PTM profiles of individual viral genomes isolated by affinity capture revealed a plethora of PTMs on viral RNAs, which far exceeds the handful of well-characterized modifications. Direct comparison of viral epitranscriptomes identified common and virus-specific PTMs. In particular, specific dimethylcytosine modifications were only present in total RNA from virus-infected cells, and in intracellular HCV RNA, and viral RNA from Zika and HCV virions. Moreover, dimethylcytosine abundance during viral infection was modulated by the cellular DEAD-box RNA helicase DDX6. By opening the Pandora's box on viral PTMs, this report presents numerous questions and hypotheses on PTM function and strongly supports PTMs as a new tier of regulation by which RNA viruses subvert the host and evade cellular surveillance systems.

Noncoding RNA danger motifs bridge innate and adaptive immunity and are potent adjuvants for vaccination

PubMed Central

Wang, Lilin; Smith, Dan; Bot, Simona; Dellamary, Luis; Bloom, Amy; Bot, Adrian

2002-01-01

The adaptive immune response is triggered by recognition of T and B cell epitopes and is influenced by “danger” motifs that act via innate immune receptors. This study shows that motifs associated with noncoding RNA are essential features in the immune response reminiscent of viral infection, mediating rapid induction of proinflammatory chemokine expression, recruitment and activation of antigen-presenting cells, modulation of regulatory cytokines, subsequent differentiation of Th1 cells, isotype switching, and stimulation of cross-priming. The heterogeneity of RNA-associated motifs results in differential binding to cellular receptors, and specifically impacts the immune profile. Naturally occurring double-stranded RNA (dsRNA) triggered activation of dendritic cells and enhancement of specific immunity, similar to selected synthetic dsRNA motifs. Based on the ability of specific RNA motifs to block tolerance induction and effectively organize the immune defense during viral infection, we conclude that such RNA species are potent danger motifs. We also demonstrate the feasibility of using selected RNA motifs as adjuvants in the context of novel aerosol carriers for optimizing the immune response to subunit vaccines. In conclusion, RNA-associated motifs produced during viral infection bridge the early response with the late adaptive phase, regulating the activation and differentiation of antigen-specific B and T cells, in addition to a short-term impact on innate immunity. PMID:12393853

Commensal–dendritic-cell interaction specifies a unique protective skin immune signature

PubMed Central

Naik, Shruti; Bouladoux, Nicolas; Linehan, Jonathan L.; Han, Seong-Ji; Harrison, Oliver J.; Wilhelm, Christoph; Conlan, Sean; Himmelfarb, Sarah; Byrd, Allyson L.; Deming, Clayton; Quinones, Mariam; Brenchley, Jason M.; Kong, Heidi H.; Tussiwand, Roxanne; Murphy, Kenneth M.; Merad, Miriam; Segre, Julia A; Belkaid, Yasmine

2015-01-01

The skin represents the primary interface between the host and the environment. This organ is also home to trillions of microorganisms that play an important role in tissue homeostasis and local immunity1–4. Skin microbial communities are highly diverse and can be remodelled over time or in response to environmental challenges5–7. How, in the context of this complexity, individual commensal microorganisms may differentially modulate skin immunity and the consequences of these responses for tissue physiology remains unclear. Here we show that defined commensals dominantly affect skin immunity and identify the cellular mediators involved in this specification. In particular, colonization with Staphylococcus epidermidis induces IL-17A+ CD8+ T cells that home to the epidermis, enhance innate barrier immunity and limit pathogen invasion. Commensal-specific T-cell responses result from the coordinated action of skin-resident dendritic cell subsets and are not associated with inflammation, revealing that tissue-resident cells are poised to sense and respond to alterations in microbial communities. This interaction may represent an evolutionary means by which the skin immune system uses fluctuating commensal signals to calibrate barrier immunity and provide heterologous protection against invasive pathogens. These findings reveal that the skin immune landscape is a highly dynamic environment that can be rapidly and specifically remodelled by encounters with defined commensals, findings that have profound implications for our understanding of tissue-specific immunity and pathologies. PMID:25539086

Quantitative phase-digital holographic microscopy: a new imaging modality to identify original cellular biomarkers of diseases

NASA Astrophysics Data System (ADS)

Marquet, P.; Rothenfusser, K.; Rappaz, B.; Depeursinge, C.; Jourdain, P.; Magistretti, P. J.

2016-03-01

Quantitative phase microscopy (QPM) has recently emerged as a powerful label-free technique in the field of living cell imaging allowing to non-invasively measure with a nanometric axial sensitivity cell structure and dynamics. Since the phase retardation of a light wave when transmitted through the observed cells, namely the quantitative phase signal (QPS), is sensitive to both cellular thickness and intracellular refractive index related to the cellular content, its accurate analysis allows to derive various cell parameters and monitor specific cell processes, which are very likely to identify new cell biomarkers. Specifically, quantitative phase-digital holographic microscopy (QP-DHM), thanks to its numerical flexibility facilitating parallelization and automation processes, represents an appealing imaging modality to both identify original cellular biomarkers of diseases as well to explore the underlying pathophysiological processes.

Elevated Serotonin Interacts with Angiotensin-II to Result in Altered Valve Interstitial Cell Contractility and Remodeling.

PubMed

Perez, Jessica; Diaz, Nancy; Tandon, Ishita; Plate, Rachel; Martindale, Christopher; Balachandran, Kartik

2018-06-01

While the valvulopathic effects of serotonin (5HT) and angiotensin-II (Ang-II) individually are known, it was not clear how 5HT and Ang-II might interact, specifically in the context of the mechanobiological responses due to altered valve mechanics potentiated by these molecules. In this context, the hypothesis of this study was that increased serotonin levels would result in accelerated progression toward disease in the presence of angiotensin-II-induced hypertension. C57/BL6 J mice were divided into four groups and subcutaneously implanted with osmotic pumps containing: PBS (control), 5HT (2.5 ng/kg/min), Ang-II (400 ng/kg/min), and 5HT + Ang-II (combination). Blood pressure was monitored using the tail cuff method. Echocardiography was performed on the mice before surgery and every week thereafter to assess ejection fraction. After three weeks, the mice were sacrificed and their hearts excised, embedded and sectioned for analysis of the aortic valves via histology and immunohistochemistry. In separate experiments, porcine valve interstitial cells (VICs) were directly stimulated with 5HT (10 -7  M), Ang-II (100 nM) or both and assayed for cellular contractility, cytoskeletal organization and collagen remodeling. After three weeks, average systolic blood pressure was significantly increased in the 5HT, Ang-II and combination groups compared to control. Echocardiographic analysis demonstrated significantly reduced ejection fraction in Ang-II and the combination groups. H&E staining demonstrated thicker leaflets in the combination groups, suggesting a more aggressive remodeling process. Picrosirius red staining and image analysis suggested that the Ang-II and combination groups had the largest proportion of thicker collagen fibers. VIC orientation, cellular contractility and collagen gene expression was highest for the 5HT + Ang-II combination treatment compared to all other groups. Overall, our results suggest that 5HT and Ang-II interact to result in significantly detrimental alteration of function and remodeling in the valve.

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.

Multiaxial behavior of foams - Experiments and modeling

NASA Astrophysics Data System (ADS)

Maheo, Laurent; Guérard, Sandra; Rio, Gérard; Donnard, Adrien; Viot, Philippe

2015-09-01

Cellular materials are strongly related to pressure level inside the material. It is therefore important to use experiments which can highlight (i) the pressure-volume behavior, (ii) the shear-shape behavior for different pressure level. Authors propose to use hydrostatic compressive, shear and combined pressure-shear tests to determine cellular materials behavior. Finite Element Modeling must take into account these behavior specificities. Authors chose to use a behavior law with a Hyperelastic, a Viscous and a Hysteretic contributions. Specific developments has been performed on the Hyperelastic one by separating the spherical and the deviatoric part to take into account volume change and shape change characteristics of cellular materials.

Physical models have gender-specific effects on student understanding of protein structure-function relationships.

PubMed

Forbes-Lorman, Robin M; Harris, Michelle A; Chang, Wesley S; Dent, Erik W; Nordheim, Erik V; Franzen, Margaret A

2016-07-08

Understanding how basic structural units influence function is identified as a foundational/core concept for undergraduate biological and biochemical literacy. It is essential for students to understand this concept at all size scales, but it is often more difficult for students to understand structure-function relationships at the molecular level, which they cannot as effectively visualize. Students need to develop accurate, 3-dimensional mental models of biomolecules to understand how biomolecular structure affects cellular functions at the molecular level, yet most traditional curricular tools such as textbooks include only 2-dimensional representations. We used a controlled, backward design approach to investigate how hand-held physical molecular model use affected students' ability to logically predict structure-function relationships. Brief (one class period) physical model use increased quiz score for females, whereas there was no significant increase in score for males using physical models. Females also self-reported higher learning gains in their understanding of context-specific protein function. Gender differences in spatial visualization may explain the gender-specific benefits of physical model use observed. © 2016 The Authors Biochemistry and Molecular Biology Education published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 44(4):326-335, 2016. © 2016 The International Union of Biochemistry and Molecular Biology.

Precision control of recombinant gene transcription for CHO cell synthetic biology.

PubMed

Brown, Adam J; James, David C

2016-01-01

The next generation of mammalian cell factories for biopharmaceutical production will be genetically engineered to possess both generic and product-specific manufacturing capabilities that may not exist naturally. Introduction of entirely new combinations of synthetic functions (e.g. novel metabolic or stress-response pathways), and retro-engineering of existing functional cell modules will drive disruptive change in cellular manufacturing performance. However, before we can apply the core concepts underpinning synthetic biology (design, build, test) to CHO cell engineering we must first develop practical and robust enabling technologies. Fundamentally, we will require the ability to precisely control the relative stoichiometry of numerous functional components we simultaneously introduce into the host cell factory. In this review we discuss how this can be achieved by design of engineered promoters that enable concerted control of recombinant gene transcription. We describe the specific mechanisms of transcriptional regulation that affect promoter function during bioproduction processes, and detail the highly-specific promoter design criteria that are required in the context of CHO cell engineering. The relative applicability of diverse promoter development strategies are discussed, including re-engineering of natural sequences, design of synthetic transcription factor-based systems, and construction of synthetic promoters. This review highlights the potential of promoter engineering to achieve precision transcriptional control for CHO cell synthetic biology. Copyright © 2015. Published by Elsevier Inc.

[Cellular microparticles, potential useful biomarkers in the identification of cerebrovascular accidents].

PubMed

Anglés-Cano, Eduardo; Vivien, Denis

2009-10-01

The clinical utility of biomarkers depends on their ability to identify high-risk individuals in order to establish preventive, diagnostic or therapeutic measures. Currently, no practical, rapid and sensitive test is available for the diagnosis of acute ischemic stroke. A number of soluble molecules have been identified that are merely associated to these cerebrovascular accidents. Despite this association not a single molecule has the characteristics of a true biomarker directly involved in the pathophysiology of ischemic stroke-none of them is organ-specific and may therefore be elevated in the context of medical comorbidities. When explored as a combination of biomarkers, e.g. matrix metalloproteinase 9, brain natriuretic protein, D-dimer, protein S100B, the question still remains whether serial biomarker analysis provides additional prognostic information. Even S100B, a glial activation protein, has a low specificity for acute ischemic stroke because it may originate from extracranial sources. Current knowledge from the field of cell-derived microparticles suggests that these membrane fragments may represent reliable biomarkers as they are cell-specific and are released early in the pathophysiological cascade of a disease. These microparticles can be found not only in the cerebrospinal fluid but also in tears and circulating blood in case of blood-brain barrier dysfunction. They represent a new challenge in stroke diagnosis and management.

Ligand-free palladium-mediated site-specific protein labeling inside gram-negative bacterial pathogens.

PubMed

Li, Jie; Lin, Shixian; Wang, Jie; Jia, Shang; Yang, Maiyun; Hao, Ziyang; Zhang, Xiaoyu; Chen, Peng R

2013-05-15

Palladium, a key transition metal in advancing modern organic synthesis, mediates diverse chemical conversions including many carbon-carbon bond formation reactions between organic compounds. However, expanding palladium chemistry for conjugation of biomolecules such as proteins, particularly within their native cellular context, is still in its infancy. Here we report the site-specific protein labeling inside pathogenic Gram-negative bacterial cells via a ligand-free palladium-mediated cross-coupling reaction. Two rationally designed pyrrolysine analogues bearing an aliphatic alkyne or an iodophenyl handle were first encoded in different enteric bacteria, which offered two facial handles for palladium-mediated Sonogashira coupling reaction on proteins within these pathogens. A GFP-based bioorthogonal reaction screening system was then developed, allowing evaluation of both the efficiency and the biocompatibilty of various palladium reagents in promoting protein-small molecule conjugation. The identified simple compound-Pd(NO3)2 exhibited high efficiency and biocompatibility for site-specific labeling of proteins in vitro and inside living E. coli cells. This Pd-mediated protein coupling method was further utilized to label and visualize a Type-III Secretion (T3S) toxin-OspF in Shigella cells. Our strategy may be generally applicable for imaging and tracking various virulence proteins within Gram-negative bacterial pathogens.

Sequence-dependent catalytic regulation of the SpoIIIE motor activity ensures directionality of DNA translocation.

PubMed

Chara, Osvaldo; Borges, Augusto; Milhiet, Pierre-Emmanuel; Nöllmann, Marcelo; Cattoni, Diego I

2018-03-27

Transport of cellular cargo by molecular motors requires directionality to ensure proper biological functioning. During sporulation in Bacillus subtilis, directionality of chromosome transport is mediated by the interaction between the membrane-bound DNA translocase SpoIIIE and specific octameric sequences (SRS). Whether SRS regulate directionality by recruiting and orienting SpoIIIE or by simply catalyzing its translocation activity is still unclear. By using atomic force microscopy and single-round fast kinetics translocation assays we determined the localization and dynamics of diffusing and translocating SpoIIIE complexes on DNA with or without SRS. Our findings combined with mathematical modelling revealed that SpoIIIE directionality is not regulated by protein recruitment to SRS but rather by a fine-tuned balance among the rates governing SpoIIIE-DNA interactions and the probability of starting translocation modulated by SRS. Additionally, we found that SpoIIIE can start translocation from non-specific DNA, providing an alternative active search mechanism for SRS located beyond the exploratory length defined by 1D diffusion. These findings are relevant in vivo in the context of chromosome transport through an open channel, where SpoIIIE can rapidly explore DNA while directionality is modulated by the probability of translocation initiation upon interaction with SRS versus non-specific DNA.

Renewal after the punishment of free operant behavior.

PubMed

Bouton, Mark E; Schepers, Scott T

2015-01-01

Three experiments examined the role of context in punishment learning. In Experiment 1, rats were trained to lever press for food in Context A and then punished for responding in Context B (by presenting response-contingent footshock). Punishment led to complete suppression of the response. However, when responding was tested (in extinction) in Contexts A and B, a strong renewal of responding occurred in Context A. In Experiment 2, renewal also occurred when initial reinforcement occurred in Context A, punishment occurred in Context B, and testing occurred in a new context (Context C). In both experiments, behavioral suppression and renewal were not observed in groups that received noncontingent (yoked) footshocks in Context B. In Experiment 3, 2 responses (lever press and chain pull) were separately reinforced in Contexts A and B and then punished in the opposite context. Although the procedure equated the contexts on their association with reinforcement and punishment, renewal of each response was observed when it was tested in its nonpunished context. The contexts also influenced response choice. Overall, the results suggest that punishment is specific to the context in which it is learned, and establish that its context-specificity does not depend on a simple association between the context and shock. Like extinction, punishment may involve learning to inhibit a specific response in a specific context. Implications for theories of punishment and for understanding the cessation of problematic operant behavior (e.g., drug abuse) are discussed. PsycINFO Database Record (c) 2015 APA, all rights reserved.

