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  1. Data integration reveals key homeostatic mechanisms following low dose radiation exposure

    Energy Technology Data Exchange (ETDEWEB)

    Tilton, Susan C.; Matzke, Melissa M. [Computational Biology and Bioinformatics, Pacific Northwest National Laboratory, Richland, WA 99338 (United States); Sowa, Marianne B.; Stenoien, David L.; Weber, Thomas J. [Health Impacts and Exposure Science, Pacific Northwest National Laboratory, Richland, WA 99338 (United States); Morgan, William F. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99338 (United States); Waters, Katrina M., E-mail: katrina.waters@pnnl.gov [Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99338 (United States)

    2015-05-15

    The goal of this study was to define pathways regulated by low dose radiation to understand how biological systems respond to subtle perturbations in their environment and prioritize pathways for human health assessment. Using an in vitro 3-D human full thickness skin model, we have examined the temporal response of dermal and epidermal layers to 10 cGy X-ray using transcriptomic, proteomic, phosphoproteomic and metabolomic platforms. Bioinformatics analysis of each dataset independently revealed potential signaling mechanisms affected by low dose radiation, and integrating data shed additional insight into the mechanisms regulating low dose responses in human tissue. We examined direct interactions among datasets (top down approach) and defined several hubs as significant regulators, including transcription factors (YY1, MYC and CREB1), kinases (CDK2, PLK1) and a protease (MMP2). These data indicate a shift in response across time — with an increase in DNA repair, tissue remodeling and repression of cell proliferation acutely (24–72 h). Pathway-based integration (bottom up approach) identified common molecular and pathway responses to low dose radiation, including oxidative stress, nitric oxide signaling and transcriptional regulation through the SP1 factor that would not have been identified by the individual data sets. Significant regulation of key downstream metabolites of nitrative stress was measured within these pathways. Among the features identified in our study, the regulation of MMP2 and SP1 was experimentally validated. Our results demonstrate the advantage of data integration to broadly define the pathways and networks that represent the mechanisms by which complex biological systems respond to perturbation. - Highlights: • Low dose ionizing radiation altered homeostasis in 3D skin tissue model. • Global gene/protein/metabolite data integrated using complementary statistical approaches • Time and location-specific change in matrix regulation

  2. Data Integration Reveals Key Homeostatic Mechanisms Following Low Dose Radiation Exposure

    Energy Technology Data Exchange (ETDEWEB)

    Tilton, Susan C.; Matzke, Melissa M.; Sowa, Marianne B.; Stenoien, David L.; Weber, Thomas J.; Morgan, William F.; Waters, Katrina M.

    2015-05-01

    The goal of this study was to define pathways regulated by low dose radiation to understand how biological systems respond to subtle perturbations in their environment and prioritize pathways for human health assessment. Using an in vitro 3-D human full thickness skin model, we have examined the temporal response of dermal and epidermal layers to 10 cGy X-ray using transcriptomic, proteomic, phosphoproteomic and metabolomic platforms. Bioinformatics analysis of each dataset independently revealed potential signaling mechanisms affected by low dose radiation, and integrating data shed additional insight into the mechanisms regulating low dose responses in human tissue. We examined direct interactions among datasets (top down approach) and defined several hubs as significant regulators, including transcription factors (YY1, MYC and CREB1), kinases (CDK2, PLK1) and a protease (MMP2). These data indicate a shift in response across time - with an increase in DNA repair, tissue remodeling and repression of cell proliferation acutely (24 – 72 hr). Pathway-based integration (bottom up approach) identified common molecular and pathway responses to low dose radiation, including oxidative stress, nitric oxide signaling and transcriptional regulation through the SP1 factor that would not have been identified by the individual data sets. Significant regulation of key downstream metabolites of nitrative stress were measured within these pathways. Among the features identified in our study, the regulation of MMP2 and SP1 were experimentally validated. Our results demonstrate the advantage of data integration to broadly define the pathways and networks that represent the mechanisms by which complex biological systems respond to perturbation.

  3. "Topological significance" analysis of gene expression and proteomic profiles from prostate cancer cells reveals key mechanisms of androgen response.

