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  • 1
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    Wiley
    In: BioEssays
    Publication Date: 2013-09-15
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  • 2
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    In: BioEssays
    Publication Date: 2013-09-15
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  • 3
    Publication Date: 2013-09-26
    Description: Long non-coding RNAs (lncRNAs) have recently gained increasing attention because of their crucial roles in gene regulatory processes. Functional studies using mammalian skin as a model system have revealed their role in controlling normal tissue homeostasis as well as the transition to a diseased state. Here, we describe how lncRNAs regulate differentiation to preserve an undifferentiated epidermal progenitor compartment, and to maintain a functional skin permeability barrier. Furthermore, we will reflect on recent work analyzing the impact of lncRNAs on the progression from normal epithelium to the development of skin disorders and cancer. Long non-coding RNAs (lncRNAs) have recently been shown to control a wide variety of gene regulatory processes. In mammalian skin, lncRNAs appear to regulate the intricate balance between progenitor cells undergoing continual regeneration in the basal layer and highly differentiated cells forming the epidermal permeability barrier.
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  • 4
    Publication Date: 2013-09-11
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  • 5
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    In: BioEssays
    Publication Date: 2013-09-15
    Description: General mechanism for exaggerated sexuallyselected traits. Many animals wield sexually-selected exaggerated traits. ‘Classic’ examples include the peacock's train, and the antlers observed in male deer, as well as fiddler crabs with enlarged claws, and the enlarged head-horns of rhinoceros beetles ( Trypoxylus dichotomus , cover). These traits are used for mate choice, or to deter rival males, because they act as unusually reliable signals of the condition of individual males: only the best-quality animals produce full-sized signal structures. But what keeps their expression honest? How can signal traits evolve that are resistant to invasion by cheaters who fake attractive signals? The answer may lie in the ancient insulin-like signalling pathway, which is found in all animals and directly links individual condition to growth in a dose dependent manner. Warren et al. discuss recent evidence suggesting that exaggerated sexually-selected signal traits arise when specific structures become extra-sensitive to physiological signals like insulins or insulin-like growth factors (pages 889–899 ).
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  • 6
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    Wiley
    In: BioEssays
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  • 7
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    Wiley
    In: BioEssays
    Publication Date: 2013-09-15
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  • 8
    Publication Date: 2013-09-18
    Description: mRNA synthesis in all organisms is performed by RNA polymerases, which work as nanomachines on DNA templates. The rate at which their product is made is an important parameter in gene expression. Transcription rate encompasses two related, yet different, concepts: the nascent transcription rate, which measures the in situ mRNA production by RNA polymerase, and the rate of synthesis of mature mRNA, which measures the contribution of transcription to the mRNA concentration. Both parameters are useful for molecular biologists, but they are not interchangeable and they are expressed in different units. It is important to distinguish when and where each one should be used. We propose that for functional genomics the use of nascent transcription rates should be restricted to the evaluation of the transcriptional process itself, whereas mature mRNA synthesis rates should be employed to address the transcriptional input to mRNA concentration balance leading to variation of gene expression. Transcription rate encompasses two related, yet different, concepts: the nascent transcription rate, which measures the in situ mRNA production by RNA polymerase, and the rate of synthesis of mature mRNA, which measures the contribution of transcription to the mRNA concentration. It is important to distinguish when to use each one.
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  • 9
    Publication Date: 2013-09-18
    Description: CALHM1 was recently demonstrated to be a voltage-gated ATP-permeable ion channel and to serve as a bona fide conduit for ATP release from sweet-, umami-, and bitter-sensing type II taste cells. Calhm1 is expressed in taste buds exclusively in type II cells and its product has structural and functional similarities with connexins and pannexins, two families of channel protein candidates for ATP release by type II cells. Calhm1 knockout in mice leads to loss of perception of sweet, umami, and bitter compounds and to impaired gustatory nerve responses to these tastants. These new studies validate the concept of ATP as the primary neurotransmitter from type II cells to gustatory neurons. Furthermore, they identify voltage-gated ATP release through CALHM1 as an essential molecular mechanism of ATP release in taste buds. We discuss these new findings, as well as unresolved issues in peripheral taste signaling that we hope will stimulate future research. Sweetness, umami, and bitterness are transmitted to the nervous system via ion channel-mediated ATP release from taste cells. A recent study demonstrated that CALHM1 is essential for taste cell ATP release and perception of sweetness, umami, and bitterness. We discuss the new findings and unresolved issues in peripheral taste signaling.
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  • 10
    Publication Date: 2013-09-20
    Description: The precise orchestration of two opposing protein complexes – one in the cytoplasm (β-catenin destruction complex) and the other at the plasma membrane (LRP6 signaling complex) – is critical for controlling levels of the transcriptional co-factor β-catenin, and subsequent activation of the Wnt/β-catenin signal transduction pathway. The Wnt pathway component Axin acts as an essential scaffold for the assembly of both complexes. How the β-catenin destruction and LRP6 signaling complexes are modulated following Wnt stimulation remains controversial. A recent study in Science by He and coworkers reveals an underlying logic for Wnt pathway control in which Axin phosphorylation toggles a switch between the active and inactive states. This mini-review focuses on this and two other recent studies that provide insight into the initial signaling events triggered by Wnt exposure. We emphasize regulation of the β-catenin destruction and LRP6 signaling complexes and propose a framework for future work in this area. The mechanism by which the Wnt pathway stabilizes β-catenin, a key transcriptional co-factor, remains controversial. Recent studies have revealed that the phosphorylation state of an essential regulator of the pathway, Axin, controls its conformation and, consequently, its availability to scaffold two opposing Wnt pathway protein complexes that regulate β-catenin stability.
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  • 11
    Publication Date: 2013-06-12
    Description: Metagenomics is a culture- and PCR-independent approach that is now widely exploited for directly studying microbial evolution, microbial ecology, and developing biotechnologies. Observations and discoveries are critically dependent on DNA extraction methods, sequencing technologies, and bioinformatics tools. The potential pitfalls need to be understood and, to some degree, mastered if the resulting data are to survive scrutiny. In particular, methodological variations appear to affect results from different ecosystems differently, thus increasing the risk of biological and ecological misinterpretation. Part of the difficulty is derived from the lack of knowledge concerning the true microbial diversity and because no approach can guarantee accessing microorganisms in the same proportion in which they exist in the environment. However, the variation between different approaches (e.g. DNA extraction techniques, sequence annotation systems) can be used to evaluate whether observations are meaningful. These methodological variations can be integrated into the error analysis before comparing microbial communities. Metagenomics is a powerful approach targeting environmental nucleic diversity but represents also a methodological jungle where pitfalls are challenging when quantitative observations are desired. Here we describe the effect of critical parameters (especially DNA extraction and annotation stringency) required to represent microbial communities function and structure in the metagenomic era.
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  • 12
    Publication Date: 2013-06-12
    Description: Pericytes, typically attached to the walls of microvessels in almost all organs, interact with endothelial cells and take part in diverse biological processes, e.g. blood vessel regulation and tissue repair. This suggests that pericytes harbor a remarkable degree of cellular plasticity, which could potentially be employed for the treatment of diseases affecting diverse tissues such as the skeletal muscle and the central nervous system. Here, we follow pericytes on their journey across Waddington's epigenetic landscape, descending from their origin, along a path guided by environmental signals or ectopic transcription factors, at the end of which they acquire a new identity, e.g. muscle or nerve cells. The central theme of this review is the question of whether pericytes can be enticed to differentiate into whatever cell type is needed, and thus provide an endogenous cellular source for treating as yet incurable diseases – like a magic bullet. Also watch the Video Abstract. http://youtu.be/J4b-cmRWLWI Recent studies show that microvessel-associated pericytes exhibit an unprecedented degree of plasticity and implicate these cells as a physiological cellular source or therapeutic target for tissue repair. They have potential for therapeutic applications in areas ranging from muscle degeneration to heart infarction and CNS injury.