Morphological changes of placental syncytium and their implications for the pathogenesis of preeclampsia.

PubMed

Roland, Cynthia S; Hu, Jian; Ren, Chun-E; Chen, Haibin; Li, Jinping; Varvoutis, Megan S; Leaphart, Lynn W; Byck, David B; Zhu, Xueqiong; Jiang, Shi-Wen

2016-01-01

Preeclampsia is a hypertensive disease that complicates many pregnancies, typically presenting with new-onset or worsening hypertension and proteinuria. It is well recognized that the placental syncytium plays a key role in the pathogenesis of preeclampsia. This review summarizes the findings pertaining to the structural alterations in the syncytium of preeclamptic placentas and analyzes their pathological implications for the development of preeclampsia. Changes in the trophoblastic lineage, including those in the proliferation of cytotrophoblasts, the formation of syncytiotrophoblast through cell fusion, cell apoptosis and syncytial deportation, are discussed in the context of preeclampsia. Extensive correlations are made between functional deficiencies and the alterations on the levels of gross anatomy, tissue histology, cellular events, ultrastructure, molecular pathways, and gene expression. Attention is given to the significance of dynamic changes in the syncytial turnover in preeclamptic placentas. Specifically, experimental evidences for the complex and obligatory role of syncytin-1 in cell fusion, cell-cycle regulation at the G1/S transition, and apoptosis through AIF-mediated pathway, are discussed in detail in the context of syncytium homeostasis. Finally, the recent observations on the aberrant fibrin deposition in the trophoblastic layer and the trophoblast immature phenotype in preeclamptic placentas and their potential pathogenic impact are also reviewed.

Genome-wide localization and expression profiling establish Sp2 as a sequence-specific transcription factor regulating vitally important genes

PubMed Central

Terrados, Gloria; Finkernagel, Florian; Stielow, Bastian; Sadic, Dennis; Neubert, Juliane; Herdt, Olga; Krause, Michael; Scharfe, Maren; Jarek, Michael; Suske, Guntram

2012-01-01

The transcription factor Sp2 is essential for early mouse development and for proliferation of mouse embryonic fibroblasts in culture. Yet its mechanisms of action and its target genes are largely unknown. In this study, we have combined RNA interference, in vitro DNA binding, chromatin immunoprecipitation sequencing and global gene-expression profiling to investigate the role of Sp2 for cellular functions, to define target sites and to identify genes regulated by Sp2. We show that Sp2 is important for cellular proliferation that it binds to GC-boxes and occupies proximal promoters of genes essential for vital cellular processes including gene expression, replication, metabolism and signalling. Moreover, we identified important key target genes and cellular pathways that are directly regulated by Sp2. Most significantly, Sp2 binds and activates numerous sequence-specific transcription factor and co-activator genes, and represses the whole battery of cholesterol synthesis genes. Our results establish Sp2 as a sequence-specific regulator of vitally important genes. PMID:22684502

Argonaute quenching and global changes in Dicer homeostasis caused by a pathogen-encoded GW repeat protein

PubMed Central

Azevedo, Jacinthe; Garcia, Damien; Pontier, Dominique; Ohnesorge, Stephanie; Yu, Agnes; Garcia, Shahinez; Braun, Laurence; Bergdoll, Marc; Hakimi, Mohamed Ali; Lagrange, Thierry; Voinnet, Olivier

2010-01-01

In plants and invertebrates, viral-derived siRNAs processed by the RNaseIII Dicer guide Argonaute (AGO) proteins as part of antiviral RNA-induced silencing complexes (RISC). As a counterdefense, viruses produce suppressor proteins (VSRs) that inhibit the host silencing machinery, but their mechanisms of action and cellular targets remain largely unknown. Here, we show that the Turnip crinckle virus (TCV) capsid, the P38 protein, acts as a homodimer, or multiples thereof, to mimic host-encoded glycine/tryptophane (GW)-containing proteins normally required for RISC assembly/function in diverse organisms. The P38 GW residues bind directly and specifically to Arabidopsis AGO1, which, in addition to its role in endogenous microRNA-mediated silencing, is identified as a major effector of TCV-derived siRNAs. Point mutations in the P38 GW residues are sufficient to abolish TCV virulence, which is restored in Arabidopsis ago1 hypomorphic mutants, uncovering both physical and genetic interactions between the two proteins. We further show how AGO1 quenching by P38 profoundly impacts the cellular availability of the four Arabidopsis Dicers, uncovering an AGO1-dependent, homeostatic network that functionally connects these factors together. The likely widespread occurrence and expected consequences of GW protein mimicry on host silencing pathways are discussed in the context of innate and adaptive immunity in plants and metazoans. PMID:20439431

Treatment of catastrophic antiphospholipid syndrome with defibrotide, a proposed vascular endothelial cell modulator.

PubMed

Burcoglu-O'Ral, Arsinur; Erkan, Doruk; Asherson, Ronald

2002-09-01

To define at the molecular level the vascular endothelial cell (VEC) injury characteristics of catastrophic antiphospholipid syndrome (CAPS) and to report successful therapeutic use of a VEC modulator, defibrotide. We describe a 55-year-old man with primary APS with an intractable prothrombotic state (CAPS) resistant to combined therapy with heparin, warfarin, aspirin, and dipyridamole. Treatment with defibrotide was conducted in the context of an investigational phase II protocol where the dose was regulated and individualized by disease/patient-specific molecular and clinical markers. The patient entered complete remission with defibrotide treatment. During treatment, dose dependent pharmacological actions of defibrotide and key stress markers for VEC injury were identified. Evidence of defibrotide's polypharmacology included downregulation of cytokines, notably tumor necrosis factor-alpha, as the earliest effect, cellular differentiation of VEC, possibly with direct regulatory effect over cellular genes, and the reversal of platelet consumption and prothrombotic state. Von Willebrand antigen levels were used as the sole marker to guide therapy. This case demonstrates effective remission of CAPS with defibrotide treatment. In contrast to theories that CAPS is triggered by ischemic and thrombotic tissue damage, these data present VEC injury as the primary and representative lesion of CAPS. The pathogenesis may involve concurrent impairment of different VEC functions. Achieving remission may require a polypharmacologic approach, represented here by use of defibrotide.

Generation of a stable cell line for constitutive miRNA expression.

PubMed

Lieber, Diana

2013-01-01

miRNAs have in recent years emerged as novel players in virus-host interactions. While individual miRNAs are capable of regulating many targets simultaneously, not much is known about the role of distinct host or viral miRNAs in the context of infection. Analysis of the function of a miRNA is often hampered by the complexity of virus-host interactions and the enormous changes in the host cell during infection. Many viral miRNAs as for example from Kaposi sarcoma-associated Herpesvirus (KSHV) are probably exclusively expressed in latent infection. This might lead to a steady-state situation with offense and defense mechanisms counteracting each other. Cellular miRNAs involved in defense against pathogens on the other hand might be suppressed in infection. A cell culture system allowing for constitutive expression of individual miRNAs at high levels is a useful tool to enhance miRNA-specific functions and to uncouple viral miRNA function from other infection-related mechanisms. Here, a protocol is described to generate stable cell lines for constitutive expression of single cellular or viral miRNA precursors in absence of infection. The procedure comprises cloning of the precursor sequence, generation of the lentiviral expression vector, transduction of the cells of interest, selection for polyclonal cell lines, and isolation of monoclonal cell lines by limiting dilution.

A Novel ATM/TP53/p21-Mediated Checkpoint Only Activated by Chronic γ-Irradiation

PubMed Central

Sasatani, Megumi; Iizuka, Daisuke; Masuda, Yuji; Inaba, Toshiya; Suzuki, Keiji; Ootsuyama, Akira; Umata, Toshiyuki; Kamiya, Kenji; Suzuki, Fumio

2014-01-01

Different levels or types of DNA damage activate distinct signaling pathways that elicit various cellular responses, including cell-cycle arrest, DNA repair, senescence, and apoptosis. Whereas a range of DNA-damage responses have been characterized, mechanisms underlying subsequent cell-fate decision remain elusive. Here we exposed cultured cells and mice to different doses and dose rates of γ-irradiation, which revealed cell-type-specific sensitivities to chronic, but not acute, γ-irradiation. Among tested cell types, human fibroblasts were associated with the highest levels of growth inhibition in response to chronic γ-irradiation. In this context, fibroblasts exhibited a reversible G1 cell-cycle arrest or an irreversible senescence-like growth arrest, depending on the irradiation dose rate or the rate of DNA damage. Remarkably, when the same dose of γ-irradiation was delivered chronically or acutely, chronic delivery induced considerably more cellular senescence. A similar effect was observed with primary cells isolated from irradiated mice. We demonstrate a critical role for the ataxia telangiectasia mutated (ATM)/tumor protein p53 (TP53)/p21 pathway in regulating DNA-damage-associated cell fate. Indeed, blocking the ATM/TP53/p21 pathway deregulated DNA damage responses, leading to micronucleus formation in chronically irradiated cells. Together these results provide insights into the mechanisms governing cell-fate determination in response to different rates of DNA damage. PMID:25093836

USNCTAM perspectives on mechanics in medicine

PubMed Central

Bao, Gang; Bazilevs, Yuri; Chung, Jae-Hyun; Decuzzi, Paolo; Espinosa, Horacio D.; Ferrari, Mauro; Gao, Huajian; Hossain, Shaolie S.; Hughes, Thomas J. R.; Kamm, Roger D.; Liu, Wing Kam; Marsden, Alison; Schrefler, Bernhard

2014-01-01

Over decades, the theoretical and applied mechanics community has developed sophisticated approaches for analysing the behaviour of complex engineering systems. Most of these approaches have targeted systems in the transportation, materials, defence and energy industries. Applying and further developing engineering approaches for understanding, predicting and modulating the response of complicated biomedical processes not only holds great promise in meeting societal needs, but also poses serious challenges. This report, prepared for the US National Committee on Theoretical and Applied Mechanics, aims to identify the most pressing challenges in biological sciences and medicine that can be tackled within the broad field of mechanics. This echoes and complements a number of national and international initiatives aiming at fostering interdisciplinary biomedical research. This report also comments on cultural/educational challenges. Specifically, this report focuses on three major thrusts in which we believe mechanics has and will continue to have a substantial impact. (i) Rationally engineering injectable nano/microdevices for imaging and therapy of disease. Within this context, we discuss nanoparticle carrier design, vascular transport and adhesion, endocytosis and tumour growth in response to therapy, as well as uncertainty quantification techniques to better connect models and experiments. (ii) Design of biomedical devices, including point-of-care diagnostic systems, model organ and multi-organ microdevices, and pulsatile ventricular assistant devices. (iii) Mechanics of cellular processes, including mechanosensing and mechanotransduction, improved characterization of cellular constitutive behaviour, and microfluidic systems for single-cell studies. PMID:24872502

Doctoral conceptual thresholds in cellular and molecular biology

NASA Astrophysics Data System (ADS)

Feldon, David F.; Rates, Christopher; Sun, Chongning

2017-12-01

In the biological sciences, very little is known about the mechanisms by which doctoral students acquire the skills they need to become independent scientists. In the postsecondary biology education literature, identification of specific skills and effective methods for helping students to acquire them are limited to undergraduate education. To establish a foundation from which to investigate the developmental trajectory of biologists' research skills, it is necessary to identify those skills which are integral to doctoral study and distinct from skills acquired earlier in students' educational pathways. In this context, the current study engages the framework of threshold concepts to identify candidate skills that are both obstacles and significant opportunities for developing proficiency in conducting research. Such threshold concepts are typically characterised as transformative, integrative, irreversible, and challenging. The results from interviews and focus groups with current and former doctoral students in cellular and molecular biology suggest two such threshold concepts relevant to their subfield: the first is an ability to effectively engage primary research literature from the biological sciences in a way that is critical without dismissing the value of its contributions. The second is the ability to conceptualise appropriate control conditions necessary to design and interpret the results of experiments in an efficient and effective manner for research in the biological sciences as a discipline. Implications for prioritising and sequencing graduate training experiences are discussed on the basis of the identified thresholds.

Rho2 Palmitoylation Is Required for Plasma Membrane Localization and Proper Signaling to the Fission Yeast Cell Integrity Mitogen-Activated Protein Kinase Pathway

PubMed Central

Sánchez-Mir, Laura; Franco, Alejandro; Martín-García, Rebeca; Madrid, Marisa; Vicente-Soler, Jero; Soto, Teresa; Gacto, Mariano; Pérez, Pilar

2014-01-01

The fission yeast small GTPase Rho2 regulates morphogenesis and is an upstream activator of the cell integrity pathway, whose key element, mitogen-activated protein kinase (MAPK) Pmk1, becomes activated by multiple environmental stimuli and controls several cellular functions. Here we demonstrate that farnesylated Rho2 becomes palmitoylated in vivo at cysteine-196 within its carboxyl end and that this modification allows its specific targeting to the plasma membrane. Unlike that of other palmitoylated and prenylated GTPases, the Rho2 control of morphogenesis and Pmk1 activity is strictly dependent upon plasma membrane localization and is not found in other cellular membranes. Indeed, artificial plasma membrane targeting bypassed the Rho2 need for palmitoylation in order to signal. Detailed functional analysis of Rho2 chimeras fused to the carboxyl end from the essential GTPase Rho1 showed that GTPase palmitoylation is partially dependent on the prenylation context and confirmed that Rho2 signaling is independent of Rho GTP dissociation inhibitor (GDI) function. We further demonstrate that Rho2 is an in vivo substrate for DHHC family acyltransferase Erf2 palmitoyltransferase. Remarkably, Rho3, another Erf2 target, negatively regulates Pmk1 activity in a Rho2-independent fashion, thus revealing the existence of cross talk whereby both GTPases antagonistically modulate the activity of this MAPK cascade. PMID:24820419

Entorhinal Cortical Ocean Cells Encode Specific Contexts and Drive Context-Specific Fear Memory

PubMed Central

Kitamura, Takashi; Sun, Chen; Martin, Jared; Kitch, Lacey J; Schnitzer, Mark J; Tonegawa, Susumu

2016-01-01

Summary Forming distinct representations and memories of multiple contexts and episodes is thought to be a crucial function of the hippocampal-entorhinal cortical network. The hippocampal dentate gyrus (DG) and CA3 are known to contribute to these functions but the role of the entorhinal cortex (EC) is poorly understood. Here, we show that Ocean cells, excitatory stellate neurons in the medial EC layer II projecting into DG and CA3, rapidly form a distinct representation of a novel context and drive context-specific activation of downstream CA3 cells as well as context-specific fear memory. In contrast, Island cells, excitatory pyramidal neurons in the medial EC layer II projecting into CA1, are indifferent to context-specific encoding or memory. On the other hand, Ocean cells are dispensable for temporal association learning, for which Island cells are crucial. Together, the two excitatory medial EC layer II inputs to the hippocampus have complementary roles in episodic memory. PMID:26402611

Endoplasmic reticulum mediated signaling in cellular microdomains

PubMed Central

Biwer, Lauren; Isakson, Brant E

2016-01-01

The endoplasmic reticulum (ER) is a prime mediator of cellular signaling due to its functions as an internal cellular store for calcium, as well as a site for synthesis of proteins and lipids. Its peripheral network of sheets and tubules facilitate calcium and lipid signaling, especially in areas of the cell that are more distant to the main cytoplasmic network. Specific membrane proteins shape the peripheral ER architecture and influence the network stability in order to project into restricted spaces. The signaling microdomains are anatomically separate from the cytoplasm as a whole and exhibit localized protein, ion channel and cytoskeletal element expression. Signaling can also occur between the ER and other organelles, such as the Golgi or mitochondria. Lipids made in the ER membrane can be sent to the Golgi via specialized transfer proteins and specific phospholipid synthases are enriched at ER-mitochondria junctions to more efficiently expedite phospholipid transfer. As a hub for protein and lipid synthesis, a store for intracellular calcium [Ca2+]i, and a mediator of cellular stress, the ER is an important cellular organelle. Its ability to organize into tubules and project into restricted spaces allows for discrete and temporal signaling, which is important for cellular physiology and organism homeostasis. PMID:26973141

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

Partial and specific source memory for faces associated to other- and self-relevant negative contexts.