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    Adaikkalam Vellaichamy

    Full Text Available BACKGROUND: The problem of prostate cancer progression to androgen independence has been extensively studied. Several studies systematically analyzed gene expression profiles in the context of biological networks and pathways, uncovering novel aspects of prostate cancer. Despite significant research efforts, the mechanisms underlying tumor progression are poorly understood. We applied a novel approach to reconstruct system-wide molecular events following stimulation of LNCaP prostate cancer cells with synthetic androgen and to identify potential mechanisms of androgen-independent progression of prostate cancer. METHODOLOGY/PRINCIPAL FINDINGS: We have performed concurrent measurements of gene expression and protein levels following the treatment using microarrays and iTRAQ proteomics. Sets of up-regulated genes and proteins were analyzed using our novel concept of "topological significance". This method combines high-throughput molecular data with the global network of protein interactions to identify nodes which occupy significant network positions with respect to differentially expressed genes or proteins. Our analysis identified the network of growth factor regulation of cell cycle as the main response module for androgen treatment in LNCap cells. We show that the majority of signaling nodes in this network occupy significant positions with respect to the observed gene expression and proteomic profiles elicited by androgen stimulus. Our results further indicate that growth factor signaling probably represents a "second phase" response, not directly dependent on the initial androgen stimulus. CONCLUSIONS/SIGNIFICANCE: We conclude that in prostate cancer cells the proliferative signals are likely to be transmitted from multiple growth factor receptors by a multitude of signaling pathways converging on several key regulators of cell proliferation such as c-Myc, Cyclin D and CREB1. Moreover, these pathways are not isolated but constitute an

  4. Comparative study between transcriptionally- and translationally-acting adenine riboswitches reveals key differences in riboswitch regulatory mechanisms.

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    Jean-François Lemay

    2011-01-01

    Full Text Available Many bacterial mRNAs are regulated at the transcriptional or translational level by ligand-binding elements called riboswitches. Although they both bind adenine, the adenine riboswitches of Bacillus subtilis and Vibrio vulnificus differ by controlling transcription and translation, respectively. Here, we demonstrate that, beyond the obvious difference in transcriptional and translational modulation, both adenine riboswitches exhibit different ligand binding properties and appear to operate under different regulation regimes (kinetic versus thermodynamic. While the B. subtilis pbuE riboswitch fully depends on co-transcriptional binding of adenine to function, the V. vulnificus add riboswitch can bind to adenine after transcription is completed and still perform translation regulation. Further investigation demonstrates that the rate of transcription is critical for the B. subtilis pbuE riboswitch to perform efficiently, which is in agreement with a co-transcriptional regulation. Our results suggest that the nature of gene regulation control, that is transcription or translation, may have a high importance in riboswitch regulatory mechanisms.

  5. In-vivo quantitative proteomics reveals a key contribution of post-transcriptional mechanisms to the circadian regulation of liver metabolism.

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    Maria S Robles

    2014-01-01

    Full Text Available Circadian clocks are endogenous oscillators that drive the rhythmic expression of a broad array of genes, orchestrating metabolism and physiology. Recent evidence indicates that post-transcriptional and post-translational mechanisms play essential roles in modulating temporal gene expression for proper circadian function, particularly for the molecular mechanism of the clock. Due to technical limitations in large-scale, quantitative protein measurements, it remains unresolved to what extent the circadian clock regulates metabolism by driving rhythms of protein abundance. Therefore, we aimed to identify global circadian oscillations of the proteome in the mouse liver by applying in vivo SILAC mouse technology in combination with state of the art mass spectrometry. Among the 3000 proteins accurately quantified across two consecutive cycles, 6% showed circadian oscillations with a defined phase of expression. Interestingly, daily rhythms of one fifth of the liver proteins were not accompanied by changes at the transcript level. The oscillations of almost half of the cycling proteome were delayed by more than six hours with respect to the corresponding, rhythmic mRNA. Strikingly we observed that the length of the time lag between mRNA and protein cycles varies across the day. Our analysis revealed a high temporal coordination in the abundance of proteins involved in the same metabolic process, such as xenobiotic detoxification. Apart from liver specific metabolic pathways, we identified many other essential cellular processes in which protein levels are under circadian control, for instance vesicle trafficking and protein folding. Our large-scale proteomic analysis reveals thus that circadian post-transcriptional and post-translational mechanisms play a key role in the temporal orchestration of liver metabolism and physiology.

  6. Role of the key mutation in the selective binding of avian and human influenza hemagglutinin to sialosides revealed by quantum-mechanical calculations.