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  • 13
    Publication Date: 2013-04-03
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  • 14
    Publication Date: 2013-04-11
    Description: Factors affecting transcriptional elongation have been characterized extensively in in vitro, single cell (yeast) and cell culture systems; however, data from the context of multicellular organisms has been relatively scarce. While studies in homogeneous cell populations have been highly informative about the underlying molecular mechanisms and prevalence of polymerase pausing, they do not reveal the biological impact of perturbing this regulation in an animal. The core components regulating pausing are expressed in all animal cells and are recruited to the majority of genes, however, disrupting their function often results in discrete phenotypic effects. Mutations in genes encoding key regulators of transcriptional pausing have been recovered from several genetic screens for specific phenotypes or interactions with specific factors in mice, zebrafish and flies. Analysis of these mutations has revealed that control of transcriptional pausing is critical for a diverse range of biological pathways essential for animal development and survival. Animal studies reveal that correct regulation of promoter proximal pausing is critical for a diverse range of biological pathways during embryo development and also for health in adult life. This regulation facilitates fine control of gene expression levels and may also act as a barrier to uncontrolled cell proliferation.
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  • 15
    Publication Date: 2013-04-11
    Description: Phosphatidylinositol 4,5-bisphosphate (PI4,5P 2 ) is a key lipid signaling molecule that regulates a vast array of biological activities. PI4,5P 2 can act directly as a messenger or can be utilized as a precursor to generate other messengers: inositol trisphosphate, diacylglycerol, or phosphatidylinositol 3,4,5-trisphosphate. PI4,5P 2 interacts with hundreds of different effector proteins. The enormous diversity of PI4,5P 2 effector proteins and the spatio-temporal control of PI4,5P 2 generation allow PI4,5P 2 signaling to control a broad spectrum of cellular functions. PI4,5P 2 is synthesized by phosphatidylinositol phosphate kinases (PIPKs). The array of PIPKs in cells enables their targeting to specific subcellular compartments through interactions with targeting factors that are often PI4,5P 2 effectors. These interactions are a mechanism to define spatial and temporal PI4,5P 2 synthesis and the specificity of PI4,5P 2 signaling. In turn, the regulation of PI4,5P 2 effectors at specific cellular compartments has implications for understanding how PI4,5P 2 controls cellular processes and its role in diseases. Site-directed synthesis of phosphatidylinositol-4,5-bisphosphate (PI4,5P 2 ) at distinct sub-cellular compartments mediates a variety of events, such as migration, cell-cell adhesion, transcription and vesicle trafficking. PI4,5P 2 regulated processes are critical for function at the cellular level, which is evident in neurons, platelet, and macrophage function.
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  • 16
    Publication Date: 2013-04-11
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  • 17
    Publication Date: 2013-04-11
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  • 18
    Publication Date: 2013-04-11
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  • 19
    Publication Date: 2013-04-11
    Description: The increased incidence of morbidity and mortality due to Clostridium difficile infection, had led to the emergence of fecal microbial transplantation (FMT) as a highly successful treatment. From this, a 32 strain stool substitute has been derived, and successfully tested in a pilot human study. These approaches could revolutionize not only medical care of infectious diseases, but potentially many other conditions linked to the human microbiome. But a second revolution may be needed in order for regulatory agencies, society and medical practitioners to accept and utilize these interventions, monitor their long term effects, have a degree of control over their use, or at a minimum provide guidelines for donors and recipients. If a simple replacement of your gut microbiota by someone else's could improve your health and ability to function, would you do it? How would you select the donor and would the “authorities” let you perform the transplant? The age of the microbiome is here, but is society ready?
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  • 20
    Publication Date: 2013-09-10
    Description: Many species maintain cytosine DNA methyltransferase (MTase) genes belonging to the Dnmt2 family. Prokaryotic modification-restriction systems utilize DNA methylation to distinguish between self and foreign DNA, and cytosine methylation in eukaryotic DNA contributes to epigenetic mechanisms that control gene expression. However, Dnmt2 proteins display only low or no DNA MTase activity, making this protein family the odd and enigmatic family member. Recent evidence showed that Dnmt2 proteins are not DNA but RNA MTases with functions in biological processes as diverse as stress responses and RNA-mediated inheritance. These observations not only raise profound questions regarding the perceived substrate specificities of cytosine MTase, but also suggest links between DNA and RNA modification systems. Here, we speculate that Dnmt2 proteins might be part of an ancient cytosine modification toolbox that is used to successfully respond to environmental challenges, including constantly evolving RNA and DNA substrates. Recent observations indicated that Dnmt2 proteins, which belong to a highly conserved DNA cytosine methyltransferase family, are important for the response to RNA-based stressors, including transposons, viruses, and experimentally introduced small RNAs. Is Dnmt2 part of an ancient defense system that employs nucleotide modifications against invading pathogens?
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  • 21
    Publication Date: 2013-09-15
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  • 22
    Publication Date: 2013-09-21
    Description: Inflammatory immune cells, when activated, display much the same metabolic profile as a glycolytic tumor cell. This involves a shift in metabolism away from oxidative phosphorylation towards aerobic glycolysis, a phenomenon known as the Warburg effect. The result of this change in macrophages is to rapidly provide ATP and metabolic intermediates for the biosynthesis of immune and inflammatory proteins. In addition, a rise in certain tricarboxylic acid cycle intermediates occurs notably in citrate for lipid biosynthesis, and succinate, which activates the transcription factor Hypoxia-inducible factor. In this review we take a look at the emerging evidence for a role for the Warburg effect in the immune and inflammatory responses. The reprogramming of metabolic pathways in macrophages, dendritic cells, and T cells could have relevance in the pathogenesis of inflammatory and metabolic diseases and might provide novel therapeutic strategies. Recent studies reveal that inflammatory cells, when activated, display similar metabolic traits as cancer cells. During an inflammatory response or infection pro-inflammatory immune cells can shift their metabolism away from oxidative phosphorylation towards a high rate of glycolysis, a phenomenon known as the Warburg effect.
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  • 23
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    Wiley
    In: BioEssays
    Publication Date: 2013-09-21
    Description: The fast advancing RNA-seq technology has unveiled an unexpected diversity and expression specificity of 3′ untranslated regions (3′UTRs) of mRNAs. In particular, neural mRNAs seem to express significantly longer 3′UTRs, some of which are over 10 kb in length. The extensive elongation of 3′UTRs in neural tissues provides intriguing possibilities for cell type-specific regulations that are governed by miRNAs, RNA-binding proteins and ribonucleoprotein aggregates. In this article, we review recent progress in the characterization of mRNA 3′UTRs and discuss their implications in the understanding of 3′UTR-mediated gene regulation. Differential expression of short and long 3′UTRs, especially in neurons, presents technical challenges for experimental characterization. Such dynamic expression of 3′UTR isoforms, however, provides a mechanism to regulate protein production of both individual and a pool of transcripts in physiological and pathological conditions.
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  • 24
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    Wiley
    In: BioEssays
    Publication Date: 2014-12-18
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  • 25
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    In: BioEssays
    Publication Date: 2014-11-11
    Description: Dynamic interactions with DNA allow replication protein A to direct single-stranded DNA-intermediates into different pathways for synthesis or repair. On pages 1156–1161 , Chen and Wold review recent discoveries that show that replication protein A (RPA), the major eukaryotic single-stranded DNA-binding protein, binds DNA dynamically and that this is important for correct processing of DNA intermediates. The cover shows a model of human RPA interacting with ssDNA based on the known structures of the domains of human RPA and the structure of Ustalago RPA bound to DNA. The three subunits of RPA are shown in blue (RPA1), red (RPA2), and green (RPA3) with ssDNA shown in black.