PubMed

Bell, Raoul; Giang, Trang; Buchner, Axel

2012-01-01

Previous research has shown a source memory advantage for faces presented in negative contexts. As yet it remains unclear whether participants remember the specific type of context in which the faces were presented or whether they can only remember that the face was associated with negative valence. In the present study, participants saw faces together with descriptions of two different types of negative behaviour and neutral behaviour. In Experiment 1, we examined whether the participants were able to discriminate between two types of other-relevant negative context information (cheating and disgusting behaviour) in a source memory test. In Experiment 2, we assessed source memory for other-relevant negative (threatening) context information (other-aggressive behaviour) and self-relevant negative context information (self-aggressive behaviour). A multinomial source memory model was used to separately assess partial source memory for the negative valence of the behaviour and specific source memory for the particular type of negative context the face was associated with. In Experiment 1, source memory was specific for the particular type of negative context presented (i.e., cheating or disgusting behaviour). Experiment 2 showed that source memory for other-relevant negative information was more specific than source memory for self-relevant information. Thus, emotional source memory may vary in specificity depending on the degree to which the negative emotional context is perceived as threatening.

CAM: A high-performance cellular-automaton machine

NASA Astrophysics Data System (ADS)

Toffoli, Tommaso

1984-01-01

CAM is a high-performance machine dedicated to the simulation of cellular automata and other distributed dynamical systems. Its speed is about one-thousand times greater than that of a general-purpose computer programmed to do the same task; in practical terms, this means that CAM can show the evolution of cellular automata on a color monitor with an update rate, dynamic range, and spatial resolution comparable to those of a Super-8 movie, thus permitting intensive interactive experimentation. Machines of this kind can open up novel fields of research, and in this context it is important that results be easy to obtain, reproduce, and transmit. For these reasons, in designing CAM it was important to achieve functional simplicity, high flexibility, and moderate production cost. We expect that many research groups will be able to own their own copy of the machine to do research with.

Driven to distraction: dual-Task studies of simulated driving and conversing on a cellular telephone.

PubMed

Strayer, D L; Johnston, W A

2001-11-01

Dual-task studies assessed the effects of cellular-phone conversations on performance of a simulated driving task. Performance was not disrupted by listening to radio broadcasts or listening to a book on tape. Nor was it disrupted by a continuous shadowing task using a handheld phone, ruling out, in this case, dual-task interpretations associated with holding the phone, listening, or speaking, However significant interference was observed in a word-generation variant of the shadowing task, and this deficit increased with the difficulty of driving. Moreover unconstrained conversations using either a handheld or a hands-free cell phone resulted in a twofold increase in the failure to detect simulated traffic signals and slower reactions to those signals that were detected. We suggest that cellular-phone use disrupts performance by diverting attention to an engaging cognitive context other than the one immediately associated with driving.

Geminiviruses and Plant Hosts: A Closer Examination of the Molecular Arms Race.

PubMed

Ramesh, Shunmugiah V; Sahu, Pranav P; Prasad, Manoj; Praveen, Shelly; Pappu, Hanu R

2017-09-15

Geminiviruses are plant-infecting viruses characterized by a single-stranded DNA (ssDNA) genome. Geminivirus-derived proteins are multifunctional and effective regulators in modulating the host cellular processes resulting in successful infection. Virus-host interactions result in changes in host gene expression patterns, reprogram plant signaling controls, disrupt central cellular metabolic pathways, impair plant's defense system, and effectively evade RNA silencing response leading to host susceptibility. This review summarizes what is known about the cellular processes in the continuing tug of war between geminiviruses and their plant hosts at the molecular level. In addition, implications for engineered resistance to geminivirus infection in the context of a greater understanding of the molecular processes are also discussed. Finally, the prospect of employing geminivirus-based vectors in plant genome engineering and the emergence of powerful genome editing tools to confer geminivirus resistance are highlighted to complete the perspective on geminivirus-plant molecular interactions.

Spatial reconstruction of single-cell gene expression data.

PubMed

Satija, Rahul; Farrell, Jeffrey A; Gennert, David; Schier, Alexander F; Regev, Aviv

2015-05-01

Spatial localization is a key determinant of cellular fate and behavior, but methods for spatially resolved, transcriptome-wide gene expression profiling across complex tissues are lacking. RNA staining methods assay only a small number of transcripts, whereas single-cell RNA-seq, which measures global gene expression, separates cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos and generated a transcriptome-wide map of spatial patterning. We confirmed Seurat's accuracy using several experimental approaches, then used the strategy to identify a set of archetypal expression patterns and spatial markers. Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems.

Protein design in systems metabolic engineering for industrial strain development.

PubMed

Chen, Zhen; Zeng, An-Ping

2013-05-01

Accelerating the process of industrial bacterial host strain development, aimed at increasing productivity, generating new bio-products or utilizing alternative feedstocks, requires the integration of complementary approaches to manipulate cellular metabolism and regulatory networks. Systems metabolic engineering extends the concept of classical metabolic engineering to the systems level by incorporating the techniques used in systems biology and synthetic biology, and offers a framework for the development of the next generation of industrial strains. As one of the most useful tools of systems metabolic engineering, protein design allows us to design and optimize cellular metabolism at a molecular level. Here, we review the current strategies of protein design for engineering cellular synthetic pathways, metabolic control systems and signaling pathways, and highlight the challenges of this subfield within the context of systems metabolic engineering. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Kinesins and Myosins: Molecular Motors that Coordinate Cellular Functions in Plants.

PubMed

Nebenführ, Andreas; Dixit, Ram

2018-04-29

Kinesins and myosins are motor proteins that can move actively along microtubules and actin filaments, respectively. Plants have evolved a unique set of motors that function as regulators and organizers of the cytoskeleton and as drivers of long-distance transport of various cellular components. Recent progress has established the full complement of motors encoded in plant genomes and has revealed valuable insights into the cellular functions of many kinesin and myosin isoforms. Interestingly, several of the motors were found to functionally connect the two cytoskeletal systems and thereby to coordinate their activities. In this review, we discuss the available genetic, cell biological, and biochemical data for each of the plant kinesin and myosin families from the context of their subcellular mechanism of action as well as their physiological function in the whole plant. We particularly emphasize work that illustrates mechanisms by which kinesins and myosins coordinate the activities of the cytoskeletal system.

Motifs, modules and games in bacteria.

PubMed

Wolf, Denise M; Arkin, Adam P

2003-04-01

Global explorations of regulatory network dynamics, organization and evolution have become tractable thanks to high-throughput sequencing and molecular measurement of bacterial physiology. From these, a nascent conceptual framework is developing, that views the principles of regulation in term of motifs, modules and games. Motifs are small, repeated, and conserved biological units ranging from molecular domains to small reaction networks. They are arranged into functional modules, genetically dissectible cellular functions such as the cell cycle, or different stress responses. The dynamical functioning of modules defines the organism's strategy to survive in a game, pitting cell against cell, and cell against environment. Placing pathway structure and dynamics into an evolutionary context begins to allow discrimination between those physical and molecular features that particularize a species to its surroundings, and those that provide core physiological function. This approach promises to generate a higher level understanding of cellular design, pathway evolution and cellular bioengineering.

Cellular Mechanisms of Somatic Stem Cell Aging

PubMed Central

Jung, Yunjoon

2014-01-01

Tissue homeostasis and regenerative capacity rely on rare populations of somatic stem cells endowed with the potential to self-renew and differentiate. During aging, many tissues show a decline in regenerative potential coupled with a loss of stem cell function. Cells including somatic stem cells have evolved a series of checks and balances to sense and repair cellular damage to maximize tissue function. However, during aging the mechanisms that protect normal cell function begin to fail. In this review, we will discuss how common cellular mechanisms that maintain tissue fidelity and organismal lifespan impact somatic stem cell function. We will highlight context-dependent changes and commonalities that define aging, by focusing on three age-sensitive stem cell compartments: blood, neural, and muscle. Understanding the interaction between extrinsic regulators and intrinsic effectors that operate within different stem cell compartments is likely to have important implications for identifying strategies to improve health span and treat age-related degenerative diseases. PMID:24439814

In silico method for modelling metabolism and gene product expression at genome scale

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

Lerman, Joshua A.; Hyduke, Daniel R.; Latif, Haythem

2012-07-03

Transcription and translation use raw materials and energy generated metabolically to create the macromolecular machinery responsible for all cellular functions, including metabolism. A biochemically accurate model of molecular biology and metabolism will facilitate comprehensive and quantitative computations of an organism's molecular constitution as a function of genetic and environmental parameters. Here we formulate a model of metabolism and macromolecular expression. Prototyping it using the simple microorganism Thermotoga maritima, we show our model accurately simulates variations in cellular composition and gene expression. Moreover, through in silico comparative transcriptomics, the model allows the discovery of new regulons and improving the genome andmore » transcription unit annotations. Our method presents a framework for investigating molecular biology and cellular physiology in silico and may allow quantitative interpretation of multi-omics data sets in the context of an integrated biochemical description of an organism.« less

Motifs, modules and games in bacteria

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

Wolf, Denise M.; Arkin, Adam P.

2003-04-01

Global explorations of regulatory network dynamics, organization and evolution have become tractable thanks to high-throughput sequencing and molecular measurement of bacterial physiology. From these, a nascent conceptual framework is developing, that views the principles of regulation in term of motifs, modules and games. Motifs are small, repeated, and conserved biological units ranging from molecular domains to small reaction networks. They are arranged into functional modules, genetically dissectible cellular functions such as the cell cycle, or different stress responses. The dynamical functioning of modules defines the organism's strategy to survive in a game, pitting cell against cell, and cell against environment.more » Placing pathway structure and dynamics into an evolutionary context begins to allow discrimination between those physical and molecular features that particularize a species to its surroundings, and those that provide core physiological function. This approach promises to generate a higher level understanding of cellular design, pathway evolution and cellular bioengineering.« less

Geminiviruses and Plant Hosts: A Closer Examination of the Molecular Arms Race

PubMed Central

Ramesh, Shunmugiah V.; Sahu, Pranav P.; Prasad, Manoj; Praveen, Shelly; Pappu, Hanu R.

2017-01-01

Geminiviruses are plant-infecting viruses characterized by a single-stranded DNA (ssDNA) genome. Geminivirus-derived proteins are multifunctional and effective regulators in modulating the host cellular processes resulting in successful infection. Virus-host interactions result in changes in host gene expression patterns, reprogram plant signaling controls, disrupt central cellular metabolic pathways, impair plant’s defense system, and effectively evade RNA silencing response leading to host susceptibility. This review summarizes what is known about the cellular processes in the continuing tug of war between geminiviruses and their plant hosts at the molecular level. In addition, implications for engineered resistance to geminivirus infection in the context of a greater understanding of the molecular processes are also discussed. Finally, the prospect of employing geminivirus-based vectors in plant genome engineering and the emergence of powerful genome editing tools to confer geminivirus resistance are highlighted to complete the perspective on geminivirus-plant molecular interactions. PMID:28914771

The interferon-induced antiviral protein PML (TRIM19) promotes the restriction and transcriptional silencing of lentiviruses in a context-specific, isoform-specific fashion.

PubMed

Masroori, Nasser; Merindol, Natacha; Berthoux, Lionel

2016-03-22

The promyelocytic leukemia (PML) protein, a type I interferon (IFN-I)-induced gene product and a member of the tripartite motif (TRIM) family, modulates the transcriptional activity of viruses belonging to various families. Whether PML has an impact on the replication of HIV-1 has not been fully addressed, but recent studies point to its possible involvement in the restriction of HIV-1 in human cells and in the maintenance of transcriptional latency in human cell lines in which HIV-1 is constitutively repressed. We investigated further the restriction of HIV-1 and a related lentivirus, SIVmac, by PML in murine cells and in a lymphocytic human cell line. In particular, we studied the relevance of PML to IFN-I-mediated inhibition and the role of individual human isoforms. We demonstrate that both human PML (hPML) and murine PML (mPML) inhibit the early post-entry stages of the replication of HIV-1 and a related lentivirus, SIVmac. In addition, HIV-1 was transcriptionally silenced by mPML and by hPML isoforms I, II, IV and VI in MEFs. This PML-mediated transcriptional repression was attenuated in presence of the histone deacetylase inhibitor SAHA. In contrast, depletion of PML had no effect on HIV-1 gene expression in a human T cell line. PML was found to contribute to the inhibition of HIV-1 by IFN-I. Specifically, IFN-α and IFN-β treatments of MEFs enhanced the PML-dependent inhibition of HIV-1 early replication stages. We show that PML can inhibit HIV-1 and other lentiviruses as part of the IFN-I-mediated response. The restriction takes place at two distinct steps, i.e. reverse transcription and transcription, and in an isoform-specific, cellular context-specific fashion. Our results support a model in which PML activates innate immune antilentiviral effectors. These data are relevant to the development of latency reversal-inducing pharmacological agents, since PML was previously proposed as a pharmacological target for such inhibitors. This study also has implications for the development of murine models of HIV-1.

Persistence at one year of age of antigen-induced cellular immune responses in preterm infants vaccinated against whooping cough: comparison of three different vaccines and effect of a booster dose.

PubMed

Vermeulen, Françoise; Dirix, Violette; Verscheure, Virginie; Damis, Eliane; Vermeylen, Danièle; Locht, Camille; Mascart, Françoise

2013-04-08

Due to their high risk of developing severe Bordetella pertussis (Bp) infections, it is recommended to immunize preterm infants at their chronological age. However, little is known about the persistence of their specific immune responses, especially of the cellular responses recognized to play a role in protection. We compared here the cellular immune responses to two major antigens of Bp between three groups of one year-old children born prematurely, who received for their primary vaccination respectively the whole cell vaccine Tetracoq(®) (TC), the acellular vaccine Tetravac(®) (TV), or the acellular vaccine Infanrix-hexa(®) (IR). Whereas most children had still detectable IFN-γ responses at one year of age, they were lower in the IR-vaccinated children compared to the two other groups. In contrast, both the TV- and the IR-vaccinated children displayed higher Th2-type immune responses, resulting in higher antigen-specific IFN-γ/IL-5 ratios in TC- than in TV- or IR-vaccinated children. The IFN-γ/IL-5 ratio of mitogen-induced cytokines was also lower in IR- compared to TC- or TV-vaccinated children. No major differences in the immune responses were noted after the booster compared to the pre-booster responses for each vaccine. The IR-vaccinated children had a persistently low specific Th1-type immune response associated with high specific Th2-type immune responses, resulting in lower antigen-specific IFN-γ/IL-5 ratios compared to the two other groups. We conclude that antigen-specific cellular immune responses persisted in one year-old children born prematurely and vaccinated during infancy at their chronological age, that a booster dose did not significantly boost the cellular immune responses, and that the Th1/Th2 balance of the immune responses is modulated by the different vaccines. Copyright © 2013 Elsevier Ltd. All rights reserved.