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    Sawada, Toshihiko; Fedorov, Dmitri G; Kitaura, Kazuo

    2010-12-01

    The selective binding between avian and human influenza A viral hemagglutinins (HA) subtype H3 and Neu5Acα2-3 and α2-6Gal (avian α2-3, human α2-6) is qualitatively rationalized by the fragment molecular orbital (FMO) method. We suggest a general model of analyzing protein-ligand interactions based on the electrostatic, polarization, dispersion, and desolvation components obtained from quantum-mechanical calculations at the MP2/6-31G(d) level with the polarizable continuum model of solvation. The favorable avian H3 (A/duck/Ukraine/1963)-avian α2-3 binding arises from the hydrophilic interaction between Gal-4 OH and side-chain NH(2)CO on Gln226, which is supported by the intermolecular hydrogen-bond network to the 1-COO group on Neu5Ac moiety. A substitution of Gln226Leu in the avian H3 HA1 domain increases the binding affinity to human α2-6 due to the Leu226···human α2-6 dispersion with a small entropic penalty during the complex formation. The remarkable human H3 (A/Aichi/2/1968)-human α2-6 binding is not governed by the Ser228-OH···OH-9 Neu5Ac hydrogen bond. These fragment-based chemical aspects can help design monovalent inhibitors of the influenza viral HA-sialoside binding and the simulation studies on the viral HAs-human α2-6 binding.

  7. In vitro degradation and cytocompatibility of dicalcium phosphate dihydrate cements prepared using the monocalcium phosphate monohydrate/hydroxyapatite system reveals rapid conversion to HA as a key mechanism.

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    Alge, Daniel L; Goebel, W Scott; Chu, Tien-Min Gabriel

    2012-04-01

    We previously showed that dicalcium phosphate dihydrate (DCPD) cements can be prepared using monocalcium phosphate monohydrate (MCPM) and hydroxyapatite (HA). In this study, we have characterized the degradation properties and biocompatibility of these novel cements. To study the degradation properties, cements were prepared using MCPM:HA molar ratios of 4:1, 2:1, 2:3, and 2:5. Degradation was evaluated in vitro by static soaking in PBS, and changes in pH, mass, compressive strength, and composition were monitored. Conversion of DCPD to HA was noted in the 4:1 group, which initially consisted of pure DCPD. However, the 2:1 group, which initially consisted of DCPD and an intermediate amount of unreacted HA, underwent rapid conversion to HA associated with significantly greater pH drop and mass loss as well as a complete loss of mechanical integrity. On the basis of these results, we directly compared the cytocompatibility of 2:1 MCPM:HA cements to DCPD cements prepared with an equivalent percent molar excess of β-tricalcium phosphate (β-TCP) using an in vitro cell viability assay. Viability of cells co-cultured with 2:1 MCPM:HA cements was significantly reduced after just 48 h, while viability of cells cultured with the β-TCP-based cements was no different from control cells. In conclusion, this study demonstrates that conversion to HA plays an important role in the degradation of DCPD cements prepared with the MCPM/HA system, affecting both physical properties and cytocompatibility. These results could have important clinical implications for MCPM/HA cements.

  8. State Key Laboratory of Mechanical Transmission

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    @@1 General introduction The State Key Laboratory of Mechanical Transmission (SLMT), located at Chongqing University,was founded upon the approval of the State Planning Commission in 1988, and began to open to scholarsat home and abroad in August 1990 and passed the approval check in 1991. Since then, a great deal offruits have been reaped in scientific researches, personnel training and academic exchange. In 1997, itpassed the evaluation organized by the National Natural Science Foundation of China entrusted by theState Planning Commission and the State Science and Technology Commission. Now, Prof. Qin Datong isthe director of the laboratory. Academicians Lei Tianjue and Zhang Qixian are the honorary chairmen andAcademician Yang Shuzi holds the chairman post of the Laboratory' s Academic Committee which consistsof 18 famous experts in the field of mechanical engineering. There are six doctoral supervisors in the Lab-oratory: Prof. Liang Xichang, Prof. Zhang Guanghui, Prof. Li Runfang, Prof. Liu Fei, Prof. Qin Da-tong and Prof. Wei Yunlong. There are 25 permanent staff members altogether, including 20 researchers(16 with senior technical title, 4 with middle technical title), 3 technicians and 2 administrators. 2 Major research areas The main research areas of the Laboratory include: (i) basic theory and application of mechanicaltransmission, including gear geometry, failure theory, manufacturing technology, establishment of newdrive systems and design estimating systems; (ii) modem transmission theory and technology, includingcombination of mechanical transmission with new technology and high technology such as micro-electron-ics, computer, automatic controlling, etc; (iii) information and controlling of machinery and transmis-sion system such as integrated system of computer aided engineering, computer integrated manufacturingsystem, fault diagnosis and state monitoring. In recent years, 13 national key projects, 9 national hightechnology development program