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  • 26
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    Wiley
    In: BioEssays
    Publication Date: 2014-11-11
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  • 27
    Publication Date: 2014-11-11
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  • 28
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    In: BioEssays
    Publication Date: 2014-11-11
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  • 29
    Publication Date: 2014-12-19
    Description: Ramathal et al. have employed an elegant xenotransplantation technique to study the fate of human induced pluripotent stem cells (hiPSCs) from fertile males and from males carrying Y chromosome deletions of the azoospermia factor ( AZF ) region. When placed in a mouse testis niche, hiPSCs from fertile males differentiate into germ cell-like cells (GCLCs). Highlighting the crucial role of cell autonomous factors in male sterility, hiPSCs derived from azoospermic males prove to be less successful under similar circumstances. Their studies argue that the agametic “Sertoli cell only” phenotype of two of the AZF deletions likely arises from a defect in the maintenance of germline stem cells (GSCs) rather than from a defect in their specification. These observations underscore the importance of the dialogue between the somatic niche and its inhabitant stem cells, and open up interesting questions concerning the functioning of the somatic niche and how it communicates to the GSCs. In Ramathal et al., human induced pluripotent stem cells (hiPSCs) from wild type and azoospermic men adopt germ cell fate in vitro. Upon xenotransplantation into mouse seminiferous tubules, these hiPSCs acquired germ cell-like (GCL) fate. Remarkably, samples from azoospermic males were less efficient in both contexts.
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  • 30
    Publication Date: 2014-12-03
    Description: Gene transcription is strictly controlled by the interplay of regulatory events at gene promoters and gene-distal regulatory elements called enhancers. Despite extensive studies of enhancers, we still have a very limited understanding of their mechanisms of action and their restricted spatio-temporal activities. A better understanding would ultimately lead to fundamental insights into the control of gene transcription and the action of regulatory genetic variants involved in disease. Here, I review and discuss pros and cons of state-of-the-art genomics methods to localize and infer the activity of enhancers. Among the different approaches, profiling of enhancer RNAs yields the highest specificity and may be superior in detecting in vivo activity. I discuss their apparent similarities to promoters, which challenge the established view of enhancers and promoters as distinct entities, and present a unifying model of regulatory elements in transcriptional regulation, in which activity, transcriptional output and regulatory function is context specific. Profiling of enhancer RNAs (eRNAs) likely represents a paradigm shift in enhancer genomics, yielding high specificity in the localization of active enhancers. The similarities between enhancers and promoters are discussed and their established distinctions are deconstructed. Based on this, a unified model of regulatory elements in transcriptional regulation is presented.
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  • 31
    Publication Date: 2012-12-28
    Description: Mitochondrial function is achieved through the cooperative interaction of two genomes: one nuclear (nuDNA) and the other mitochondrial (mtDNA). The unusual transmission of mtDNA, predominantly maternal without recombination is predicted to affect the fitness of male offspring. Recent research suggests the strong sexual dimorphism in aging is one such fitness consequence. The uniparental inheritance of mtDNA results in a selection asymmetry; mutations that affect only males will not respond to natural selection, imposing a male-specific mitochondrial mutation load. Prior work has implicated this male-specific mutation load in disease and infertility, but new data from fruit flies suggests a prominent role for mtDNA in aging; across many taxa males almost invariably live shorter lives than females. Here we discuss this new work and identify some areas of future research that might now be encouraged to explore what may be the underpinning cause of the strong sexual dimorphism in aging. Editor's suggested further reading in BioEssays: Mitonuclear match: Optimizing fitness and fertility over generations drives ageing within generations Abstract Mitochondrial manoeuvres: Latest insights and hypotheses on mitochondrial partitioning during mitosis in Saccharomyces cerevisiae Abstract Mitochondria and the culture of the Borg Abstract The maternal inheritance of mtDNA results in a selection asymmetry; natural selection cannot act on mutations that affect only males. This asymmetry imposes a male-specific mutation load previously implicated in male disease and infertility. New work suggests this asymmetry underpins the strong sexual dimorphism in aging observed across taxa.
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  • 32
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    In: BioEssays
    Publication Date: 2013-01-16
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  • 33
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    In: BioEssays
    Publication Date: 2013-01-16
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  • 34
    Publication Date: 2013-01-16
    Description: Simple DNA repeats (trinucleotide repeats, micro- and minisatellites) are prone to expansion/contraction via formation of secondary structures during DNA synthesis. Such structures both inhibit replication forks and create opportunities for template-primer slippage, making these repeats unstable. Certain aspects of simple repeat instability, however, suggest additional mechanisms of replication inhibition dependent on the primary DNA sequence, rather than on secondary structure formation. I argue that expanded simple repeats, due to their lower DNA complexity, should transiently inhibit DNA synthesis by locally depleting specific DNA precursors. Such transient inhibition would promote formation of secondary structures and would stabilize these structures, facilitating strand slippage. Thus, replication problems at simple repeats could be explained by potentiated toxicity, where the secondary structure-driven repeat instability is enhanced by DNA polymerase stalling at the low complexity template DNA. This minireview is dedicated to the FASEB-2012 meeting “Dynamic DNA Structures in Biology”, organized by Nancy Maizels and Sergei Mirkin. Expanded trinucleotide repeats are inherently unstable due to their propensity for strand slippage. I hypothesize that strand slippage is further promoted by local transient depletion of specific dNTPs during synthesis across this low-complexity DNA. Possible hairpin extrusion between polymerase and the clamp (the scheme) may explain saltatory repeat expansion.
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  • 35
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    In: BioEssays
    Publication Date: 2013-01-16
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  • 36
    Publication Date: 2013-02-27
    Description: Clathrin-mediated endocytosis is a major route for the retrieval of plasma-membrane cargoes, and defects of this process can cause catastrophic human dysfunctions. However, the processes governing how a clathrin-coated profile (ccp) is initiated are still murky. Despite an ever-growing cast of molecules proposed as triggers of ccp nucleation and increasingly sophisticated bioimaging techniques examining clathrin-mediated endocytosis, it is yet unknown if ccp formation is governed by a universal mechanism. A recent paper by Cocucci et al. has tracked single-molecule events to identify that stable accumulation of ccps requires the near-simultaneous arrival of two AP2 adaptors bridged by one clathrin triskelion. This commentary examines the role of AP2 in cargo-mediated endocytosis in the light of recent advances in biophotonics, chemical inhibitors and genetics, examines the claims of other molecules to be the initiators of ccp formation and proposes future directions in research into this topic. Editor's suggested further reading in BioEssays: The evolution of dynamin to regulate clathrin-mediated endocytosis Abstract Clathrin-mediated endocytosis: What works for small, also works for big Abstract Coccuci et al. propose that clathrin coated pits (ccps) randomly nucleate via simultaneous arrival of two AP2 complexes and a clathrin triskelion at the plasma membrane. The nascent ccp stabilizes by incorporating more clathrin/AP2 complexes and early factors such as FCHo1/2, before integrating transmembrane cargoes and acquiring curvature.