Oxidative stress, a trigger of hepatitis C and B virus-induced liver carcinogenesis

PubMed Central

Ivanov, Alexander V.; Valuev-Elliston, Vladimir T.; Tyurina, Daria A.; Ivanova, Olga N.; Kochetkov, Sergey N.; Bartosch, Birke; Isaguliants, Maria G.

2017-01-01

Virally induced liver cancer usually evolves over long periods of time in the context of a strongly oxidative microenvironment, characterized by chronic liver inflammation and regeneration processes. They ultimately lead to oncogenic mutations in many cellular signaling cascades that drive cell growth and proliferation. Oxidative stress, induced by hepatitis viruses, therefore is one of the factors that drives the neoplastic transformation process in the liver. This review summarizes current knowledge on oxidative stress and oxidative stress responses induced by human hepatitis B and C viruses. It focuses on the molecular mechanisms by which these viruses activate cellular enzymes/systems that generate or scavenge reactive oxygen species (ROS) and control cellular redox homeostasis. The impact of an altered cellular redox homeostasis on the initiation and establishment of chronic viral infection, as well as on the course and outcome of liver fibrosis and hepatocarcinogenesis will be discussed The review neither discusses reactive nitrogen species, although their metabolism is interferes with that of ROS, nor antioxidants as potential therapeutic remedies against viral infections, both subjects meriting an independent review. PMID:27965466

Adoptive cellular therapy for chronic lymphocytic leukemia and B cell malignancies. CARs and more.

PubMed

Castro, Januario E; Kipps, Thomas J

2016-03-01

Treatment of patients with chronic lymphocytic leukemia and other B cell malignancies is evolving very rapidly. We have observed the quick transition during the last couple of years, from chemo-immunotherapy based treatments to oral targeted therapies based on B cell receptor signaling and Bcl-2 inhibitors, as well as the increasing use of second generation glyco-engineered antibodies. The next wave of revolution in the treatment for this conditions is approaching and it will be based on strategies that harness the power of the immune system to fight cancer. In the center of this biotechnological revolution is cellular engineering, the field that had made possible to redirect the immune system effector cells to achieve a more effective and targeted adoptive cellular therapy. In this chapter, we will review the historical context of these scientific developments, the most recent basic and clinical research in the field and some opinions regarding the future of adoptive cellular therapy in CLL and other B cell malignancies. Copyright © 2016 Elsevier Ltd. All rights reserved.

A comparative cellular and molecular biology of longevity database.

PubMed

Stuart, Jeffrey A; Liang, Ping; Luo, Xuemei; Page, Melissa M; Gallagher, Emily J; Christoff, Casey A; Robb, Ellen L

2013-10-01

Discovering key cellular and molecular traits that promote longevity is a major goal of aging and longevity research. One experimental strategy is to determine which traits have been selected during the evolution of longevity in naturally long-lived animal species. This comparative approach has been applied to lifespan research for nearly four decades, yielding hundreds of datasets describing aspects of cell and molecular biology hypothesized to relate to animal longevity. Here, we introduce a Comparative Cellular and Molecular Biology of Longevity Database, available at ( http://genomics.brocku.ca/ccmbl/ ), as a compendium of comparative cell and molecular data presented in the context of longevity. This open access database will facilitate the meta-analysis of amalgamated datasets using standardized maximum lifespan (MLSP) data (from AnAge). The first edition contains over 800 data records describing experimental measurements of cellular stress resistance, reactive oxygen species metabolism, membrane composition, protein homeostasis, and genome homeostasis as they relate to vertebrate species MLSP. The purpose of this review is to introduce the database and briefly demonstrate its use in the meta-analysis of combined datasets.

Specific RNP capture with antisense LNA/DNA mixmers

PubMed Central

Rogell, Birgit; Fischer, Bernd; Rettel, Mandy; Krijgsveld, Jeroen; Castello, Alfredo; Hentze, Matthias W.

2017-01-01

RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe “specific ribonucleoprotein (RNP) capture,” a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein–RNA interactions taking place at “zero distance.” Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins. PMID:28476952

Specific RNP capture with antisense LNA/DNA mixmers.

PubMed

Rogell, Birgit; Fischer, Bernd; Rettel, Mandy; Krijgsveld, Jeroen; Castello, Alfredo; Hentze, Matthias W

2017-08-01

RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture," a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein-RNA interactions taking place at "zero distance." Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins. © 2017 Rogell et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

The cellular immune response of Daphnia magna under host-parasite genetic variation and variation in initial dose

PubMed Central

Auld, Stuart K. J. R; Edel, Kai H.; Little, Tom J.

2013-01-01

In invertebrate-parasite systems, the likelihood of infection following parasite exposure is often dependent on the specific combination of host and parasite genotypes (termed genetic specificity). Genetic specificity can maintain diversity in host and parasite populations and is a major component of the Red Queen hypothesis. However, invertebrate immune systems are thought to only distinguish between broad classes of parasite. Using a natural host-parasite system with a well-established pattern of genetic specificity, the crustacean Daphnia magna and its bacterial parasite Pasteuria ramosa, we found that only hosts from susceptible host-parasite genetic combinations mounted a cellular response following exposure to the parasite. These data are compatible with the hypothesis that genetic specificity is attributable to barrier defenses at the site of infection (the gut), and that the systemic immune response is general, reporting the number of parasite spores entering the hemocoel. Further supporting this, we found that larger cellular responses occurred at higher initial parasite doses. By studying the natural infection route, where parasites must pass barrier defenses before interacting with systemic immune responses, these data shed light on which components of invertebrate defense underlie genetic specificity. PMID:23025616

Contextual analysis of immunological response through whole-organ fluorescent imaging.

PubMed

Woodruff, Matthew C; Herndon, Caroline N; Heesters, B A; Carroll, Michael C

2013-09-01

As fluorescent microscopy has developed, significant insights have been gained into the establishment of immune response within secondary lymphoid organs, particularly in draining lymph nodes. While established techniques such as confocal imaging and intravital multi-photon microscopy have proven invaluable, they provide limited insight into the architectural and structural context in which these responses occur. To interrogate the role of the lymph node environment in immune response effectively, a new set of imaging tools taking into account broader architectural context must be implemented into emerging immunological questions. Using two different methods of whole-organ imaging, optical clearing and three-dimensional reconstruction of serially sectioned lymph nodes, fluorescent representations of whole lymph nodes can be acquired at cellular resolution. Using freely available post-processing tools, images of unlimited size and depth can be assembled into cohesive, contextual snapshots of immunological response. Through the implementation of robust iterative analysis techniques, these highly complex three-dimensional images can be objectified into sortable object data sets. These data can then be used to interrogate complex questions at the cellular level within the broader context of lymph node biology. By combining existing imaging technology with complex methods of sample preparation and capture, we have developed efficient systems for contextualizing immunological phenomena within lymphatic architecture. In combination with robust approaches to image analysis, these advances provide a path to integrating scientific understanding of basic lymphatic biology into the complex nature of immunological response.

Discrete Circuits Support Generalized versus Context-Specific Vocal Learning in the Songbird.

PubMed

Tian, Lucas Y; Brainard, Michael S

2017-12-06

Motor skills depend on the reuse of individual gestures in multiple sequential contexts (e.g., a single phoneme in different words). Yet optimal performance requires that a given gesture be modified appropriately depending on the sequence in which it occurs. To investigate the neural architecture underlying such context-dependent modifications, we studied Bengalese finch song, which, like speech, consists of variable sequences of "syllables." We found that when birds are instructed to modify a syllable in one sequential context, learning generalizes across contexts; however, if unique instruction is provided in different contexts, learning is specific for each context. Using localized inactivation of a cortical-basal ganglia circuit specialized for song, we show that this balance between generalization and specificity reflects a hierarchical organization of neural substrates. Primary motor circuitry encodes a core syllable representation that contributes to generalization, while top-down input from cortical-basal ganglia circuitry biases this representation to enable context-specific learning. Copyright © 2017 Elsevier Inc. All rights reserved.

From Syncitium to Regulated Pump: A Cardiac Muscle Cellular Update

ERIC Educational Resources Information Center

Korzick, Donna H.

2011-01-01

The primary purpose of this article is to present a basic overview of some key teaching concepts that should be considered for inclusion in an six- to eight-lecture introductory block on the regulation of cardiac performance for graduate students. Within the context of cardiac excitation-contraction coupling, this review incorporates information…

Cellular Basis of Mechanotransduction

NASA Technical Reports Server (NTRS)

Ingber, Donald E.

1996-01-01

Physical forces, such as those due to gravity are fundamental regulators of tissue development. To influence morphogenesis, mechanical forces must alter growth and function. Yet little is known about how cells convert mechanical signals into a chemical response. This presentation attempts to place the potential molecular mediators of mechanotransduction within the context of the structural complexity of living cells.

Postdoctoral Fellow | Center for Cancer Research

Cancer.gov

The Women's Malignancies Branch (WMB), Center for Cancer Research (CCR), National Cancer Institute (NCI) of the National Institutes of Health (NIH) is seeking outstanding postdoctoral candidates interested in studying DNA repair and cell cycle pathways in the context of ovarian cancer and drug resistance. Our broad goal is to explore the molecular and cellular mechanisms of

Transcriptional Enhancers Induce Insertional Gene Deregulation Independently From the Vector Type and Design

PubMed Central

Maruggi, Giulietta; Porcellini, Simona; Facchini, Giulia; Perna, Serena K; Cattoglio, Claudia; Sartori, Daniela; Ambrosi, Alessandro; Schambach, Axel; Baum, Christopher; Bonini, Chiara; Bovolenta, Chiara; Mavilio, Fulvio; Recchia, Alessandra

2009-01-01

The integration characteristics of retroviral (RV) vectors increase the probability of interfering with the regulation of cellular genes, and account for a tangible risk of insertional mutagenesis in treated patients. To assess the potential genotoxic risk of conventional or self-inactivating (SIN) γ-RV and lentiviral (LV) vectors independently from the biological consequences of the insertion event, we developed a quantitative assay based on real-time reverse transcriptase—PCR on low-density arrays to evaluate alterations of gene expression in individual primary T-cell clones. We show that the Moloney leukemia virus long terminal repeat (LTR) enhancer has the strongest activity in both a γ-RV and a LV vector context, while an internal cellular promoter induces deregulation of gene expression less frequently, at a shorter range and to a lower extent in both vector types. Downregulation of gene expression was observed only in the context of LV vectors. This study indicates that insertional gene activation is determined by the characteristics of the transcriptional regulatory elements carried by the vector, and is largely independent from the vector type or design. PMID:19293778

A Comparative Analysis of Drug-Induced Hepatotoxicity in Clinically Relevant Situations

PubMed Central

Thiel, Christoph; Cordes, Henrik; Fabbri, Lorenzo; Aschmann, Hélène Eloise; Baier, Vanessa; Atkinson, Francis; Blank, Lars Mathias; Kuepfer, Lars

2017-01-01

Drug-induced toxicity is a significant problem in clinical care. A key problem here is a general understanding of the molecular mechanisms accompanying the transition from desired drug effects to adverse events following administration of either therapeutic or toxic doses, in particular within a patient context. Here, a comparative toxicity analysis was performed for fifteen hepatotoxic drugs by evaluating toxic changes reflecting the transition from therapeutic drug responses to toxic reactions at the cellular level. By use of physiologically-based pharmacokinetic modeling, in vitro toxicity data were first contextualized to quantitatively describe time-resolved drug responses within a patient context. Comparatively studying toxic changes across the considered hepatotoxicants allowed the identification of subsets of drugs sharing similar perturbations on key cellular processes, functional classes of genes, and individual genes. The identified subsets of drugs were next analyzed with regard to drug-related characteristics and their physicochemical properties. Toxic changes were finally evaluated to predict both molecular biomarkers and potential drug-drug interactions. The results may facilitate the early diagnosis of adverse drug events in clinical application. PMID:28151932

Monitoring developmental force distributions in reconstituted embryonic epithelia.

PubMed

Przybyla, L; Lakins, J N; Sunyer, R; Trepat, X; Weaver, V M

2016-02-01

The way cells are organized within a tissue dictates how they sense and respond to extracellular signals, as cues are received and interpreted based on expression and organization of receptors, downstream signaling proteins, and transcription factors. Part of this microenvironmental context is the result of forces acting on the cell, including forces from other cells or from the cellular substrate or basement membrane. However, measuring forces exerted on and by cells is difficult, particularly in an in vivo context, and interpreting how forces affect downstream cellular processes poses an even greater challenge. Here, we present a simple method for monitoring and analyzing forces generated from cell collectives. We demonstrate the ability to generate traction force data from human embryonic stem cells grown in large organized epithelial sheets to determine the magnitude and organization of cell-ECM and cell-cell forces within a self-renewing colony. We show that this method can be used to measure forces in a dynamic hESC system and demonstrate the ability to map intracolony protein localization to force organization. Copyright © 2015 Elsevier Inc. All rights reserved.

Neuro-glial crosstalk in inflammatory bowel disease.

PubMed

Neunlist, M; Van Landeghem, L; Bourreille, A; Savidge, T

2008-06-01

Inflammatory bowel disease (IBD) is a multifactorial disease in which environmental, immune and genetic factors are involved in the pathogenesis. Although biological therapies (antibodies anti-tumour necrosis factor-alpha or anti-integrin) have considerably improved the symptoms and quality of life of IBD patients, some drawbacks have emerged limiting their long-term use. In addition, prevention of relapses and treatment of resistant ulcers remains a clinical challenge. In this context, a better understanding of the pathophysiology of IBD and the development of novel therapeutic intervention would benefit from further basic and preclinical research into the role of the cellular microenvironment and the interaction between its cellular constituents. In this context, the role of the enteric nervous system (ENS) in the regulation of the intestinal epithelial barrier (IEB) and the gut immune response has fuelled an increased interest in the last few years. Recent advances, summarized in this review, have highlighted the ENS as playing a key role in the control of IEB functions and gut immune homeostasis, and that alterations of the ENS could be directly associated in the development of IBD and its associated symptoms.

Predicting multicellular function through multi-layer tissue networks

PubMed Central

Zitnik, Marinka; Leskovec, Jure

2017-01-01

Abstract Motivation: Understanding functions of proteins in specific human tissues is essential for insights into disease diagnostics and therapeutics, yet prediction of tissue-specific cellular function remains a critical challenge for biomedicine. Results: Here, we present OhmNet, a hierarchy-aware unsupervised node feature learning approach for multi-layer networks. We build a multi-layer network, where each layer represents molecular interactions in a different human tissue. OhmNet then automatically learns a mapping of proteins, represented as nodes, to a neural embedding-based low-dimensional space of features. OhmNet encourages sharing of similar features among proteins with similar network neighborhoods and among proteins activated in similar tissues. The algorithm generalizes prior work, which generally ignores relationships between tissues, by modeling tissue organization with a rich multiscale tissue hierarchy. We use OhmNet to study multicellular function in a multi-layer protein interaction network of 107 human tissues. In 48 tissues with known tissue-specific cellular functions, OhmNet provides more accurate predictions of cellular function than alternative approaches, and also generates more accurate hypotheses about tissue-specific protein actions. We show that taking into account the tissue hierarchy leads to improved predictive power. Remarkably, we also demonstrate that it is possible to leverage the tissue hierarchy in order to effectively transfer cellular functions to a functionally uncharacterized tissue. Overall, OhmNet moves from flat networks to multiscale models able to predict a range of phenotypes spanning cellular subsystems. Availability and implementation: Source code and datasets are available at http://snap.stanford.edu/ohmnet. Contact: jure@cs.stanford.edu PMID:28881986

Dendritic Cells in the Context of Human Tumors: Biology and Experimental Tools.