  9. Transcriptome analysis reveals key differentially expressed genes involved in wheat grain development

    Institute of Scientific and Technical Information of China (English)

    Yonglong Yu; Dong Zhu; Chaoying Ma; Hui Cao; Yaping Wang; Yanhao Xu; Wenying Zhang; Yueming Yan

    2016-01-01

    Wheat seed development is an important physiological process of seed maturation and directly affects wheat yield and quality. In this study, we performed dynamic transcriptome microarray analysis of an elite Chinese bread wheat cultivar (Jimai 20) during grain development using the GeneChip Wheat Genome Array. Grain morphology and scanning electron microscope observations showed that the period of 11–15 days post-anthesis (DPA) was a key stage for the synthesis and accumulation of seed starch. Genome-wide transcriptional profiling and significance analysis of microarrays revealed that the period from 11 to 15 DPA was more important than the 15–20 DPA stage for the synthesis and accumulation of nutritive reserves. Series test of cluster analysis of differential genes revealed five statistically significant gene expression profiles. Gene ontology annotation and enrichment analysis gave further informa-tion about differentially expressed genes, and MapMan analysis revealed expression changes within functional groups during seed development. Metabolic pathway network analysis showed that major and minor metabolic pathways regulate one another to ensure regular seed development and nutritive reserve accumulation. We performed gene co-expression network analysis to identify genes that play vital roles in seed development and identified several key genes involved in important metabolic pathways. The transcriptional expression of eight key genes involved in starch and protein synthesis and stress defense was further validated by qRT-PCR. Our results provide new insight into the molecular mechanisms of wheat seed development and the determinants of yield and quality.

  10. Transcriptome analysis reveals key differentially expressed genes involved in wheat grain development

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    Yonglong Yu

    2016-04-01

    Full Text Available Wheat seed development is an important physiological process of seed maturation and directly affects wheat yield and quality. In this study, we performed dynamic transcriptome microarray analysis of an elite Chinese bread wheat cultivar (Jimai 20 during grain development using the GeneChip Wheat Genome Array. Grain morphology and scanning electron microscope observations showed that the period of 11–15 days post-anthesis (DPA was a key stage for the synthesis and accumulation of seed starch. Genome-wide transcriptional profiling and significance analysis of microarrays revealed that the period from 11 to 15 DPA was more important than the 15–20 DPA stage for the synthesis and accumulation of nutritive reserves. Series test of cluster analysis of differential genes revealed five statistically significant gene expression profiles. Gene ontology annotation and enrichment analysis gave further information about differentially expressed genes, and MapMan analysis revealed expression changes within functional groups during seed development. Metabolic pathway network analysis showed that major and minor metabolic pathways regulate one another to ensure regular seed development and nutritive reserve accumulation. We performed gene co-expression network analysis to identify genes that play vital roles in seed development and identified several key genes involved in important metabolic pathways. The transcriptional expression of eight key genes involved in starch and protein synthesis and stress defense was further validated by qRT-PCR. Our results provide new insight into the molecular mechanisms of wheat seed development and the determinants of yield and quality.

  11. Statistical mechanics approach to lock-key supramolecular chemistry interactions.

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    Odriozola, Gerardo; Lozada-Cassou, Marcelo

    2013-03-08

    In the supramolecular chemistry field, intuitive concepts such as molecular complementarity and molecular recognition are used to explain the mechanism of lock-key associations. However, these concepts lack a precise definition, and consequently this mechanism is not well defined and understood. Here we address the physical basis of this mechanism, based on formal statistical mechanics, through Monte Carlo simulation and compare our results with recent experimental data for charged or uncharged lock-key colloids. We find that, given the size range of the molecules involved in these associations, the entropy contribution, driven by the solvent, rules the interaction, over that of the enthalpy. A universal behavior for the uncharged lock-key association is found. Based on our results, we propose a supramolecular chemistry definition.