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  • 37
    Publication Date: 2012-11-14
    Description: While innovations in modern microscopy, spectroscopy, and nanoscopy techniques have made single molecule observation a standard in many laboratories, the actual design of meaningful fluorescence reporter systems now hinders major scientific breakthroughs. Even though the field of chemical biology is supercharging the fluorescence toolbox, surprisingly few strategies exist that make the transition from model systems to biologically relevant applications. At the same time, the number of microscopy techniques is growing dramatically. We explain our view on how the impact of modern technologies is influenced not only by further hard- and software developments, but also by the availability and suitability of protein-engineering tools. We identify how the largely independent research fields of chemical biology and fluorescence nanoscopy can influence each other to synergistically drive future technology that can visualize the localization, structure, and dynamics of molecular function without constraints. Fluorescence technologies provide key to study molecular structure and dynamics with super spatial and temporal resolution. We discuss how synergistic developments of novel labeling technologies combined with optical engineering can make the biggest contribution to advance state of the art tools for both, in vitro and non-invasive in vivo measurements.
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  • 38
    Publication Date: 2013-03-02
    Description: When a cell divides, it produces two daughter cells initially connected by a cytokinesis bridge, which is eventually cut through abscission. One of the two daughter cells inherits a bridge “remnant”, which has been proposed to be degraded by autophagy. The fate and function of remnants is attracting increasing attention, as their accumulation appears to influence proliferation versus differentiation of the daughter cells. Here, we present a simple model for bridge and remnant turnover in a dynamic cell population. We demonstrate that remnant proportions depend on the ratio of remnant and bridge lifetimes to the cell population doubling time. Our results yield new alternative interpretations for published experimental data, leading us to believe that autophagy-independent pathways for remnant degradation may exist. In addition, using the model, we determined experimentally inaccessible parameters such as remnant lifetime. Our model proves to be a useful tool for studying bridge and remnant populations. As cells divide, they produce bridges, which decay into remnants. Remnants are then thought to decay by autophagy. Defective remnant decay correlates with enhanced proliferation. We generated a model for remnant turnover, measured remnant lifetime, and simulated published experiments. We find that remnant decay can occur by autophagy-independent pathways.
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  • 39
    Publication Date: 2013-03-02
    Description: Culture-independent high-throughput sequencing has provided unprecedented insights into microbial ecology, particularly for Earth's most ubiquitous and diverse inhabitants – the viruses. A plethora of methods now exist for amplifying the vanishingly small amounts of nucleic acids in natural viral communities in order to sequence them, and sequencing depth is now so great that viral genomes can be detected and assembled even amid large concentrations of non-viral DNA. Complementing these advances in amplification and sequencing is the ability to physically link fluorescently labeled viruses to their host cells via high-throughput flow sorting. Sequencing of such isolated virus–host pairs facilitates cultivation-independent exploration of the natural host range of viruses. Within the next decade, as these technologies become widespread, we can expect to see a systematic expansion of our knowledge of viruses and their hosts. Recent advances in viral metagenomics include techniques for sequencing limiting amounts of viral nucleic acids, the use of ultra-deep sequencing, and viral tagging to determine virus–host interactions. Mock communities should be used to rigorously evaluate new protocols, and special consideration must be given to the storage of metagenomic data.
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  • 40
    Publication Date: 2013-03-02
    Description: The Hippo pathway, a cascade of protein kinases that inhibits the oncogenic transcriptional coactivators YAP and TAZ, was discovered in Drosophila as a major determinant of organ size in development. Known modes of regulation involve surface proteins that mediate cell-cell contact or determine epithelial cell polarity which, in a tissue-specific manner, use intracellular complexes containing FERM domain and actin-binding proteins to modulate the kinase activities or directly sequester YAP. Unexpectedly, recent work demonstrates that GPCRs, especially those signaling through Galpha 12/13 such as the protease activated receptor PAR1, cause potent YAP dephosphorylation and activation. This response requires active RhoA GTPase and increased assembly of filamentous (F-)actin. Morever, cell architectures that promote F-actin assembly per se also activate YAP by kinase-dependent and independent mechanisms. These findings unveil the ability of GPCRs to activate the YAP oncogene through a newly recognized signaling function of the actin cytoskeleton, likely to be especially important for normal and cancerous stem cells. The hippo pathway, a protein kinase cascade activated by cell contact, phosphorylates the YAP oncogene to inhibit proliferation. GPCRs like PAR1 that recruit Gα 12/13 and activate RhoA can dephosphorylate and activate YAP through an inhibition of the hippo pathway. This requires F-actin assembly, which also activates YAP through hippo-independent mechanisms.
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  • 41
    Publication Date: 2012-09-25
    Description: New genes have frequently formed and spread to fixation in a wide variety of organisms, constituting abundant sets of lineage-specific genes. It was recently reported that an excess of primate-specific and human-specific genes were upregulated in the brains of fetuses and infants, and especially in the prefrontal cortex, which is involved in cognition. These findings reveal the prevalent addition of new genetic components to the transcriptome of the human brain. More generally, these findings suggest that genomes are continually evolving in both sequence and content, eroding the conservation endowed by common ancestry. Despite increasing recognition of the importance of new genes, we highlight here that these genes are still seriously under-characterized in functional studies and that new gene annotation is inconsistent in current practice. We propose an integrative approach to annotate new genes, taking advantage of functional and evolutionary genomic methods. We finally discuss how the refinement of new gene annotation will be important for the detection of evolutionary forces governing new gene origination. Regardless of recent findings that new genes are important for human brain functions, we highlight that new genes are still generally under-characterized in functional studies and that new gene annotation is inconsistent in current practice. We propose an integrative approach to annotate new genes based on functional and evolutionary genomics.
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  • 42
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    In: BioEssays
    Publication Date: 2012-10-13
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  • 43
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    In: BioEssays
    Publication Date: 2012-10-13
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  • 44
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    In: BioEssays
    Publication Date: 2012-10-13
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  • 45
    Publication Date: 2012-10-03
    Description: Recent studies of prostate cancer and other tumor types have revealed significant support, as well as unexpected complexities, for the application of concepts from normal stem cell biology to cancer. In particular, the cell of origin and cancer stem cell models have been proposed to explain the heterogeneity of tumors during the initiation, propagation, and evolution of cancer. Thus, a basis of intertumor heterogeneity has emerged from studies investigating whether stem cells and/or non-stem cells can serve as cells of origin for cancer and give rise to tumor subtypes that vary in disease outcome. Furthermore, analyses of putative cancer stem cells have revealed the genetically diverse nature of cancers and expanded our understanding of intratumor heterogeneity and clonal evolution. Overall, the principles that have emerged from these stem cell studies highlight the challenges to be surmounted to develop effective treatment strategies for cancer. Concepts from stem cell biology can explain the basis for intertumoral heterogeneity between different tumors of the same tissue type, as well as intratumoral heterogeneity within a single tumor. In this review, we discuss the cell of origin, cancer stem cells, and clonal evolution, with a focus on prostate cancer.
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  • 46
    Publication Date: 2012-08-23
    Description: How fast? How strong? How many? So what? Why do numbers matter in biology? Chromatin binding proteins are forever in motion, exchanging rapidly between bound and free pools. How do regulatory systems whose components are in constant flux ensure stability and flexibility? This review explores the application of quantitative and mathematical approaches to mechanisms of epigenetic regulation. We discuss methods for measuring kinetic parameters and protein quantities in living cells, and explore the insights that have been gained by quantifying and modelling dynamics of chromatin binding proteins. Current models for chromatin mediated gene regulation often describe molecules as binding, modifying or recruiting other molecules, but with little reference to the quantitative differences between them. In this review we explore how quantitative and mathematical approaches can give insights into mechanisms of epigenetic regulation.