PubMed

Volovitz, Ilan; Melzer, Susanne; Amar, Sarah; Bocsi, József; Bloch, Merav; Efroni, Sol; Ram, Zvi; Tárnok, Attila

2016-01-01

Dendritic cells (DC) are the most potent and versatile antigen-presenting cells (APC) in the immune system. DC have an exceptional ability to comprehend the immune context of a captured antigen based on molecular signals identified from its vicinity. The analyzed information is then conveyed to other immune effector cells. Such capability enables DC to play a pivotal role in mediating either an immunogenic response or immune tolerance towards an acquired antigen. This review summarizes current knowledge on DC in the context of human tumors. It covers the basics of human DC biology, elaborating on the different markers, morphology and function of the different subsets of human DC. Human blood-borne DC are comprised of at least three subsets consisting of one plasmacytoid DC (pDC) and two to three myeloid DC (mDC) subsets. Some tissues have unique DC. Each subset has a different phenotype and function and may induce pro-tumoral or anti-tumoral effects. The review also discusses two methods fundamental to the research of DC on the single-cell level: multicolor flow cytometry (FCM) and image-based cytometry (IC). These methods, along with new genomics and proteomics tools, can provide high-resolution information on specific DC subsets and on immune and tumor cells with which they interact. The different layers of collected biological data may then be integrated using Immune-Cytomics modeling approaches. Such novel integrated approaches may help unravel the complex network of cellular interactions that DC carry out within tumors, and may help harness this complex immunological information into the development of more effective treatments for cancer.

Cued Reacquisition Trials during Extinction Weaken Contextual Renewal in Human Predictive Learning

ERIC Educational Resources Information Center

Effting, Marieke; Vervliet, Bram; Beckers, Tom; Kindt, Merel

2013-01-01

Extinction is generally more context specific than acquisition, as illustrated by the renewal effect. While most strategies to counteract renewal focus on decreasing the context specificity of extinction, the present work aimed at increasing the context specificity of acquisition learning. Two experiments examined whether presenting cued…

MSAT and cellular hybrid networking

NASA Astrophysics Data System (ADS)

Baranowsky, Patrick W., II

Westinghouse Electric Corporation is developing both the Communications Ground Segment and the Series 1000 Mobile Phone for American Mobile Satellite Corporation's (AMSC's) Mobile Satellite (MSAT) system. The success of the voice services portion of this system depends, to some extent, upon the interoperability of the cellular network and the satellite communication circuit switched communication channels. This paper will describe the set of user-selectable cellular interoperable modes (cellular first/satellite second, etc.) provided by the Mobile Phone and described how they are implemented with the ground segment. Topics including roaming registration and cellular-to-satellite 'seamless' call handoff will be discussed, along with the relevant Interim Standard IS-41 Revision B Cellular Radiotelecommunications Intersystem Operations and IOS-553 Mobile Station - Land Station Compatibility Specification.

Cognitive deficits are a matter of emotional context: inflexible strategy use mediates context-specific learning impairments in OCD.

PubMed

Zetsche, Ulrike; Rief, Winfried; Westermann, Stefan; Exner, Cornelia

2015-01-01

The present study examines the interplay between cognitive deficits and emotional context in obsessive-compulsive disorder (OCD) and social phobia (SP). Specifically, this study examines whether the inflexible use of efficient learning strategies in an emotional context underlies impairments in probabilistic classification learning (PCL) in OCD, and whether PCL impairments are specific to OCD. Twenty-three participants with OCD, 30 participants with SP and 30 healthy controls completed a neutral and an OCD-specific PCL task. OCD participants failed to adopt efficient learning strategies and showed fewer beneficial strategy switches than controls only in an OCD-specific context, but not in a neutral context. Additionally, OCD participants did not show any explicit memory impairments. Number of beneficial strategy switches in the OCD-specific task mediated the difference in PCL performance between OCD and control participants. Individuals with SP were impaired in both PCL tasks. In contrast to neuropsychological models postulating general cognitive impairments in OCD, the present findings suggest that it is the interaction between cognition and emotion that is impaired in OCD. Specifically, activated disorder-specific fears may impair the flexible adoption of efficient learning strategies and compromise otherwise unimpaired PCL. Impairments in PCL are not specific to OCD.

Polymeric nanocomposites loaded with fluoridated hydroxyapatite Ln3+ (Ln = Eu or Tb)/iron oxide for magnetic targeted cellular imaging

PubMed Central

Pan, Jie; Liu, Wei-Jiao; Hua, Chao; Wang, Li-Li; Wan, Dong; Gong, Jun-Bo

2015-01-01

Objective To fabricate polymeric nanocomposites with excellent photoluminescence, magnetic properties, and stability in aqueous solutions, in order to improve specificity and sensitivity of cellular imaging under a magnetic field. Methods Fluoridated Ln3+-doped HAP (Ln3+-HAP) NPs and iron oxides (IOs) can be encapsulated with biocompatible polymers via a modified solvent exaction/evaporation technique to prepare polymeric nanocomposites with fluoridated Ln3+-HAP/iron oxide. The nanocomposites were characterized for surface morphology, fluorescence spectra, magnetic properties and in vitro cytotoxicity. Magnetic targeted cellular imaging of such nanocomposites was also evaluated with confocal laser scanning microscope using A549 cells with or without magnetic field. Results The fabricated nanocomposites showed good stability and excellent luminescent properties, as well as low in vitro cytotoxicity, indicating that the nanocomposites are suitable for biological applications. Nanocomposites under magnetic field achieved much higher cellular uptake via an energy-dependent pathway than those without magnetic field. Conclusion The nanocomposites fabricated in this study will be a promising tool for magnetic targeted cellular imaging with improved specificity and enhanced selection. PMID:26487962

Giardia-specific cellular immune responses in post-giardiasis chronic fatigue syndrome.

PubMed

Hanevik, Kurt; Kristoffersen, Einar; Mørch, Kristine; Rye, Kristin Paulsen; Sørnes, Steinar; Svärd, Staffan; Bruserud, Øystein; Langeland, Nina

2017-01-28

The role of pathogen specific cellular immune responses against the eliciting pathogen in development of post-infectious chronic fatigue syndrome (PI-CFS) is not known and such studies are difficult to perform. The aim of this study was to evaluate specific anti-Giardia cellular immunity in cases that developed CFS after Giardia infection compared to cases that recovered well. Patients reporting chronic fatigue in a questionnaire study three years after a Giardia outbreak were clinically evaluated five years after the outbreak and grouped according to Fukuda criteria for CFS and idiopathic chronic fatigue. Giardia specific immune responses were evaluated in 39 of these patients by proliferation assay, T cell activation and cytokine release analysis. 20 Giardia exposed non-fatigued individuals and 10 healthy unexposed individuals were recruited as controls. Patients were clinically classified into CFS (n = 15), idiopathic chronic fatigue (n = 5), fatigue from other causes (n = 9) and recovered from fatigue (n = 10). There were statistically significant antigen specific differences between these Giardia exposed groups and unexposed controls. However, we did not find differences between the Giardia exposed fatigue classification groups with regard to CD4 T cell activation, proliferation or cytokine levels in 6 days cultured PBMCs. Interestingly, sCD40L was increased in patients with PI-CFS and other persons with fatigue after Giardia infection compared to the non-fatigued group, and correlated well with fatigue levels at the time of sampling. Our data show antigen specific cellular immune responses in the groups previously exposed to Giardia and increased sCD40L in fatigued patients.

Rab protein evolution and the history of the eukaryotic endomembrane system

PubMed Central

Brighouse, Andrew; Dacks, Joel B.

2010-01-01

Spectacular increases in the quantity of sequence data genome have facilitated major advances in eukaryotic comparative genomics. By exploiting homology with classical model organisms, this makes possible predictions of pathways and cellular functions currently impossible to address in intractable organisms. Echoing realization that core metabolic processes were established very early following evolution of life on earth, it is now emerging that many eukaryotic cellular features, including the endomembrane system, are ancient and organized around near-universal principles. Rab proteins are key mediators of vesicle transport and specificity, and via the presence of multiple paralogues, alterations in interaction specificity and modification of pathways, contribute greatly to the evolution of complexity of membrane transport. Understanding system-level contributions of Rab proteins to evolutionary history provides insight into the multiple processes sculpting cellular transport pathways and the exciting challenges that we face in delving further into the origins of membrane trafficking specificity. PMID:20582450

Drosophila atonal fully rescues the phenotype of Math1 null mice: new functions evolve in new cellular contexts

NASA Technical Reports Server (NTRS)

Wang, Vincent Y.; Hassan, Bassem A.; Bellen, Hugo J.; Zoghbi, Huda Y.

2002-01-01

Many genes share sequence similarity between species, but their properties often change significantly during evolution. For example, the Drosophila genes engrailed and orthodenticle and the onychophoran gene Ultrabithorax only partially substitute for their mouse or Drosophila homologs. We have been analyzing the relationship between atonal (ato) in the fruit fly and its mouse homolog, Math1. In flies, ato acts as a proneural gene that governs the development of chordotonal organs (CHOs), which serve as stretch receptors in the body wall and joints and as auditory organs in the antennae. In the fly CNS, ato is important not for specification but for axonal arborization. Math1, in contrast, is required for the specification of cells in both the CNS and the PNS. Furthermore, Math1 serves a role in the development of secretory lineage cells in the gut, a function that does not parallel any known to be served by ato. We wondered whether ato and Math1 might be more functionally homologous than they appear, so we expressed Math1 in ato mutant flies and ato in Math1 null mice. To our surprise, the two proteins are functionally interchangeable.

Intelligent Design of Nano-Scale Molecular Imaging Agents

PubMed Central

Kim, Sung Bae; Hattori, Mitsuru; Ozawa, Takeaki

2012-01-01

Visual representation and quantification of biological processes at the cellular and subcellular levels within living subjects are gaining great interest in life science to address frontier issues in pathology and physiology. As intact living subjects do not emit any optical signature, visual representation usually exploits nano-scale imaging agents as the source of image contrast. Many imaging agents have been developed for this purpose, some of which exert nonspecific, passive, and physical interaction with a target. Current research interest in molecular imaging has mainly shifted to fabrication of smartly integrated, specific, and versatile agents that emit fluorescence or luminescence as an optical readout. These agents include luminescent quantum dots (QDs), biofunctional antibodies, and multifunctional nanoparticles. Furthermore, genetically encoded nano-imaging agents embedding fluorescent proteins or luciferases are now gaining popularity. These agents are generated by integrative design of the components, such as luciferase, flexible linker, and receptor to exert a specific on–off switching in the complex context of living subjects. In the present review, we provide an overview of the basic concepts, smart design, and practical contribution of recent nano-scale imaging agents, especially with respect to genetically encoded imaging agents. PMID:23235326

Intelligent design of nano-scale molecular imaging agents.

PubMed

Kim, Sung Bae; Hattori, Mitsuru; Ozawa, Takeaki

2012-12-12

Visual representation and quantification of biological processes at the cellular and subcellular levels within living subjects are gaining great interest in life science to address frontier issues in pathology and physiology. As intact living subjects do not emit any optical signature, visual representation usually exploits nano-scale imaging agents as the source of image contrast. Many imaging agents have been developed for this purpose, some of which exert nonspecific, passive, and physical interaction with a target. Current research interest in molecular imaging has mainly shifted to fabrication of smartly integrated, specific, and versatile agents that emit fluorescence or luminescence as an optical readout. These agents include luminescent quantum dots (QDs), biofunctional antibodies, and multifunctional nanoparticles. Furthermore, genetically encoded nano-imaging agents embedding fluorescent proteins or luciferases are now gaining popularity. These agents are generated by integrative design of the components, such as luciferase, flexible linker, and receptor to exert a specific on-off switching in the complex context of living subjects. In the present review, we provide an overview of the basic concepts, smart design, and practical contribution of recent nano-scale imaging agents, especially with respect to genetically encoded imaging agents.

TORC2 signaling antagonizes SKN-1 to induce C. elegans mesendodermal embryonic development

PubMed Central

Ruf, Vanessa; Holzem, Christina; Peyman, Tobias; Walz, Gerd; Blackwell, T. Keith; Neumann-Haefelin, Elke

2013-01-01

The evolutionarily conserved target of rapamycin (TOR) kinase controls fundamental metabolic processes to support cell and tissue growth. TOR functions within the context of two distinct complexes, TORC1 and TORC2. TORC2, with its specific component Rictor, has been recently implicated in aging and regulation of growth and metabolism. Here, we identify rict-1/Rictor as a regulator of embryonic development in C. elegans. The transcription factor skn-1 establishes development of the mesendoderm in embryos, and is required for cellular homeostasis and longevity in adults. Loss of maternal skn-1 function leads to misspecification of the mesendodermal precursor and failure to form intestine and pharynx. We found that genetic inactivation of rict-1 suppressed skn-1-associated lethality by restoring mesendodermal specification in skn-1 deficient embryos. Inactivation of other TORC2 but not TORC1 components also partially rescued skn-1 embryonic lethality. The SGK-1 kinase mediated these functions downstream of rict-1/TORC2, as a sgk-1 gain-of-function mutant suppressed the rict-1 mutant phenotype. These data indicate that TORC2 and SGK-1 antagonize SKN-1 during embryonic development. PMID:23973804

Adoptive T-cell therapy for hematological malignancies using T cells gene-modified to express tumor antigen-specific receptors.

PubMed

Fujiwara, Hiroshi

2014-02-01

The functional properties of the adoptive immune response mediated by effector T lymphocytes are decisively regulated by their T-cell receptors (TCRs). Transfer of genes encoding target antigen-specific receptors enables polyclonal T cells to redirect toward cancer cells and virally infected cells expressing those defined antigens. Using this technology, a large population of redirected T cells displaying uniform therapeutic properties has been produced, powerfully advancing their clinical application as "cellular drugs" for adoptive immunotherapy against cancer. Clinically, anticancer adoptive immunotherapy using these genetically engineered T cells has an impressive and proven track record. Notable examples include the dramatic benefit of chimeric antigen receptor gene-modified T cells redirected towards B-cell lineage antigen CD19 in patients with chronic lymphocytic leukemia, and the impressive outcomes in the use of TCR gene-modified T cells redirected towards NY-ESO-1, a representative cancer-testis antigen, in patients with advanced melanoma and synovial cell sarcoma. In this review, we briefly overview the current status of this treatment option in the context of hematological malignancy, and discuss a number of challenges that still pose an obstacle to the full effectiveness of this strategy.

Context-specific modulation of cocaine-induced locomotor sensitization and ERK and CREB phosphorylation in rat nucleus accumbens

PubMed Central

Marin, Marcelo T.; Berkow, Alexander; Golden, Sam A.; Koya, Eisuke; Planeta, Cleopatra S.; Hope, Bruce T.