  12. Discriminative topological features reveal biological network mechanisms

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    Levovitz Chaya

    2004-11-01

    Full Text Available Abstract Background Recent genomic and bioinformatic advances have motivated the development of numerous network models intending to describe graphs of biological, technological, and sociological origin. In most cases the success of a model has been evaluated by how well it reproduces a few key features of the real-world data, such as degree distributions, mean geodesic lengths, and clustering coefficients. Often pairs of models can reproduce these features with indistinguishable fidelity despite being generated by vastly different mechanisms. In such cases, these few target features are insufficient to distinguish which of the different models best describes real world networks of interest; moreover, it is not clear a priori that any of the presently-existing algorithms for network generation offers a predictive description of the networks inspiring them. Results We present a method to assess systematically which of a set of proposed network generation algorithms gives the most accurate description of a given biological network. To derive discriminative classifiers, we construct a mapping from the set of all graphs to a high-dimensional (in principle infinite-dimensional "word space". This map defines an input space for classification schemes which allow us to state unambiguously which models are most descriptive of a given network of interest. Our training sets include networks generated from 17 models either drawn from the literature or introduced in this work. We show that different duplication-mutation schemes best describe the E. coli genetic network, the S. cerevisiae protein interaction network, and the C. elegans neuronal network, out of a set of network models including a linear preferential attachment model and a small-world model. Conclusions Our method is a first step towards systematizing network models and assessing their predictability, and we anticipate its usefulness for a number of communities.

  13. A Contents Encryption Mechanism Using Reused Key in IPTV

    Science.gov (United States)

    Jeong, Yoon-Su; Kim, Yong-Tae; Cho, Young-Bok; Lee, Ki-Jeong; Park, Gil-Cheol; Lee, Sang-Ho

    Recently IPTV is being spotlighted as a new stream service to stably provide video, audio and control signals to subscribers through the application of IP protocol. However, the IPTV system is facing more security threats than the traditional TV. This study proposes a multicasting encryption mechanism for secure transmission of the contents of IPTV by which the content provider encrypts their contents and send the encrypted contents and the key used for encryption of the contents to the user. In order to reduce the time and cost of Head-End, the proposed mechanism encrypts the media contents at the Head-End, embeds the code of the IPTV terminal used at the Head-End in the media contents for user tracking, and performs desynchronization for protection of the media contents from various attacks.

  14. Vestibular rehabilitation therapy: review of indications, mechanisms, and key exercises.

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    Han, Byung In; Song, Hyun Seok; Kim, Ji Soo

    2011-12-01

    Vestibular rehabilitation therapy (VRT) is an exercise-based treatment program designed to promote vestibular adaptation and substitution. The goals of VRT are 1) to enhance gaze stability, 2) to enhance postural stability, 3) to improve vertigo, and 4) to improve activities of daily living. VRT facilitates vestibular recovery mechanisms: vestibular adaptation, substitution by the other eye-movement systems, substitution by vision, somatosensory cues, other postural strategies, and habituation. The key exercises for VRT are head-eye movements with various body postures and activities, and maintaining balance with a reduced support base with various orientations of the head and trunk, while performing various upper-extremity tasks, repeating the movements provoking vertigo, and exposing patients gradually to various sensory and motor environments. VRT is indicated for any stable but poorly compensated vestibular lesion, regardless of the patient's age, the cause, and symptom duration and intensity. Vestibular suppressants, visual and somatosensory deprivation, immobilization, old age, concurrent central lesions, and long recovery from symptoms, but there is no difference in the final outcome. As long as exercises are performed several times every day, even brief periods of exercise are sufficient to facilitate vestibular recovery. Here the authors review the mechanisms and the key exercises for each of the VRT goals.

  15. One Time Secret Key Mechanism for Mobile Communication

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    Vasu.R

    2011-06-01

    Full Text Available Mobile communication is playing a vital role in the current technical world and becoming more popularand prevalent across the world everyday at one end and many security problems are arising at the otherend. To overcome these security problems many security mechanisms for mobile communications havebeen introduced in the literature. Among these mechanisms, authentication plays a quite important rolein the entire mobile network system and acts as the first defense against attackers since it ensures thecorrectness of the identities of distributed communication entities before they engage in any othercommunication activity. In this paper, we propose a novel authentication mechanism, called the nestedone-time secret mechanism, efficient for mobile communication environment which mainly focus on thesecurity issues of mobile communication and also overcome the problem of high computational andcommunication cost with good result in reducing the cost of computational and communication cost fromthe existing system, the cost reduction is possible by reducing the usage of the memory for key generationin the proposed scheme and the experimental result of the proposed scheme is also been showed in thispaper.