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  • 47
    Publication Date: 2012-08-23
    Description: Evolutionary theory assumed that mutations occur constantly, gradually, and randomly over time. This formulation from the “modern synthesis” of the 1930s was embraced decades before molecular understanding of genes or mutations. Since then, our labs and others have elucidated mutation mechanisms activated by stress responses. Stress-induced mutation mechanisms produce mutations, potentially accelerating evolution, specifically when cells are maladapted to their environment, that is, when they are stressed. The mechanisms of stress-induced mutation that are being revealed experimentally in laboratory settings provide compelling models for mutagenesis that propels pathogen–host adaptation, antibiotic resistance, cancer progression and resistance, and perhaps much of evolution generally. We discuss double-strand-break-dependent stress-induced mutation in Escherichia coli. Recent results illustrate how a stress response activates mutagenesis and demonstrate this mechanism's generality and importance to spontaneous mutation. New data also suggest a possible harmony between previous, apparently opposed, models for the molecular mechanism. They additionally strengthen the case for anti-evolvability therapeutics for infectious disease and cancer. Stress-induced mutation molecular mechanism in Escherichia coli : repair of DNA double-strand breaks is switched to a mutagenic mode using DinB and other error-prone DNA polymerases during stress, by the RpoS stress response. This increases genetic diversity, and the ability to evolve, when cells are maladapted to their environment: when stressed.
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  • 48
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    In: BioEssays
    Publication Date: 2012-08-21
    Description: Scientists should learn to communicate effectively with their colleagues through long-term, sustained training instead of ad hoc, one-off “interventions” that may or may not occur during graduate school or postdoctoral work. Since such training may place unreasonable demands on research advisors, institutions should create career opportunities for “peer-peer communication teachers.”
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  • 49
    Publication Date: 2012-08-21
    Description: Hedgehog is an important morphogenic signal that directs pattern formation during embryogenesis, but its activity also remains present through adult life. It is now becoming increasingly clear that during the reproductive phase of life and beyond it continues to direct cell renewal (which is essential to combat the chronic environmental stress to which the body is constantly exposed) and counteracts vascular, osteolytic and sometimes oncological insults to the body. Conversely, down-regulation of hedgehog signalling is associated with ageing-related diseases such as type 2 diabetes, neurodegeneration, atherosclerosis and osteoporosis. Hence, in this essay we argue that hedgehog signalling is not only important at the start of life, but also constitutes an important anti-geriatric influence, and that enhanced understanding of its properties may contribute to developing rational strategies for healthy ageing and prevention of ageing-related diseases. Also watch the Video Abstract Hedgehog is a morphogenic signal during embryogenesis and adult life with many vital biological functions such as neuromodulators and anti-adipogenesis. Downregulation of Hedgehog signalling is associated with ageing-related diseases like Alzheimer/Parkinson and T2D diseases and upregulation of this signalling reduces risk of these diseases. Thus, Hedgehog signalling is antagonist of ageing.
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  • 50
    Publication Date: 2012-08-21
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  • 51
    Publication Date: 2012-08-21
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  • 52
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    In: BioEssays
    Publication Date: 2012-10-13
    Description: Control of osteogenesis by the canonical Wnt and BMP pathways in vivo: The cover image shows a transverse section through a Xenopus tropicalis metamorphosing tadpole phalange. This histological section was stained with Kernechtrot to reveal cell bodies (pink) and Alcian blue to show the cartilage (blue). The circular cartilaginous shaft is surrounded by a layer of bone mineralized matrix (grey ring) secreted by adjacent osteoblasts. On pages 953–962 Marcellini et al. review recent experimental evidence showing that the canonical Wnt and BMP pathways functionally interact as cells differentiate from osteochondroprogenitors to osteoblasts and osteocytes, in the context of the developing vertebrate embryo. BMP signalling specifies multipotent mesenchymal cells into osteochondroprogenitors which are subsequently driven towards the osteoblastic fate by the Wnt pathway. In osteoblasts, both pathways promote differentiation, albeit with notable mechanistic differences. Finally, in osteocytes, the Wnt and BMP pathways exert opposite effects on the control of bone resorption by osteoclasts. Cover by S. Marcellini.
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  • 53
    Publication Date: 2012-04-14
    Description: MHC II, a major feature of the adaptive immune system, is lacking in Atlantic cod, and there are different scenarios (metabolic cost hypothesis or functional shift hypothesis) that might explain this loss. The lack of MHC II coincides with an increased number of genes for MHC I and Toll-like receptors (TLRs).
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  • 54
    Publication Date: 2012-04-15
    Description: Fluorescence microscopy is the primary tool for studying complex processes inside individual living cells. Technical advances in both molecular biology and microscopy have made it possible to image cells from many genetic and environmental backgrounds. These images contain a vast amount of information, which is often hidden behind various sources of noise, convoluted with other information and stochastic in nature. Accessing the desired biological information therefore requires new tools of computational image analysis and modeling. Here, we review some of the recent advances in computational analysis of images obtained from fluorescence microscopy, focusing on bacterial systems. We emphasize techniques that are readily available to molecular and cell biologists but also point out examples where problem-specific image analyses are necessary. Thus, image analysis is not only a toolkit to be applied to new images but also an integral part of the design and implementation of a microscopy experiment.
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  • 55
    Publication Date: 2012-04-15
    Description: The use of super-resolution imaging techniques in cell biology has yielded a wealth of information regarding cellular elements and processes that were invisible to conventional imaging. Focusing on images obtained by stimulated emission depletion (STED) microscopy, we discuss how the new high-resolution data influence the ways in which we use and interpret images in cell biology. Super-resolution images have lent support to some of our current hypotheses. But, more significantly, they have revealed unexpectedly complex processes that cannot be accounted for by the simpler models based on diffraction-limited imaging. The super-resolution imaging data challenge cell biologists to change their theoretical framework, by including, for instance, interpretations that describe multiple functions, functional errors or lack of function for cellular elements. In this context, we argue that descriptive research using super-resolution microscopy is now as necessary as hypothesis-driven research.
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  • 56
    Publication Date: 2012-04-15
    Description: The current revolution in biological microscopy stems from the realisation that advances in optics and computational tools and automation make the modern microscope an instrument that can access all scales relevant to modern biology – from individual molecules all the way to whole tissues and organisms and from single snapshots to time-lapse recordings sampling from milliseconds to days. As these and more new technologies appear, the challenges of delivering them to the community grows as well. I discuss some of these challenges, and the examples where openly shared technology have made an impact on the field.
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  • 57
    Publication Date: 2012-04-15
    Description: The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for fluorescence (or Förster) resonance energy transfer (FRET) microscopy is providing important tools for monitoring dynamic protein interactions inside living cells. The increased interest in FRET microscopy has driven the development of many different methods to measure FRET. However, the interpretation of FRET measurements is complicated by several factors including the high fluorescence background, the potential for photoconversion artifacts and the relatively low dynamic range afforded by this technique. Here, we describe the advantages and disadvantages of four methods commonly used in FRET microscopy. We then discuss the selection of FPs for the different FRET methods, identifying the most useful FP candidates for FRET microscopy. The recent success in expanding the FP color palette offers the opportunity to explore new FRET pairs.
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  • 58
    Publication Date: 2012-04-15
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  • 59
    Publication Date: 2012-04-15
    Description: Light sheet microscopy is an easy to implement and extremely powerful alternative to established fluorescence imaging techniques such as laser scanning confocal, multi-photon and spinning disk microscopy. By illuminating the sample only with a thin slice of light, photo-bleaching is reduced to a minimum, making light sheet microscopy ideal for non-destructive imaging of fragile samples over extended periods of time. Millimeter-sized samples can be imaged rapidly with high resolution and high depth penetration. A large variety of instruments have been developed and optimized for a number of different samples: Bessel beams form thin light sheets for single cells, and selective plane illumination microscopy (SPIM) offers multi-view acquisition to image entire embryos with isotropic resolution. This review explains how light sheet microscopy involves a conceptually new microscope design and how it changes modern imaging in biology.