2009-01-01

Learned associations are hypothesized to develop between drug effects and contextual stimuli during repeated drug administration to produce context-specific sensitization that is expressed only in the drug-associated environment and not in a non-drug paired environment. Neuroadaptations that mediate such context-specific behavior are largely unknown. We investigated context-specific modulation of CREB phosphorylation and four upstream kinases in nucleus accumbens that phosphorylate CREB, including ERK, PKA, CaMKII and IV. Rats received seven once daily injections of cocaine or saline in one of two distinct environments outside their home cages. Seven days later, test injections of cocaine or saline were administered in either the Paired or the Non-paired environment. CREB and ERK phosphorylation were assessed with immunohistochemistry while phosphorylation of the remaining kinases, as well as CREB and ERK, were assessed by Western blotting. Repeated cocaine administration produced context-specific sensitized locomotor responses accompanied by context-specific enhancement of the number of cocaine-induced phosphoCREB and phosphoERK immunoreactive nuclei in a minority of neurons. In contrast, CREB and CaMKIV phosphorylation in nucleus accumbens homogenates were decreased by cocaine test injections. We have recently shown that a small number of cocaine-activated accumbens neurons mediate the learned association between cocaine effects and the drug administration environment to produce context-specific sensitization. The corresponding cocaine and context-specific phosphorylation of ERK and CREB in cocaine-activated accumbens neurons in the present study suggests that this signal transduction pathway is also selectively activated in the same set of accumbens neurons. PMID:19912338

Cellular entry of G3.5 poly (amido amine) dendrimers by clathrin- and dynamin-dependent endocytosis promotes tight junctional opening in intestinal epithelia.

PubMed

Goldberg, Deborah S; Ghandehari, Hamidreza; Swaan, Peter W

2010-08-01

This study investigates the mechanisms of G3.5 poly (amido amine) dendrimer cellular uptake, intracellular trafficking, transepithelial transport and tight junction modulation in Caco-2 cells in the context of oral drug delivery. Chemical inhibitors blocking clathrin-, caveolin- and dynamin-dependent endocytosis pathways were used to investigate the mechanisms of dendrimer cellular uptake and transport across Caco-2 cells using flow cytometry and confocal microscopy. Dendrimer cellular uptake was found to be dynamin-dependent and was reduced by both clathrin and caveolin endocytosis inhibitors, while transepithelial transport was only dependent on dynamin- and clathrin-mediated endocytosis. Dendrimers were quickly trafficked to the lysosomes after 15 min of incubation and showed increased endosomal accumulation at later time points, suggesting saturation of this pathway. Dendrimers were unable to open tight junctions in cell monolayers treated with dynasore, a selective inhibitor of dynamin, confirming that dendrimer internalization promotes tight junction modulation. G3.5 PAMAM dendrimers take advantage of several receptor-mediated endocytosis pathways for cellular entry in Caco-2 cells. Dendrimer internalization by dynamin-dependent mechanisms promotes tight junction opening, suggesting that dendrimers act on intracellular cytoskeletal proteins to modulate tight junctions, thus catalyzing their own transport via the paracellular route.

Transcriptome-wide analysis of alternative RNA splicing events in Epstein-Barr virus-associated gastric carcinomas

PubMed Central

Armero, Victoria E. S.; Tremblay, Marie-Pier; Allaire, Andréa; Boudreault, Simon; Martenon-Brodeur, Camille; Duval, Cyntia; Durand, Mathieu; Lapointe, Elvy; Thibault, Philippe; Tremblay-Létourneau, Maude; Perreault, Jean-Pierre; Scott, Michelle S.

2017-01-01

Multiple human diseases including cancer have been associated with a dysregulation in RNA splicing patterns. In the current study, modifications to the global RNA splicing landscape of cellular genes were investigated in the context of Epstein-Barr virus-associated gastric cancer. Global alterations to the RNA splicing landscape of cellular genes was examined in a large-scale screen from 295 primary gastric adenocarcinomas using high-throughput RNA sequencing data. RT-PCR analysis, mass spectrometry, and co-immunoprecipitation studies were also used to experimentally validate and investigate the differential alternative splicing (AS) events that were observed through RNA-seq studies. Our study identifies alterations in the AS patterns of approximately 900 genes such as tumor suppressor genes, transcription factors, splicing factors, and kinases. These findings allowed the identification of unique gene signatures for which AS is misregulated in both Epstein-Barr virus-associated gastric cancer and EBV-negative gastric cancer. Moreover, we show that the expression of Epstein–Barr nuclear antigen 1 (EBNA1) leads to modifications in the AS profile of cellular genes and that the EBNA1 protein interacts with cellular splicing factors. These findings provide insights into the molecular differences between various types of gastric cancer and suggest a role for the EBNA1 protein in the dysregulation of cellular AS. PMID:28493890

Transcriptome-wide analysis of alternative RNA splicing events in Epstein-Barr virus-associated gastric carcinomas.

PubMed

Armero, Victoria E S; Tremblay, Marie-Pier; Allaire, Andréa; Boudreault, Simon; Martenon-Brodeur, Camille; Duval, Cyntia; Durand, Mathieu; Lapointe, Elvy; Thibault, Philippe; Tremblay-Létourneau, Maude; Perreault, Jean-Pierre; Scott, Michelle S; Bisaillon, Martin

2017-01-01

Multiple human diseases including cancer have been associated with a dysregulation in RNA splicing patterns. In the current study, modifications to the global RNA splicing landscape of cellular genes were investigated in the context of Epstein-Barr virus-associated gastric cancer. Global alterations to the RNA splicing landscape of cellular genes was examined in a large-scale screen from 295 primary gastric adenocarcinomas using high-throughput RNA sequencing data. RT-PCR analysis, mass spectrometry, and co-immunoprecipitation studies were also used to experimentally validate and investigate the differential alternative splicing (AS) events that were observed through RNA-seq studies. Our study identifies alterations in the AS patterns of approximately 900 genes such as tumor suppressor genes, transcription factors, splicing factors, and kinases. These findings allowed the identification of unique gene signatures for which AS is misregulated in both Epstein-Barr virus-associated gastric cancer and EBV-negative gastric cancer. Moreover, we show that the expression of Epstein-Barr nuclear antigen 1 (EBNA1) leads to modifications in the AS profile of cellular genes and that the EBNA1 protein interacts with cellular splicing factors. These findings provide insights into the molecular differences between various types of gastric cancer and suggest a role for the EBNA1 protein in the dysregulation of cellular AS.

Carbon Ion-Irradiated Hepatoma Cells Exhibit Coupling Interplay between Apoptotic Signaling and Morphological and Mechanical Remodeling

PubMed Central

Zhang, Baoping; Li, Long; Li, Zhiqiang; Liu, Yang; Zhang, Hong; Wang, Jizeng

2016-01-01

A apoptotic model was established based on the results of five hepatocellular carcinoma cell (HCC) lines irradiated with carbon ions to investigate the coupling interplay between apoptotic signaling and morphological and mechanical cellular remodeling. The expression levels of key apoptotic proteins and the changes in morphological characteristics and mechanical properties were systematically examined in the irradiated HCC lines. We observed that caspase-3 was activated and that the Bax/Bcl-2 ratio was significantly increased over time. Cellular morphology and mechanics analyses indicated monotonic decreases in spatial sizes, an increase in surface roughness, a considerable reduction in stiffness, and disassembly of the cytoskeletal architecture. A theoretical model of apoptosis revealed that mechanical changes in cells induce the characteristic cellular budding of apoptotic bodies. Statistical analysis indicated that the projected area, stiffness, and cytoskeletal density of the irradiated cells were positively correlated, whereas stiffness and caspase-3 expression were negatively correlated, suggesting a tight coupling interplay between the cellular structures, mechanical properties, and apoptotic protein levels. These results help to clarify a novel arbitration mechanism of cellular demise induced by carbon ions. This biomechanics strategy for evaluating apoptosis contributes to our understanding of cancer-killing mechanisms in the context of carbon ion radiotherapy. PMID:27731354

The Next Frontier: Quantitative Biochemistry in Living Cells.

PubMed

Honigmann, Alf; Nadler, André

2018-01-09

Researchers striving to convert biology into an exact science foremost rely on structural biology and biochemical reconstitution approaches to obtain quantitative data. However, cell biological research is moving at an ever-accelerating speed into areas where these approaches lose much of their edge. Intrinsically unstructured proteins and biochemical interaction networks composed of interchangeable, multivalent, and unspecific interactions pose unique challenges to quantitative biology, as do processes that occur in discrete cellular microenvironments. Here we argue that a conceptual change in our way of conducting biochemical experiments is required to take on these new challenges. We propose that reconstitution of cellular processes in vitro should be much more focused on mimicking the cellular environment in vivo, an approach that requires detailed knowledge of the material properties of cellular compartments, essentially requiring a material science of the cell. In a similar vein, we suggest that quantitative biochemical experiments in vitro should be accompanied by corresponding experiments in vivo, as many newly relevant cellular processes are highly context-dependent. In essence, this constitutes a call for chemical biologists to convert their discipline from a proof-of-principle science to an area that could rightfully be called quantitative biochemistry in living cells. In this essay, we discuss novel techniques and experimental strategies with regard to their potential to fulfill such ambitious aims.

Using Generic and Context-Specific Scaffolding to Support Authentic Science Inquiry

ERIC Educational Resources Information Center

Belland, Brian R.; Gu, Jiangyue; Armbrust, Sara; Cook, Brant

2013-01-01

In this conceptual paper, we propose an heuristic to balance context-specific and generic scaffolding, as well as computer-based and teacher scaffolding, during instruction centered on authentic, scientific problems. This paper is novel in that many researchers ask a dichotomous question of whether generic or context-specific scaffolding is best,…

Cellular Automata Generalized To An Inferential System

NASA Astrophysics Data System (ADS)

Blower, David J.

2007-11-01

Stephen Wolfram popularized elementary one-dimensional cellular automata in his book, A New Kind of Science. Among many remarkable things, he proved that one of these cellular automata was a Universal Turing Machine. Such cellular automata can be interpreted in a different way by viewing them within the context of the formal manipulation rules from probability theory. Bayes's Theorem is the most famous of such formal rules. As a prelude, we recapitulate Jaynes's presentation of how probability theory generalizes classical logic using modus ponens as the canonical example. We emphasize the important conceptual standing of Boolean Algebra for the formal rules of probability manipulation and give an alternative demonstration augmenting and complementing Jaynes's derivation. We show the complementary roles played in arguments of this kind by Bayes's Theorem and joint probability tables. A good explanation for all of this is afforded by the expansion of any particular logic function via the disjunctive normal form (DNF). The DNF expansion is a useful heuristic emphasized in this exposition because such expansions point out where relevant 0s should be placed in the joint probability tables for logic functions involving any number of variables. It then becomes a straightforward exercise to rely on Boolean Algebra, Bayes's Theorem, and joint probability tables in extrapolating to Wolfram's cellular automata. Cellular automata are seen as purely deductive systems, just like classical logic, which probability theory is then able to generalize. Thus, any uncertainties which we might like to introduce into the discussion about cellular automata are handled with ease via the familiar inferential path. Most importantly, the difficult problem of predicting what cellular automata will do in the far future is treated like any inferential prediction problem.

Premature aging/senescence in cancer cells facing therapy: good or bad?

PubMed

Gonzalez, Llilians Calvo; Ghadaouia, Sabrina; Martinez, Aurélie; Rodier, Francis

2016-02-01

Normal and cancer cells facing their demise following exposure to radio-chemotherapy can actively participate in choosing their subsequent fate. These programmed cell fate decisions include true cell death (apoptosis-necroptosis) and therapy-induced cellular senescence (TIS), a permanent "proliferative arrest" commonly portrayed as premature cellular aging. Despite a permanent loss of proliferative potential, senescent cells remain viable and are highly bioactive at the microenvironment level, resulting in a prolonged impact on tissue architecture and functions. Cellular senescence is primarily documented as a tumor suppression mechanism that prevents cellular transformation. In the context of normal tissues, cellular senescence also plays important roles in tissue repair, but contributes to age-associated tissue dysfunction when senescent cells accumulate. Theoretically, in multi-step cancer progression models, cancer cells have already bypassed cellular senescence during their immortalization step (see hallmarks of cancer). It is then perhaps surprising to find that cancer cells often retain the ability to undergo TIS, or premature aging. This occurs because cellular senescence results from multiple signalling pathways, some retained in cancer cells, aiming to prevent cell cycle progression in damaged cells. Since senescent cancer cells persist after therapy and secrete an array of cytokines and growth factors that can modulate the tumor microenvironment, these cells may have beneficial and detrimental effects regarding immune modulation and survival of remaining proliferation-competent cancer cells. Similarly, while normal cells undergoing senescence are believed to remain indefinitely growth arrested, whether this is true for senescent cancer cells remains unclear, raising the possibility that these cells may represent a reservoir for cancer recurrence after treatment. This review discusses our current knowledge on cancer cell senescence and highlight questions that must be addressed to fully understand the beneficial and detrimental impacts of cellular senescence during cancer therapy.

Activation of context-specific attentional control sets by exogenous allocation of visual attention to the context?

PubMed

Gottschalk, Caroline; Fischer, Rico

2017-03-01

Different contexts with high versus low conflict frequencies require a specific attentional control involvement, i.e., strong attentional control for high conflict contexts and less attentional control for low conflict contexts. While it is assumed that the corresponding control set can be activated upon stimulus presentation at the respective context (e.g., upper versus lower location), the actual features that trigger control set activation are to date not described. Here, we ask whether the perceptual priming of the location context by an abrupt onset of irrelevant stimuli is sufficient in activating the context-specific attentional control set. For example, the mere onset of a stimulus might disambiguate the relevant location context and thus, serve as a low-level perceptual trigger mechanism that activates the context-specific attentional control set. In Experiment 1 and 2, the onsets of task-relevant and task-irrelevant (distracter) stimuli were manipulated at each context location to compete for triggering the activation of the appropriate control set. In Experiment 3, a prior training session enabled distracter stimuli to establish contextual control associations of their own before entering the test session. Results consistently showed that the mere onset of a task-irrelevant stimulus (with or without a context-control association) is not sufficient to activate the context-associated attentional control set by disambiguating the relevant context location. Instead, we argue that the identification of the relevant stimulus at the respective context is a precondition to trigger the activation of the context-associated attentional control set.

High-Fidelity Tissue Engineering of Patient-Specific Auricles for Reconstruction of Pediatric Microtia and Other Auricular Deformities

PubMed Central

Reiffel, Alyssa J.; Kafka, Concepcion; Hernandez, Karina A.; Popa, Samantha; Perez, Justin L.; Zhou, Sherry; Pramanik, Satadru; Brown, Bryan N.; Ryu, Won Seuk; Bonassar, Lawrence J.; Spector, Jason A.