  16. Genetic and Ultrastructural Analysis Reveals the Key Players and Initial Steps of Bacterial Magnetosome Membrane Biogenesis

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    Kolinko, Isabel; Uebe, René; Schüler, Dirk

    2016-01-01

    Magnetosomes of magnetotactic bacteria contain well-ordered nanocrystals for magnetic navigation and have recently emerged as the most sophisticated model system to study the formation of membrane bounded organelles in prokaryotes. Magnetosome biosynthesis is thought to begin with the formation of a dedicated compartment, the magnetosome membrane (MM), in which the biosynthesis of a magnetic mineral is strictly controlled. While the biomineralization of magnetosomes and their subsequent assembly into linear chains recently have become increasingly well studied, the molecular mechanisms and early stages involved in MM formation remained poorly understood. In the Alphaproteobacterium Magnetospirillum gryphiswaldense, approximately 30 genes were found to control magnetosome biosynthesis. By cryo-electron tomography of several key mutant strains we identified the gene complement controlling MM formation in this model organism. Whereas the putative magnetosomal iron transporter MamB was most crucial for the process and caused the most severe MM phenotype upon elimination, MamM, MamQ and MamL were also required for the formation of wild-type-like MMs. A subset of seven genes (mamLQBIEMO) combined within a synthetic operon was sufficient to restore the formation of intracellular membranes in the absence of other genes from the key mamAB operon. Tracking of de novo magnetosome membrane formation by genetic induction revealed that magnetosomes originate from unspecific cytoplasmic membrane locations before alignment into coherent chains. Our results indicate that no single factor alone is essential for MM formation, which instead is orchestrated by the cumulative action of several magnetosome proteins. PMID:27286560

  17. Multi-Omics Reveals that Lead Exposure Disturbs Gut Microbiome Development, Key Metabolites and Metabolic Pathways.

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    Gao, Bei; Chi, Liang; Mahbub, Ridwan; Bian, Xiaoming; Tu, Pengcheng; Ru, Hongyu; Lu, Kun

    2017-02-24

    Lead exposure remains as a global public health issue and recent Flint water crisis has again raised concern about lead toxicity in the public. The toxicity of lead has been well established in a variety of systems and organs. It has been increasingly appreciated that gut microbiome is highly involved in many critical physiological processes, such as food digestion, immune system development, and metabolic homeostasis, etc. However, despite the key role of gut microbiome in human health, the functional impact of lead exposure on gut microbiome has not been studied yet. This study aims at defining gut microbiome toxicity induced by lead exposure in C57BL/6 mice by multi-omics approaches including 16S rRNA sequencing, whole genome metagenomics sequencing and gas chromatography-mass spectrometry (GC-MS) metabolomics profiling. 16S rRNA sequencing revealed that lead exposure altered the gut microbiome trajectory and phylogenetic diversity. Metagenomics sequencing and metabolomics profiling showed that numerous metabolic pathways, including vitamin E and bile acids, nitrogen metabolism, energy metabolism, oxidative stress and defense/detoxification mechanism, were significantly disturbed by lead exposure. These perturbed molecules and pathways may have important implications in lead toxicity in the host. Taken together, we have demonstrated that lead exposure not only alters the gut microbiome community structures/diversity, but also largely affects its metabolic functions, leading to gut microbiome toxicity.

  18. Mechanical sensitivity reveals evolutionary dynamics of mechanical systems.

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    Anderson, P S L; Patek, S N

    2015-04-07

    A classic question in evolutionary biology is how form-function relationships promote or limit diversification. Mechanical metrics, such as kinematic transmission (KT) in linkage systems, are useful tools for examining the evolution of form and function in a comparative context. The convergence of disparate systems on equivalent metric values (mechanical equivalence) has been highlighted as a source of potential morphological diversity under the assumption that morphology can evolve with minimal impact on function. However, this assumption does not account for mechanical sensitivity-the sensitivity of the metric to morphological changes in individual components of a structure. We examined the diversification of a four-bar linkage system in mantis shrimp (Stomatopoda), and found evidence for both mechanical equivalence and differential mechanical sensitivity. KT exhibited variable correlations with individual linkage components, highlighting the components that influence KT evolution, and the components that are free to evolve independently from KT and thereby contribute to the observed pattern of mechanical equivalence. Determining the mechanical sensitivity in a system leads to a deeper understanding of both functional convergence and morphological diversification. This study illustrates the importance of multi-level analyses in delineating the factors that limit and promote diversification in form-function systems. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

  19. Valuing snorkeling visits to the Florida Keys with stated and revealed preference models.

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    Park, Timothy; Bowker, J M; Leeworthy, Vernon R