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  • 60
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    In: BioEssays
    Publication Date: 2012-04-15
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  • 61
    Publication Date: 2012-04-15
    Description: The role of the major histocompatibility complex (MHC) in mate choice in humans is controversial. Nowadays, the availability of genetic variation data at genomic scales allows for a careful assessment of this question. In 2008, Chaix et al. reported evidence for MHC-dependent mate choice among European American spouses from the HapMap 2 dataset. Recently, Derti et al. suggested that this observation was not robust. Furthermore, when Derti et al. applied similar analyses to the HapMap 3 European American samples, they did not see a significant effect. Although some of the points raised by Derti et al. are relevant, we disagree with the reported absence of evidence for MHC-dependent mate choice within the HapMap samples. More precisely, we show here that the MHC dissimilarity among HapMap 3 European American spouses is still extreme in comparison to the rest of the genome, even after multiple testing correction. This finding supports the hypothesis of MHC-dependent mate choice in some human populations. The MHC may influence mate choice in some human populations also depending on other variables such as socio-cultural factors, the level of genetic diversity and the strength of pathogenic pressures.
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  • 62
    Publication Date: 2012-04-15
    Description: Is a two-fold approach — preliminary studies based on small samples followed by a large-sample study to check reproducibility — in the search for biomarkers really prudent?
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  • 63
    Publication Date: 2012-04-15
    Description: Here we elucidate a paradox: how a single chemoattractant-receptor system in two individuals is used for communication despite the seeming inevitability of self-excitation. In the filamentous fungus Neurospora crassa , genetically identical cells that produce the same chemoattractant fuse via the homing of individual cell protrusions toward each other. This is achieved via a recently described “ping-pong” pulsatile communication. Using a generic activator-inhibitor model of excitable behavior, we demonstrate that the pulse exchange can be fully understood in terms of two excitable systems locked into a stable oscillatory pattern of mutual excitation. The most puzzling properties of this communication are the sudden onset of oscillations with final amplitude, and the absence of seemingly inevitable self-excitation. We show that these properties result directly from both the excitability threshold and refractory period characteristic of excitable systems. Our model suggests possible molecular mechanisms for the ping-pong communication. Fusion of genetically identical cells in Neurospora crassa is achieved by the cells taking turns in releasing a chemoattractant and sensing it.
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  • 64
    Publication Date: 2012-04-15
    Description: Food turns out to be not only the nutrient supplier for our body but also a carrier of regulatory information. Interestingly, a recent study made the discovery that some plant/food-derived microRNAs (miRNAs) accumulate in the serum of humans or plant-feeding animals, and regulate mammalian gene expression in a sequence-specific manner. The authors provided striking evidence that miRNAs could function as active signaling molecules to transport information across distinct species or even kingdoms. Although the mechanism of how miRNAs are shuttled between different organisms is still not well characterized, initial results point to the involvement of microvesicles and specific RNA-transporter-like proteins. These findings raise both speculation about the potential impact that plants may have on animal physiology at the molecular level, and an appealing possibility that food-derived miRNAs may offer us another means to deliver necessary nutrients or therapeutics to our bodies. There is recent evidence that microRNAs derived from ingested food sources can be taken up, packaged into microvesicles and, upon reaching the final recipient cells, regulate a target gene in a sequence-specific manner.
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  • 65
    Publication Date: 2012-04-15
    Description: Cancer is generally defined as uncontrollable growth of cells caused by genetic aberrations and/or environmental factors. Yet contagious cancers also occur. The recent emergence of a contagious cancer in Tasmanian devils has reignited interest in transmissible cancers. Two naturally occurring transmissible cancers are known: devil facial tumour disease and canine transmissible venereal tumour. Both cancers evolved once and have then been transmitted from one individual to another as clonal cell lines. The dog cancer is ancient; having evolved more than 6,000 years ago, while the devil disease was first seen in 1996. In this review I will compare and contrast the two diseases focusing on the life histories of the clonal cell lines, their evolutionary trajectories and the mechanisms by which they have achieved immune tolerance. A greater understanding of these contagious cancers will provide unique insights into the role of the immune system in shaping tumour evolution and may uncover novel approaches for treating human cancer. A better understanding of the two known transmissible cancers (Tasmanian devil facial tumour and canine transmissible venereal tumour) may provide unique insights into the role of the immune system in shaping tumour evolution and may uncover novel approaches for treating human cancer.
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  • 66
    Publication Date: 2012-04-15
    Description: Balancing self-renewal and differentiation of stem cells is an important issue in stem cell and cancer biology. Recently, the Drosophila neuroblast (NB), neural stem cell has emerged as an excellent model for stem cell self-renewal and tumorigenesis. It is of great interest to understand how defects in the asymmetric division of neural stem cells lead to tumor formation. Here, we review recent advances in asymmetric division and the self-renewal control of Drosophila NBs. We summarize molecular mechanisms of asymmetric cell division and discuss how the defects in asymmetric division lead to tumor formation. Gain-of-function or loss-of-function of various proteins in the asymmetric machinery can drive NB overgrowth and tumor formation. These proteins control either the asymmetric protein localization or mitotic spindle orientation of NBs. We also discuss other mechanisms of brain tumor suppression that are beyond the control of asymmetric division. Drosophila neuroblasts represent an excellent model system to study the molecular mechanisms of asymmetric divisions during stem cell self-renewal and how defects in this system lead to tumourigenesis.
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  • 67
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    In: BioEssays
    Publication Date: 2012-04-15
    Description: Cell interdivision periods (IDP) in homogenous cell populations vary stochastically. Another aspect of probabilistic cell behavior is randomness in cell differentiation. These features are suggested to result from competing stochastic events of assembly/disassembly of the transcription pre-initiation complex (PIC) at gene promoters. The time needed to assemble a proper PIC from different proteins, which must be numerous enough to make their combination gene specific, may be comparable to the IDP. Nascent mRNA visualization at defined genes and inferences from protein level fluctuations in single cells suggest that some genes do operate in this way. The onset of mRNA production by such genes may miss the time windows provided by the cell cycle, resulting in cells differentiating into those in which the respective mRNAs are either present or absent. This creates a way to generate cell phenotype diversity in multicellular organisms.
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  • 68
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    In: BioEssays
    Publication Date: 2012-04-17
    Description: Light Microscopy: From imaging probes to high resolution techniques andcomputational image analysis . On pages 333–340 Jason Swedlow gives a nice overview over the topics covered in this Special Issue. Advances in optics, automation, computational tools and imaging probes allow the visualization of ever smaller structures at increasing resolution. Furthermore, these techniques can be used for a wide variety of applications from basic research to clinical domains, e.g. by employing imaging techniques for screening. The image shows a section of mouse small intestine stained with DAPI (blue), anti-tubulin (red) and phalloidin (green).
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  • 69
    Publication Date: 2012-04-17
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  • 70
    Publication Date: 2012-04-15
    Description: Second harmonic generating (SHG) nanoprobes have recently emerged as versatile and durable labels suitable for in vivo imaging, circumventing many of the inherent drawbacks encountered with classical fluorescent probes. Since their nanocrystalline structure lacks a central point of symmetry, they are capable of generating second harmonic signal under intense illumination – converting two photons into one photon of half the incident wavelength – and can be detected by conventional two-photon microscopy. Because the optical signal of SHG nanoprobes is based on scattering, rather than absorption as in the case of fluorescent probes, they neither bleach nor blink, and the signal does not saturate with increasing illumination intensity. When SHG nanoprobes are used to image live tissue, the SHG signal can be detected with little background signal, and they are physiologically inert, showing excellent long-term photostability. Because of their photophysical properties, SHG nanoprobes provide unique advantages for molecular imaging of living cells and tissues with unmatched sensitivity and temporal resolution.