2013-01-01

Introduction Autologous techniques for the reconstruction of pediatric microtia often result in suboptimal aesthetic outcomes and morbidity at the costal cartilage donor site. We therefore sought to combine digital photogrammetry with CAD/CAM techniques to develop collagen type I hydrogel scaffolds and their respective molds that would precisely mimic the normal anatomy of the patient-specific external ear as well as recapitulate the complex biomechanical properties of native auricular elastic cartilage while avoiding the morbidity of traditional autologous reconstructions. Methods Three-dimensional structures of normal pediatric ears were digitized and converted to virtual solids for mold design. Image-based synthetic reconstructions of these ears were fabricated from collagen type I hydrogels. Half were seeded with bovine auricular chondrocytes. Cellular and acellular constructs were implanted subcutaneously in the dorsa of nude rats and harvested after 1 and 3 months. Results Gross inspection revealed that acellular implants had significantly decreased in size by 1 month. Cellular constructs retained their contour/projection from the animals' dorsa, even after 3 months. Post-harvest weight of cellular constructs was significantly greater than that of acellular constructs after 1 and 3 months. Safranin O-staining revealed that cellular constructs demonstrated evidence of a self-assembled perichondrial layer and copious neocartilage deposition. Verhoeff staining of 1 month cellular constructs revealed de novo elastic cartilage deposition, which was even more extensive and robust after 3 months. The equilibrium modulus and hydraulic permeability of cellular constructs were not significantly different from native bovine auricular cartilage after 3 months. Conclusions We have developed high-fidelity, biocompatible, patient-specific tissue-engineered constructs for auricular reconstruction which largely mimic the native auricle both biomechanically and histologically, even after an extended period of implantation. This strategy holds immense potential for durable patient-specific tissue-engineered anatomically proper auricular reconstructions in the future. PMID:23437148

Primary Respiratory Chain Disease Causes Tissue-Specific Dysregulation of the Global Transcriptome and Nutrient-Sensing Signaling Network

PubMed Central

Zhang, Zhe; Tsukikawa, Mai; Peng, Min; Polyak, Erzsebet; Nakamaru-Ogiso, Eiko; Ostrovsky, Julian; McCormack, Shana; Place, Emily; Clarke, Colleen; Reiner, Gail; McCormick, Elizabeth; Rappaport, Eric; Haas, Richard; Baur, Joseph A.; Falk, Marni J.

2013-01-01

Primary mitochondrial respiratory chain (RC) diseases are heterogeneous in etiology and manifestations but collectively impair cellular energy metabolism. Mechanism(s) by which RC dysfunction causes global cellular sequelae are poorly understood. To identify a common cellular response to RC disease, integrated gene, pathway, and systems biology analyses were performed in human primary RC disease skeletal muscle and fibroblast transcriptomes. Significant changes were evident in muscle across diverse RC complex and genetic etiologies that were consistent with prior reports in other primary RC disease models and involved dysregulation of genes involved in RNA processing, protein translation, transport, and degradation, and muscle structure. Global transcriptional and post-transcriptional dysregulation was also found to occur in a highly tissue-specific fashion. In particular, RC disease muscle had decreased transcription of cytosolic ribosomal proteins suggestive of reduced anabolic processes, increased transcription of mitochondrial ribosomal proteins, shorter 5′-UTRs that likely improve translational efficiency, and stabilization of 3′-UTRs containing AU-rich elements. RC disease fibroblasts showed a strikingly similar pattern of global transcriptome dysregulation in a reverse direction. In parallel with these transcriptional effects, RC disease dysregulated the integrated nutrient-sensing signaling network involving FOXO, PPAR, sirtuins, AMPK, and mTORC1, which collectively sense nutrient availability and regulate cellular growth. Altered activities of central nodes in the nutrient-sensing signaling network were validated by phosphokinase immunoblot analysis in RC inhibited cells. Remarkably, treating RC mutant fibroblasts with nicotinic acid to enhance sirtuin and PPAR activity also normalized mTORC1 and AMPK signaling, restored NADH/NAD+ redox balance, and improved cellular respiratory capacity. These data specifically highlight a common pathogenesis extending across different molecular and biochemical etiologies of individual RC disorders that involves global transcriptome modifications. We further identify the integrated nutrient-sensing signaling network as a common cellular response that mediates, and may be amenable to targeted therapies for, tissue-specific sequelae of primary mitochondrial RC disease. PMID:23894440

The BRAIN Initiative Provides a Unifying Context for Integrating Core STEM Competencies into a Neurobiology Course.

PubMed

Schaefer, Jennifer E

2016-01-01

The Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative introduced by the Obama Administration in 2013 presents a context for integrating many STEM competencies into undergraduate neuroscience coursework. The BRAIN Initiative core principles overlap with core STEM competencies identified by the AAAS Vision and Change report and other entities. This neurobiology course utilizes the BRAIN Initiative to serve as the unifying theme that facilitates a primary emphasis on student competencies such as scientific process, scientific communication, and societal relevance while teaching foundational neurobiological content such as brain anatomy, cellular neurophysiology, and activity modulation. Student feedback indicates that the BRAIN Initiative is an engaging and instructional context for this course. Course module organization, suitable BRAIN Initiative commentary literature, sample primary literature, and important assignments are presented.

Podocytes populate cellular crescents in a murine model of inflammatory glomerulonephritis.

PubMed

Moeller, Marcus J; Soofi, Abdulsalaam; Hartmann, Inge; Le Hir, Michel; Wiggins, Roger; Kriz, Wilhelm; Holzman, Lawrence B

2004-01-01

Cellular crescents are a defining histologic finding in many forms of inflammatory glomerulonephritis. Despite numerous studies, the origin of glomerular crescents remains unresolved. A genetic cell lineage-mapping study with a novel transgenic mouse model was performed to investigate whether visceral glomerular epithelial cells, termed podocytes, are precursors of cells that populate cellular crescents. The podocyte-specific 2.5P-Cre mouse line was crossed with the ROSA26 reporter line, resulting in irreversible constitutive expression of beta-galactosidase in doubly transgenic 2.5P-Cre/ROSA26 mice. In these mice, crescentic glomerulonephritis was induced with a previously described rabbit anti-glomerular basement membrane antiserum nephritis approach. Interestingly, beta-galactosidase-positive cells derived from podocytes adhered to the parietal basement membrane and populated glomerular crescents during the early phases of cellular crescent formation, accounting for at least one-fourth of the total cell mass. In cellular crescents, the proliferation marker Ki-67 was expressed in beta-galactosidase-positive and beta-galactosidase-negative cells, indicating that both cell types contributed to the formation of cellular crescents through proliferation in situ. Podocyte-specific antigens, including WT-1, synaptopodin, nephrin, and podocin, were not expressed by any cells in glomerular crescents, suggesting that podocytes underwent profound phenotypic changes in this nephritis model.

A SIMPLE CELLULAR AUTOMATON MODEL FOR HIGH-LEVEL VEGETATION DYNAMICS

EPA Science Inventory

We have produced a simple two-dimensional (ground-plan) cellular automata model of vegetation dynamics specifically to investigate high-level community processes. The model is probabilistic, with individual plant behavior determined by physiologically-based rules derived from a w...

CMV-specific T cell isolation from G-CSF mobilized peripheral blood: depletion of myeloid progenitors eliminates non-specific binding of MHC-multimers.

PubMed

Beloki, Lorea; Ciaurriz, Miriam; Mansilla, Cristina; Zabalza, Amaya; Perez-Valderrama, Estela; Samuel, Edward R; Lowdell, Mark W; Ramirez, Natalia; Olavarria, Eduardo

2014-11-19

Cytomegalovirus (CMV)-specific T cell infusion to immunocompromised patients following allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) is able to induce a successful anti-viral response. These cells have classically been manufactured from steady-state apheresis samples collected from the donor in an additional harvest prior to G-CSF mobilization, treatment that induces hematopoietic stem cell (HSC) mobilization to the periphery. However, two closely-timed cellular collections are not usually available in the unrelated donor setting, which limits the accessibility of anti-viral cells for adoptive immunotherapy. CMV-specific cytotoxic T cell (CTL) manufacture from the same G-CSF mobilized donor stem cell harvest offers great regulatory advantages, but the isolation using MHC-multimers is hampered by the high non-specific binding to myeloid progenitors, which reduces the purity of the cellular product. In the present study we describe an easy and fast method based on plastic adherence to remove myeloid cell subsets from 11 G-CSF mobilized donor samples. CMV-specific CTLs were isolated from the non-adherent fraction using pentamers and purity and yield of the process were compared to products obtained from unmanipulated samples. After the elimination of unwanted cell subtypes, non-specific binding of pentamers was notably reduced. Accordingly, following the isolation process the purity of the obtained cellular product was significantly improved. G-CSF mobilized leukapheresis samples can successfully be used to isolate antigen-specific T cells with MHC-multimers to be adoptively transferred following allo-HSCT, widening the accessibility of this therapy in the unrelated donor setting. The combination of the clinically translatable plastic adherence process to the antigen-specific cell isolation using MHC-multimers improves the quality of the therapeutic cellular product, thereby reducing the clinical negative effects associated with undesired alloreactive cell infusion.

Evaluation of the health impact of nanoparticles emitted from combustion sources: Comprehensive characterization of the physicochemical properties of nanoparticle emissions from wood combustion compliances, car- and ship diesel-engines as well as investigation of their toxicological effects on human lung cells and macrophages.

NASA Astrophysics Data System (ADS)

Zimmermann, R.; Dittmar, G.; Kanashova, T.; Buters, J.; Öder, S.; Paur, H. R.; Mülhopt, S.; Dilger, M.; Weiss, C.; Harndorf, H.; Stengel, B.; Hirvonen, M. R.; Jokiniemi, J.; Hiller, K.; Sapcariu, S.; Sippula, O.; Streibel, T.; Karg, E.; Weggler, B.; Schnelle-Kreis, J.; Lintelmann, J.; Sklorz, M.; Orasche, J.; Müller, L.; Passig, J.; Gröger, T.; BéruBé, K.; Krebs, T.

2016-12-01

Combustion emissions cause health effects. The HICE-Aerosol and Health project team studies the physicochemical properties as well as biological and toxicological effects on lung cells of combustion particle emissions. The chemical composition and physical parameters thoroughly characterized. Human lung cells are exposed to the diluted combustion exhaust fumes at the air-liquid interface (ALI), allowing a realistic lung-cell exposure by simulation of the lung situation. After exposure, cellular responses of the exposed lung cells are studied by multi-omics molecular biological analyses on transcriptomic, proteomic and metabolomic level. Emissions of wood combustion (log wood, pellet heater), ship diesel engines and car gasoline engines are addressed. Special field deployable ALI-exposition systems in a mobile S2-biological laboratory were set up and applied. Human alveolar epithelial cells (A549, BEAS2B and primary cells) as well as murine macrophages were ALI-exposed to diluted emissions. The cellular effects were then comprehensively characterized (viability, cyto-toxicology, multi-omics effects monitoring) and put in context with the chemical and physical aerosol data. The following order of overall cellular response-strength was observed: A relatively mild cellular effect is observed for the diluted wood combustion emissions. Interestingly the effects-strength for log-wood and pellet burner emissions are similar, although PM-concentrations are much higher for the log-wood heater. Similar mild biological effects are observed for the gasoline car emissions. The ship diesel engine emissions induced the most intense biological responses. A surprising result in this context is, that heavy fuel oil (HFO)-emissions showed lower biological effect strengths than the supposedly cleaner diesel fuel emissions (DF). The HFO-emission contain high concentrations of known toxicants (transition metals, polycyclic aromatics). This result was recently confirmed by experiments with murine RAW macrophages. Detailed analyses of the activated cellular response pathways, such as pro-inflammatory responses, xenobiotic metabolism, phagocytosis and oxidative stress were performed. The data is suggesting a large difference in relative toxicity for different combustion sources.

Curative or pre-emptive adenovirus-specific T cell transfer from matched unrelated or third party haploidentical donors after HSCT, including UCB transplantations: a successful phase I/II multicenter clinical trial.

PubMed

Qian, Chongsheng; Campidelli, Arnaud; Wang, Yingying; Cai, Huili; Venard, Véronique; Jeulin, Hélène; Dalle, Jean Hugues; Pochon, Cécile; D'aveni, Maud; Bruno, Benedicte; Paillard, Catherine; Vigouroux, Stéphane; Jubert, Charlotte; Ceballos, Patrice; Marie-Cardine, Aude; Galambrun, Claire; Cholle, Clément; Clerc Urmes, Isabelle; Petitpain, Nadine; De Carvalho Bittencourt, Marcelo; Decot, Véronique; Reppel, Loïc; Salmon, Alexandra; Clement, Laurence; Bensoussan, Danièle

2017-05-08

Allogeneic hematopoietic stem cell transplantation (HSCT), the most widely used potentially curable cellular immunotherapeutic approach in the treatment of hematological malignancies, is limited by life-threatening complications: graft versus host disease (GVHD) and infections especially viral infections refractory to antiviral drugs. Adoptive transfer of virus-specific T cells is becoming an alternative treatment for infections following HSCT. We report here the results of a phase I/II multicenter study which includes a series of adenovirus-specific T cell (ADV-VST) infusion either from the HSCT donor or from a third party haploidentical donor for patients transplanted with umbilical cord blood (UCB). Fourteen patients were eligible and 11 patients received infusions of ADV-VST generated by interferon (IFN)-γ-based immunomagnetic isolation from a leukapheresis from their original donor (42.9%) or a third party haploidentical donor (57.1%). One patient resolved ADV infection before infusion, and ADV-VST could not reach release or infusion criteria for two patients. Two patients received cellular immunotherapy alone without antiviral drugs as a pre-emptive treatment. One patient with adenovirus infection and ten with adenovirus disease were infused with ADV-VST (mean 5.83 ± 8.23 × 10 3 CD3+IFN-γ+ cells/kg) up to 9 months after transplantation. The 11 patients showed in vivo expansion of specific T cells up to 60 days post-infusion, associated with adenovirus load clearance in ten of the patients (91%). Neither de novo GVHD nor side effects were observed during the first month post-infusion, but GVHD reactivations occurred in three patients, irrespective of the type of leukapheresis donor. For two of these patients, GVHD reactivation was controlled by immunosuppressive treatment. Four patients died during follow-up, one due to refractory ADV disease. Adoptive transfer of rapidly isolated ADV-VST is an effective therapeutic option for achieving in vivo expansion of specific T cells and clearance of viral load, even as a pre-emptive treatment. Our study highlights that third party haploidentical donors are of great interest for ADV-VST generation in the context of UCB transplantation. (N° Clinical trial.gov: NCT02851576, retrospectively registered).

Time-Dependent Effects of Prazosin on the Development of Methamphetamine Conditioned Hyperactivity and Context-Specific Sensitization in Mice

PubMed Central

White, André O.; Rauhut, Anthony S.

2014-01-01

The present experiments examined the effects of prazosin, a selective α1-adrenergic receptor antagonist, on the development of methamphetamine conditioned hyperactivity and context-specific sensitization. Mice received an injection of vehicle (distilled water) or prazosin (0.5, 1.0 or 2.0 mg/kg) 30 minutes prior to a second injection of vehicle (saline) or methamphetamine (1.0 mg/kg) during the conditioning sessions (Experiment 1). Following the conditioning sessions, mice were tested for conditioned hyperactivity and then tested for context-specific sensitization. In subsequent experiments, mice received an injection of vehicle (distilled water) or prazosin (2.0 mg/kg) immediately (Experiment 2) or 24 hours (Experiment 3) after the conditioning sessions and then tested for conditioned hyperactivity and context-specific sensitization. Prazosin dose-dependently blocked the development of methamphetamine conditioned hyperactivity and context-specific sensitization when administered prior to the methamphetamine during the conditioning phase; however nonspecific motor impairments also were observed (Experiment 1). Immediate (Experiment 2), but not the 24-hour delay (Experiment 3), post-session administration of prazosin attenuated the development of methamphetamine conditioned hyperactivity and context-specific sensitization. Nonspecific motor impairments were not observed in these latter experiments. Collectively, these results suggest that the α1-adrenergic receptor mediates the development of methamphetamine-conditioned hyperactivity and context-specific sensitization, perhaps by altering memory consolidation and/or reconsolidation processes. PMID:24487011

Improved Cellular Specificity of Plasmonic Nanobubbles versus Nanoparticles in Heterogeneous Cell Systems

PubMed Central

Lukianova-Hleb, Ekaterina Y.; Ren, Xiaoyang; Constantinou, Pamela E.; Danysh, Brian P.; Shenefelt, Derek L.; Carson, Daniel D.; Farach-Carson, Mary C.; Kulchitsky, Vladimir A.; Wu, Xiangwei; Wagner, Daniel S.; Lapotko, Dmitri O.