    2002-07-01

    Coastal coral reefs, especially in the Florida Keys, are declining at a disturbing rate. Marine ecologists and reef scientists have emphasized the importance of establishing nonmarket values of coral reefs to assess the cost effectiveness of coral reef management and remediation programs. The purpose of this paper is to develop a travel cost-contingent valuation model of demand for trips to the Florida Keys focusing on willingness to pay (WTP) to preserve the current water quality and health of the coral reefs. The stated and revealed preference models allow the marginal valuation of recreationists to adjust depending on current and planned trip commitments in valuing nonmarginal policy changes in recreational opportunities. The integrated model incorporates key factors for establishing baseline amenity values for tourist dive sites, including perceptions of reef quality and dive conditions, the role of substitute sites, and the quality and availability of tourist facilities and recreation opportunities. The travel cost and WTP model differ in identifying critical variables and provide insight into the adjustment of trip decisions across alternative destination sites and the valuation of trips. In contrast to the travel cost model, a measure of the availability of substitute sites and total recreation activities does not have a significant impact on WTP valuations reported by snorkelers. Snorkelers engage in a relatively focused set of activities, suggesting that these recreationists may not shift expenditures to other sites or other recreation activities in the Florida Keys when confronted with increased access costs for the snorkeling experience.

  20. Revealing the Differences Between Free and Complexed Enzyme Mechanisms and Factors Contributing to Cell Wall Recalcitrance

    Energy Technology Data Exchange (ETDEWEB)

    Resch, Michael G.; Donohoe, Byron; Ciesielski, Peter; Nill, Jennifer; McKinney, Kellene; Mittal, Ashutosh; Katahira, Rui; Himmel, Michael; Biddy, Mary; Beckham, Gregg; Decker, Steve

    2014-09-08

    Enzymatic depolymerization of polysaccharides is a key step in the production of fuels and chemicals from lignocellulosic biomass, and discovery of synergistic biomass-degrading enzyme paradigms will enable improved conversion processes. Historically, revealing insights into enzymatic saccharification mechanisms on plant cell walls has been hindered by uncharacterized substrates and low resolution.

  1. Proteomic analysis reveals key proteins and phosphoproteins upon seed germination of wheat (Triticum aestivum L.

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    Kun eDong

    2015-11-01

    Full Text Available Wheat (Triticum aestivum L. is one of the oldest cultivated crops and the second most important food crop in the world. Seed germination is the key developmental process in plant growth and development, and poor germination directly affects plant growth and subsequent grain yield. In this study, we performed the first dynamic proteome analysis of wheat seed germination using a two-dimensional differential gel electrophoresis (2D-DIGE-based proteomic approach. A total of 166 differentially expressed protein (DEP spots representing 73 unique proteins were identified, which are mainly involved in storage, stress/defense/detoxification, carbohydrate metabolism, photosynthesis, cell metabolism, and transcription/translation/ transposition. The identified DEPs and their dynamic expression profiles generally correspond to three distinct seed germination phases after imbibition: storage degradation, physiological processes/morphogenesis, and photosynthesis. Some key DEPs involved in storage substance degradation and plant defense mechanisms, such as globulin 3, sucrose synthase type I, serpin, beta-amylase, and plastid ADP-glucose pyrophosphorylase (AGPase small subunit, were found to be phosphorylated during seed germination. Particularly, the phosphorylation site Ser355 was found to be located in the enzyme active region of beta-amylase, which promotes substrate binding. Phosphorylated modification of several proteins could promote storage substance degradation and environmental stress defense during seed germination. The central metabolic pathways involved in wheat seed germination are proposed herein, providing new insights into the molecular mechanisms of cereal seed germination.

  2. Same pattern, different mechanism: Locking onto the role of key species in seafloor ecosystem process

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    Woodin, Sarah Ann; Volkenborn, Nils; Pilditch, Conrad A.; Lohrer, Andrew M.; Wethey, David S.; Hewitt, Judi E.; Thrush, Simon F.