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  • 71
    Publication Date: 2012-04-15
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  • 72
    Publication Date: 2012-04-15
    Description: New imaging methodologies in quantitative fluorescence microscopy, such as Förster resonance energy transfer (FRET), have been developed in the last few years and are beginning to be extensively applied to biological problems. FRET is employed for the detection and quantification of protein interactions, and of biochemical activities. Herein, we review the different methods to measure FRET in microscopy, and more importantly, their strengths and weaknesses. In our opinion, fluorescence lifetime imaging microscopy (FLIM) is advantageous for detecting inter-molecular interactions quantitatively, the intensity ratio approach representing a valid and straightforward option for detecting intra-molecular FRET. Promising approaches in single molecule techniques and data analysis for quantitative and fast spatio-temporal protein-protein interaction studies open new avenues for FRET in biological research.
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  • 73
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    In: BioEssays
    Publication Date: 2012-04-15
    Description: The Tasmanian devil is threatened by the incidence of a transmissible cancer (devil facial tumour disease, DFTD). On pages 285–292 Katherine Belov compares and contrasts this disease with canine transmissible venereal tumour (CTVT), focusing on the life histories of the clonal cell lines, their evolutionary trajectories and the mechanisms by which they have achieved immune tolerance. These two diseases provide unique models for understanding cancer biology and immunobiology, particularly the evolution of immune evasion strategies. Recently, whole-genome sequences of the Tasmanian devil, and of two distinct cancer subclones, have been published; these data provide further information about the pattern of evolution and spread of this parasitic clonal disease. Cover photo © melanieplusdaniel.de - Fotolia.com
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  • 74
    Publication Date: 2012-04-15
    Description: Powerful ultrastructural tools are providing new insights into neuronal circuits, revealing a wealth of anatomically-defined synaptic connections. These wiring diagrams are incomplete, however, because functional connectivity is actively shaped by neuromodulators that modify neuronal dynamics, excitability, and synaptic function. Studies of defined neural circuits in crustaceans, C. elegans , Drosophila , and the vertebrate retina have revealed the ability of modulators and sensory context to reconfigure information processing by changing the composition and activity of functional circuits. Each ultrastructural connectivity map encodes multiple circuits, some of which are active and some of which are latent at any given time.
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  • 75
    Publication Date: 2012-04-15
    Description: New C. elegans studies imply that lipases and lipid desaturases can mediate signaling effects on aging. But why might fat homeostasis be critical to aging? Could problems with fat handling compromise health in nematodes as they do in mammals? The study of signaling pathways that control longevity could provide the key to one of the great unsolved mysteries of biology: the mechanism of aging. But as our view of the regulatory pathways that control aging grows ever clearer, the nature of aging itself has, if anything, grown more obscure. In particular, focused investigations of the oxidative damage theory have raised questions about an old assumption: that a fundamental cause of aging is accumulation of molecular damage. Could fat dyshomeostasis instead be critical?
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  • 76
    Publication Date: 2012-04-15
    Description: Fluorescence correlation spectroscopy (FCS) is a powerful technique to measure concentrations, mobilities, and interactions of fluorescent biomolecules. It can be applied to various biological systems such as simple homogeneous solutions, cells, artificial, or cellular membranes and whole organisms. Here, we introduce the basic principle of FCS, discuss its application to biological questions as well as its limitations and challenges, present an overview of novel technical developments to overcome those challenges, and conclude with speculations about the future applications of fluorescence fluctuation spectroscopy.
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  • 77
    Publication Date: 2012-04-15
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  • 78
    Publication Date: 2012-04-15
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  • 79
    Publication Date: 2012-04-15
    Description: Soft X-ray tomography (SXT) is an imaging technique capable of characterizing and quantifying the structural phenotype of cells. In particular, SXT is used to visualize the internal architecture of fully hydrated, intact eukaryotic and prokaryotic cells at high spatial resolution (50 nm or better). Image contrast in SXT is derived from the biochemical composition of the cell, and obtained without the need to use potentially damaging contrast-enhancing agents, such as heavy metals. The cells are simply cryopreserved prior to imaging, and are therefore imaged in a near-native state. As a complement to structural imaging by SXT, the same specimen can now be imaged by correlated cryo-light microscopy. By combining data from these two modalities specific molecules can be localized directly within the framework of a high-resolution, three-dimensional reconstruction of the cell. This combination of data types allows sophisticated analyses to be carried out on the impact of environmental and/or genetic factors on cell phenotypes. Soft X-ray tomography is an imaging technique that can be used to visualize the internal architecture of fully hydrated, intact eukaryotic and prokaryotic cells at high spatial resolution.
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  • 80
    Publication Date: 2012-04-17
    Description: Three-dimensional structured illumination microscopy (3D-SIM) has opened up new possibilities to study nuclear architecture at the ultrastructural level down to the ∼100 nm range. We present first results and assess the potential using 3D-SIM in combination with 3D fluorescence in situ hybridization (3D-FISH) for the topographical analysis of defined nuclear targets. Our study also deals with the concern that artifacts produced by FISH may counteract the gain in resolution. We address the topography of DAPI-stained DNA in nuclei before and after 3D-FISH, nuclear pores and the lamina, chromosome territories, chromatin domains, and individual gene loci. We also look at the replication patterns of chromocenters and the topographical relationship of Xist -RNA within the inactive X-territory. These examples demonstrate that an appropriately adapted 3D-FISH/3D-SIM approach preserves key characteristics of the nuclear ultrastructure and that the gain in information obtained by 3D-SIM yields new insights into the functional nuclear organization. 3D-structured illumination microscopy (3D-SIM) can be used to study nuclear architecture at the ultrastructural level.This method can also be combined with 3D fluorescence in situ hybridization (3D-FISH) for the topographical analysis of defined nuclear targets. The proof of principle of this combination of methods lies in the high degree of structural preservation of chromatin presented here.
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  • 81
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    In: BioEssays
    Publication Date: 2012-04-17
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  • 82
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    In: BioEssays
    Publication Date: 2012-04-17
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  • 83
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    Wiley
    In: BioEssays
    Publication Date: 2012-04-17
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  • 84
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    Wiley
    In: BioEssays
    Publication Date: 2012-04-17
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  • 85
    Publication Date: 2012-04-11
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  • 86
    Publication Date: 2012-04-11
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  • 87
    Publication Date: 2012-04-15
    Description: MHC II, a major feature of the adaptive immune system, is lacking in Atlantic cod, and there are different scenarios (metabolic cost hypothesis or functional shift hypothesis) that might explain this loss. The lack of MHC II coincides with an increased number of genes for MHC I and Toll-like receptors (TLRs).
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  • 88
    Publication Date: 2012-04-15
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  • 89
    Publication Date: 2012-04-15
    Description: Genes are gained and lost over the course of evolution. A recent study found that over 1,800 new genes have appeared during primate evolution and that an unexpectedly high proportion of these genes are expressed in the human brain. But what are the molecular functions of newly evolved genes and what is their impact on an organism's fitness? The acquisition of new genes may provide a rich source of genetic diversity that fuels evolutionary innovation. Although gene manipulation experiments are not feasible in humans, studies in model organisms, such as Drosophila melanogaster , have shown that new genes can quickly become integrated into genetic networks and become essential for survival or fertility. Future studies of new genes, especially chimeric genes, and their functions will help determine the role of genetic novelty in the adaptation and diversification of species.