2012-01-01

The limited specificity of nanoparticle (NP) uptake by target cells associated with a disease is one of the principal challenges of nanomedicine. Using the threshold mechanism of plasmonic nanobubble (PNB) generation and enhanced accumulation and clustering of gold nanoparticles in target cells, we increased the specificity of PNB generation and detection in target versus non-target cells by more than one order of magnitude compared to the specificity of NP uptake by the same cells. This improved cellular specificity of PNBs was demonstrated in six different cell models representing diverse molecular targets such as epidermal growth factor receptor, CD3 receptor, prostate specific membrane antigen and mucin molecule MUC1. Thus PNBs may be a universal method and nano-agent that overcome the problem of non-specific uptake of NPs by non-target cells and improve the specificity of NP-based diagnostics, therapeutics and theranostics at the cell level. PMID:22509318

Improved cellular specificity of plasmonic nanobubbles versus nanoparticles in heterogeneous cell systems.

PubMed

Lukianova-Hleb, Ekaterina Y; Ren, Xiaoyang; Constantinou, Pamela E; Danysh, Brian P; Shenefelt, Derek L; Carson, Daniel D; Farach-Carson, Mary C; Kulchitsky, Vladimir A; Wu, Xiangwei; Wagner, Daniel S; Lapotko, Dmitri O

2012-01-01

The limited specificity of nanoparticle (NP) uptake by target cells associated with a disease is one of the principal challenges of nanomedicine. Using the threshold mechanism of plasmonic nanobubble (PNB) generation and enhanced accumulation and clustering of gold nanoparticles in target cells, we increased the specificity of PNB generation and detection in target versus non-target cells by more than one order of magnitude compared to the specificity of NP uptake by the same cells. This improved cellular specificity of PNBs was demonstrated in six different cell models representing diverse molecular targets such as epidermal growth factor receptor, CD3 receptor, prostate specific membrane antigen and mucin molecule MUC1. Thus PNBs may be a universal method and nano-agent that overcome the problem of non-specific uptake of NPs by non-target cells and improve the specificity of NP-based diagnostics, therapeutics and theranostics at the cell level.

The genome-wide DNA sequence specificity of the anti-tumour drug bleomycin in human cells.

PubMed

Murray, Vincent; Chen, Jon K; Tanaka, Mark M

2016-07-01

The cancer chemotherapeutic agent, bleomycin, cleaves DNA at specific sites. For the first time, the genome-wide DNA sequence specificity of bleomycin breakage was determined in human cells. Utilising Illumina next-generation DNA sequencing techniques, over 200 million bleomycin cleavage sites were examined to elucidate the bleomycin genome-wide DNA selectivity. The genome-wide bleomycin cleavage data were analysed by four different methods to determine the cellular DNA sequence specificity of bleomycin strand breakage. For the most highly cleaved DNA sequences, the preferred site of bleomycin breakage was at 5'-GT* dinucleotide sequences (where the asterisk indicates the bleomycin cleavage site), with lesser cleavage at 5'-GC* dinucleotides. This investigation also determined longer bleomycin cleavage sequences, with preferred cleavage at 5'-GT*A and 5'- TGT* trinucleotide sequences, and 5'-TGT*A tetranucleotides. For cellular DNA, the hexanucleotide DNA sequence 5'-RTGT*AY (where R is a purine and Y is a pyrimidine) was the most highly cleaved DNA sequence. It was striking that alternating purine-pyrimidine sequences were highly cleaved by bleomycin. The highest intensity cleavage sites in cellular and purified DNA were very similar although there were some minor differences. Statistical nucleotide frequency analysis indicated a G nucleotide was present at the -3 position (relative to the cleavage site) in cellular DNA but was absent in purified DNA.

A Jagged 1-Notch 4 molecular switch mediates airway inflammation induced by ultrafine particles.

PubMed

Xia, Mingcan; Harb, Hani; Saffari, Arian; Sioutas, Constantinos; Chatila, Talal A

2018-04-05

Exposure to traffic-related particulate matter promotes asthma and allergic diseases. However, the precise cellular and molecular mechanisms by which particulate matter exposure acts to mediate these effects remain unclear. We sought to elucidate the cellular targets and signaling pathways critical for augmentation of allergic airway inflammation induced by ambient ultrafine particles (UFP). We used in vitro cell-culture assays with lung-derived antigen-presenting cells and allergen-specific T cells and in vivo mouse models of allergic airway inflammation with myeloid lineage-specific gene deletions, cellular reconstitution approaches, and antibody inhibition studies. We identified lung alveolar macrophages (AM) as the key cellular target of UFP in promoting airway inflammation. Aryl hydrocarbon receptor-dependent induction of Jagged 1 (Jag1) expression in AM was necessary and sufficient for augmentation of allergic airway inflammation by UFP. UFP promoted T H 2 and T H 17 cell differentiation of allergen-specific T cells in a Jag1- and Notch 4-dependent manner. Treatment of mice with an anti-Notch 4 antibody abrogated exacerbation of allergic airway inflammation induced by UFP. UFP exacerbate allergic airway inflammation by promoting a Jag1-Notch 4-dependent interaction between AM and allergen-specific T cells, leading to augmented T H cell differentiation. Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Membrane Curvature Sensing by Amphipathic Helices Is Modulated by the Surrounding Protein Backbone.

PubMed

Doucet, Christine M; Esmery, Nina; de Saint-Jean, Maud; Antonny, Bruno

2015-01-01

Membrane curvature is involved in numerous biological pathways like vesicle trafficking, endocytosis or nuclear pore complex assembly. In addition to its topological role, membrane curvature is sensed by specific proteins, enabling the coordination of biological processes in space and time. Amongst membrane curvature sensors are the ALPS (Amphipathic Lipid Packing Sensors). ALPS motifs are short peptides with peculiar amphipathic properties. They are found in proteins targeted to distinct curved membranes, mostly in the early secretory pathway. For instance, the ALPS motif of the golgin GMAP210 binds trafficking vesicles, while the ALPS motif of Nup133 targets nuclear pores. It is not clear if, besides curvature sensitivity, ALPS motifs also provide target specificity, or if other domains in the surrounding protein backbone are involved. To elucidate this aspect, we studied the subcellular localization of ALPS motifs outside their natural protein context. The ALPS motifs of GMAP210 or Nup133 were grafted on artificial fluorescent probes. Importantly, ALPS motifs are held in different positions and these contrasting architectures were mimicked by the fluorescent probes. The resulting chimeras recapitulated the original proteins localization, indicating that ALPS motifs are sufficient to specifically localize proteins. Modulating the electrostatic or hydrophobic content of Nup133 ALPS motif modified its avidity for cellular membranes but did not change its organelle targeting properties. In contrast, the structure of the backbone surrounding the helix strongly influenced targeting. In particular, introducing an artificial coiled-coil between ALPS and the fluorescent protein increased membrane curvature sensitivity. This coiled-coil domain also provided membrane curvature sensitivity to the amphipathic helix of Sar1. The degree of curvature sensitivity within the coiled-coil context remains correlated to the natural curvature sensitivity of the helices. This suggests that the chemistry of ALPS motifs is a key parameter for membrane curvature sensitivity, which can be further modulated by the surrounding protein backbone.

Context-specific control and the Stroop negative priming effect.

PubMed

Milliken, Bruce; Thomson, David R; Bleile, Karmen; MacLellan, Ellen; Giammarco, Maria

2012-01-01

The present study highlights the utility of context-specific learning for different probe types in accounting for the commonly observed dependence of negative priming on probe selection. Using a Stroop priming procedure, Experiments 1a and 1b offered a demonstration that Stroop priming effects can differ qualitatively for selection and no-selection probes when probe selection is manipulated between subjects, but not when it is manipulated randomly from trial to trial within subject (see also Moore, 1994). In Experiments 2 and 3, selection and no-selection probes served as two contexts that varied randomly from trial to trial, but for which proportion repeated was manipulated separately. A context-specific proportion repeated effect was observed in Experiment 2, characterized by modest quantitative shifts in the repetition effects as a function of the context-specific proportion repeated manipulation. However, with a longer intertrial interval in Experiment 3, a context-specific proportion repeated manipulation that focused on the no-selection probes changed the repetition effect qualitatively, from negative priming when the proportion repeated was .25 to positive priming when the proportion repeated was .75. The results are discussed with reference to the role of rapid, context-specific learning processes in the integration of prior experiences with current perception and action.

What Purposes, Specifically? Re-Thinking Purposes and Specificity in the Context of the "New Vocationalism"

ERIC Educational Resources Information Center

Dovey, Teresa

2006-01-01

Discipline-specific approaches to English for Academic Purposes (EAP) tend to overlook the purposes of the disciplines themselves and the issue of transferability from academic to professional contexts. This becomes problematic in the context of the "new knowledge economy" and emergent pedagogies in higher education, which are increasingly focused…

The emerging role of nuclear viral DNA sensors.

PubMed

Diner, Benjamin A; Lum, Krystal K; Cristea, Ileana M

2015-10-30

Detecting pathogenic DNA by intracellular receptors termed "sensors" is critical toward galvanizing host immune responses and eliminating microbial infections. Emerging evidence has challenged the dogma that sensing of viral DNA occurs exclusively in sub-cellular compartments normally devoid of cellular DNA. The interferon-inducible protein IFI16 was shown to bind nuclear viral DNA and initiate immune signaling, culminating in antiviral cytokine secretion. Here, we review the newly characterized nucleus-originating immune signaling pathways, their links to other crucial host defenses, and unique mechanisms by which viruses suppress their functions. We frame these findings in the context of human pathologies associated with nuclear replicating DNA viruses. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Cell biology of spinocerebellar ataxia.

PubMed

Orr, Harry T

2012-04-16

Ataxia is a neurological disorder characterized by loss of control of body movements. Spinocerebellar ataxia (SCA), previously known as autosomal dominant cerebellar ataxia, is a biologically robust group of close to 30 progressive neurodegenerative diseases. Six SCAs, including the more prevalent SCA1, SCA2, SCA3, and SCA6 along with SCA7 and SCA17 are caused by expansion of a CAG repeat that encodes a polyglutamine tract in the affected protein. How the mutated proteins in these polyglutamine SCAs cause disease is highly debated. Recent work suggests that the mutated protein contributes to pathogenesis within the context of its "normal" cellular function. Thus, understanding the cellular function of these proteins could aid in the development of therapeutics.

Do Memory CD4 T Cells Keep Their Cell-Type Programming: Plasticity versus Fate Commitment? Epigenome: A Dynamic Vehicle for Transmitting and Recording Cytokine Signaling.

PubMed

Johnson, John L; Vahedi, Golnaz

2018-03-01

CD4 + T cells are critical for the elimination of an immense array of microbial pathogens. Although there are aspects of helper T-cell differentiation that can be modeled as a classic cell-fate commitment, CD4 + T cells also maintain considerable flexibility in their transcriptional program. Here, we present an overview of chromatin biology during cellular reprogramming and, within this context, envision how the scope of cellular reprogramming may be expanded to further our understanding of the controversy surrounding CD4 + T lymphocyte plasticity or determinism. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Crowding in Cellular Environments at an Atomistic Level from Computer Simulations

PubMed Central

2017-01-01

The effects of crowding in biological environments on biomolecular structure, dynamics, and function remain not well understood. Computer simulations of atomistic models of concentrated peptide and protein systems at different levels of complexity are beginning to provide new insights. Crowding, weak interactions with other macromolecules and metabolites, and altered solvent properties within cellular environments appear to remodel the energy landscape of peptides and proteins in significant ways including the possibility of native state destabilization. Crowding is also seen to affect dynamic properties, both conformational dynamics and diffusional properties of macromolecules. Recent simulations that address these questions are reviewed here and discussed in the context of relevant experiments. PMID:28666087

A Tale of Two Sites: Cellphones, Participatory Video and Indigeneity in Community-Based Research

ERIC Educational Resources Information Center

Schwab-Cartas, Joshua; Mitchell, Claudia

2014-01-01

This polyvocal text is both a narrative and a dialogue between two scholar-activist researchers working in rural communities in distinct parts of the world--South Africa and Southern Mexico--sharing their experiences of using cellular phone and camcorders, while also exploring the potential sustainability of these technologies in the context of…

PML, SUMOylation, and Senescence

PubMed Central

Ivanschitz, Lisa; De Thé, Hugues; Le Bras, Morgane

2013-01-01

Since its discovery, 25 years ago, promyelocytic leukemia (PML) has been an enigma. Implicated in the oncogenic PML/RARA fusion, forming elusive intranuclear domains, triggering cell death or senescence, controlled by and perhaps controlling SUMOylation… there are multiple PML-related issues. Here we review the reciprocal interactions between PML, senescence, and SUMOylation, notably in the context of cellular transformation. PMID:23847762

Developing the Ability to Recontextualise Cellular Respiration: An Explorative Study in Recontextualising Biological Concepts

ERIC Educational Resources Information Center

Wierdsma, Menno; Boersma, Kerst Th.; Knippels, Marie-Christine; van Oers, Bert

2016-01-01

In many science education practices, students are expected to develop an understanding of scientific knowledge without being allowed a view of the practices and cultures that have developed and use this knowledge. Therefore, students should be allowed to develop scientific concepts in relation to the contexts in which those concepts are used.…

Changing appetites: The adaptive advantages of fuel choice

PubMed Central

Stanley, Illana A.; Ribeiro, Sofia M.; Giménez-Cassina, Alfredo; Norberg, Erik; Danial, Nika N.

2013-01-01

Cells are capable of metabolizing a variety of carbon substrates, including glucose, fatty acids, ketone bodies, and amino acids. Cellular fuel choice not only fulfills specific biosynthetic needs, but also enables programmatic adaptations to stress conditions beyond compensating for changes in nutrient availability. Emerging evidence indicates that specific switches from utilization of one substrate to another can have protective or permissive roles in disease pathogenesis. Understanding the molecular determinants of cellular fuel preference may provide insights into the homeostatic control of stress responses, and unveil therapeutic targets. Here, we highlight overarching themes encompassing cellular fuel choice, its link to cell fate and function, its advantages in stress protection, and its contribution to metabolic dependencies and maladaptations in pathologic conditions. PMID:24018218

Context-specific control and context selection in conflict tasks.

PubMed

Schouppe, Nathalie; Ridderinkhof, K Richard; Verguts, Tom; Notebaert, Wim

2014-02-01

This study investigated whether participants prefer contexts with relatively little cognitive conflict and whether this preference is related to context-specific control. A conflict selection task was administered in which participants had to choose between two categories that contained different levels of conflict. One category was associated with 80% congruent Stroop trials and 20% incongruent Stroop trials, while the other category was associated with only 20% congruent Stroop trials and 80% incongruent Stroop trials. As predicted, participants selected the low-conflict category more frequently, indicating that participants avoid contexts with high-conflict likelihood. Furthermore, we predicted a correlation between this preference for the low-conflict category and the control implementation associated with the categories (i.e., context-specific proportion congruency effect, CSPC effect). Results however did not show such a correlation, thereby failing to support a relationship between context control and context selection. Copyright © 2013 Elsevier B.V. All rights reserved.