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  • 90
    Publication Date: 2012-04-15
    Description: Fluorescence microscopy has provided a route to qualitatively analyze features of nuclear structures and chromatin domains with increasing resolution. However, it is becoming increasingly important to develop tools for quantitative analysis. Here, we present an automated method to quantitatively determine the enrichment of several endogenous factors, immunostained in pericentric heterochromatin domains in mouse cells. We show that this method permits an unbiased characterization of changes in the enrichment of several factors with statistical significance from a large number of nuclei. Furthermore, the nuclei can be sorted according to the enrichment value of these factors. This method should prove useful to monitor events related to changes in the amount, rather than the presence or absence, of any factor. By adapting a few parameters, it could be extended to other nuclear structures and the benefit of using available software will permit its use in many biological labs. This method allows the quantitative analysis of the localisation of several endogenous markers (e.g. heterochromatin protein 1, HP1) at pericentric heterochromatin domains imaged via immunofluorescence microscopy.
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  • 91
    Publication Date: 2012-04-15
    Description: Pheomelanogenesis may have evolved as an excretory mechanism to remove excess cysteine, and in humans this might potentially confer a greater ability to avoid disease such as Parkinson's and Alzheimer's disease, in which excess cysteine is a contributory cause.
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  • 92
    Publication Date: 2012-04-15
    Description: Pointillistic based super-resolution techniques, such as photoactivated localization microscopy (PALM), involve multiple cycles of sequential activation, imaging, and precise localization of single fluorescent molecules. A super-resolution image, having nanoscopic structural information, is then constructed by compiling all the image sequences. Because the final image resolution is determined by the localization precision of detected single molecules and their density, accurate image reconstruction requires imaging of biological structures labeled with fluorescent molecules at high density. In such image datasets, stochastic variations in photon emission and intervening dark states lead to uncertainties in identification of single molecules. This, in turn, prevents the proper utilization of the wealth of information on molecular distribution and quantity. A recent strategy for overcoming this problem is pair-correlation analysis applied to PALM. Using rigorous statistical algorithms to estimate the number of detected proteins, this approach allows the spatial organization of molecules to be quantitatively described.
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  • 93
    Publication Date: 2012-04-15
    Description: A recent study in the lepidopteran Ostrinia scapulalis shows that endosymbionts can actively manipulate the sex determination mechanism of their host. Wolbachia bacteria alter the sex-specific splicing of the doublesex master switch gene. In ZZ males of this female heterogametic system, the female isoform of doublesex is produced in the presence of the bacteria. The effect is a lethal feminization of genotypic males. Curing of ZW females leads to males that die, indicating that the bacteria have an obligate role in proper sex determination and development of their host. Microbial intervention with host sex determination may be a driving force behind the evolutionary turnover of sex determination mechanisms. Sex determination in insects is achieved through the action of a cascade of genes, and symbionts may interfere with their host at different levels of this cascade. One example is the lepidopteran Ostrinia scapulalis , where Wolbachia bacteria alter the sex-specific splicing of the doublesex master switch gene.
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  • 94
    Publication Date: 2012-04-15
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  • 95
    Publication Date: 2012-04-15
    Description: Reprogramming of somatic cells to a pluripotent state holds huge potentials for regenerative medicine. However, a debate over which method is better, somatic cell nuclear transfer (SCNT) or induced pluripotent stem (iPS) cells, still persists. Both approaches have the potential to generate patient-specific pluripotent stem cells for replacement therapy. Yet, although SCNT has been successfully applied in various vertebrates, no human pluripotent stem cells have been generated by SCNT due to technical, legal and ethical difficulties. On the other hand, human iPS cell lines have been reported from both healthy and diseased individuals. A recent study reported the generation of triploid human pluripotent stem cells by transferring somatic nuclei into oocytes, a variant form of SCNT. In this essay, we discuss this progress and the potentials of these two reprogramming approaches for regenerative medicine. The generation of patient-specific pluripotent stem cells holds great potential for regenerative medicine and can be achieved by somatic cell nuclear transfer (SCNT) or by the forced expression of a just a few transcription factors in somatic cells to generate induced pluripotent stem (iPS) cells.
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  • 96
    Publication Date: 2012-04-15
    Description: The most enigmatic feature of polytene chromosomes is their banding pattern, the genetic organization of which has been a very attractive puzzle for many years. Recent genome-wide protein mapping efforts have produced a wealth of data for the chromosome proteins of Drosophila cells. Based on their specific protein composition, the chromosomes comprise two types of bands, as well as interbands. These differ in terms of time of replication and specific types of proteins. The interbands are characterized by their association with “active” chromatin proteins, nucleosome remodeling, and origin recognition complexes, and so they have three functions: acting as binding sites for RNA pol II, initiation of replication and nucleosome remodeling of short fragments of DNA. The borders and organization of the same band and interband regions are largely identical, irrespective of the cell type studied. This demonstrates that the banding pattern is a universal principle of the organization of interphase polytene and non-polytene chromosomes. Editor's suggested further reading in BioEssays Caught in the act: Rapid, symbiont-driven evolution Abstract Function and evolution of sex determination mechanisms, genes and pathways in insects Abstract Recent genome-wide protein mapping efforts in Drosophila have provided information on the proteins associated with interbands as well as on the correspondence between the genomic maps and the polytene chromosome structure, and have shown that the polytene chromosome structure is likely to provide a good representation of the organisation of interphase chromatin in diploid cells.
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  • 97
    Publication Date: 2012-04-15
    Description: Apoptosis is not only involved in patterning by removal of tissue (destructive apoptotic patterning), but it can also function in signalling the site of de novo tissue generation via morphogenic signals (instructive apoptotic patterning).
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  • 98
    Publication Date: 2012-04-15
    Description: Molecular interactions are at the origin of life. How molecules get at different locations in the cell and how they locate their partners is a major and partially unresolved question in biology that is paramount to signaling. Spatio-temporal correlations of fluctuating fluorescently tagged molecules reveal how they move, interact, and bind in the different cellular compartments. Methods based on fluctuations represent a remarkable technical advancement in biological imaging. Here we discuss image analysis methods based on spatial and temporal correlation of fluctuations, raster image correlation spectroscopy, number and brightness, and spatial cross-correlations that give us information about how individual molecules move in cells and interact with partners at the single molecule level. These methods can be implemented with a standard laser scanning microscope and produce a cellular level spatio-temporal map of molecular interactions.
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  • 99
    Publication Date: 2012-04-15
    Description: Once regarded as cellular ‘debris’ extracellular vesicles (EVs) emerge as one of the most intriguing entities in cancer pathogenesis. Intercellular trafficking of EVs challenges the notion of cancer cell autonomy, and highlights the multicellular nature of such fundamental processes as stem cell niche formation, tumour stroma generation, angiogenesis, inflammation or immunity. Recent studies reveal that intercellular exchange mediated by EVs runs deeper than expected, and includes molecules causative for cancer progression, such as oncogenes (epidermal growth factor receptor, Ras), and tumour suppressors (PTEN). The uptake of oncogenic EVs (oncosomes) by various cells may profoundly change their biology, signalling patterns and gene expression, and in some cases cause their overt tumorigenic conversion. Moreover, EVs circulating in blood and present in body fluids provide an unprecedented access to the molecular circuitry driving cancer cells, and new technologies are being developed to exploit this property as a source of unique cancer biomarkers. Extracellular vesicles (EVs) are capable of transferring a wide spectrum of macromolecules, such as oncogenes and tumour suppressors, from one cell to another (e.g. also from a mutant cancer cell to a normal one). The molecular analysis of EV-derived material holds potential to be used for clinical purposes.
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  • 100
    Publication Date: 2012-04-15
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