    2016-05-01

    Seafloor biodiversity is a key mediator of ecosystem functioning, but its role is often excluded from global budgets or simplified to black boxes in models. New techniques allow quantification of the behavior of animals living below the sediment surface and assessment of the ecosystem consequences of complex interactions, yielding a better understanding of the role of seafloor animals in affecting key processes like primary productivity. Combining predictions based on natural history, behavior of key benthic species and environmental context allow assessment of differences in functioning and process, even when the measured ecosystem property in different systems is similar. Data from three sedimentary systems in New Zealand illustrate this. Analysis of the behaviors of the infaunal ecosystem engineers in each system revealed three very different mechanisms driving ecosystem function: density and excretion, sediment turnover and surface rugosity, and hydraulic activities and porewater bioadvection. Integrative metrics of ecosystem function in some cases differentiate among the systems (gross primary production) and in others do not (photosynthetic efficiency). Analyses based on behaviors and activities revealed important ecosystem functional differences and can dramatically improve our ability to model the impact of stressors on ecosystem and global processes.

  3. Novel key metabolites reveal further branching of the roquefortine/meleagrin biosynthetic pathway.

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    Ries, Marco I; Ali, Hazrat; Lankhorst, Peter P; Hankemeier, Thomas; Bovenberg, Roel A L; Driessen, Arnold J M; Vreeken, Rob J

    2013-12-27

    Metabolic profiling and structural elucidation of novel secondary metabolites obtained from derived deletion strains of the filamentous fungus Penicillium chrysogenum were used to reassign various previously ascribed synthetase genes of the roquefortine/meleagrin pathway to their corresponding products. Next to the structural characterization of roquefortine F and neoxaline, which are for the first time reported for P. chrysogenum, we identified the novel metabolite roquefortine L, including its degradation products, harboring remarkable chemical structures. Their biosynthesis is discussed, questioning the exclusive role of glandicoline A as key intermediate in the pathway. The results reveal that further enzymes of this pathway are rather unspecific and catalyze more than one reaction, leading to excessive branching in the pathway with meleagrin and neoxaline as end products of two branches.

  4. Systematic prioritization and integrative analysis of copy number variations in schizophrenia reveal key schizophrenia susceptibility genes.

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    Luo, Xiongjian; Huang, Liang; Han, Leng; Luo, Zhenwu; Hu, Fang; Tieu, Roger; Gan, Lin

    2014-11-01

    Schizophrenia is a common mental disorder with high heritability and strong genetic heterogeneity. Common disease-common variants hypothesis predicts that schizophrenia is attributable in part to common genetic variants. However, recent studies have clearly demonstrated that copy number variations (CNVs) also play pivotal roles in schizophrenia susceptibility and explain a proportion of missing heritability. Though numerous CNVs have been identified, many of the regions affected by CNVs show poor overlapping among different studies, and it is not known whether the genes disrupted by CNVs contribute to the risk of schizophrenia. By using cumulative scoring, we systematically prioritized the genes affected by CNVs in schizophrenia. We identified 8 top genes that are frequently disrupted by CNVs, including NRXN1, CHRNA7, BCL9, CYFIP1, GJA8, NDE1, SNAP29, and GJA5. Integration of genes affected by CNVs with known schizophrenia susceptibility genes (from previous genetic linkage and association studies) reveals that many genes disrupted by CNVs are also associated with schizophrenia. Further protein-protein interaction (PPI) analysis indicates that protein products of genes affected by CNVs frequently interact with known schizophrenia-associated proteins. Finally, systematic integration of CNVs prioritization data with genetic association and PPI data identifies key schizophrenia candidate genes. Our results provide a global overview of genes impacted by CNVs in schizophrenia and reveal a densely interconnected molecular network of de novo CNVs in schizophrenia. Though the prioritized top genes represent promising schizophrenia risk genes, further work with different prioritization methods and independent samples is needed to confirm these findings. Nevertheless, the identified key candidate genes may have important roles in the pathogenesis of schizophrenia, and further functional characterization of these genes may provide pivotal targets for future therapeutics and

  5. Vestibular Rehabilitation Therapy: Review of Indications, Mechanisms, and Key Exercises

    OpenAIRE

    Han, Byung In; Song, Hyun Seok; Kim, Ji Soo

    2011-01-01

    Vestibular rehabilitation therapy (VRT) is an exercise-based treatment program designed to promote vestibular adaptation and substitution. The goals of VRT are 1) to enhance gaze stability, 2) to enhance postural stability, 3) to improve vertigo, and 4) to improve activities of daily living. VRT facilitates vestibular recovery mechanisms: vestibular adaptation, substitution by the other eye-movement systems, substitution by vision, somatosensory cues, other postural strategies, and habituatio...

  6. Phosphodiesterase 1 regulation is a key mechanism in vascular aging

    DEFF Research Database (Denmark)

    Niño, Paula K Bautista; Durik, Matej; Danser, A H Jan

    2015-01-01

    Reduced nitric oxide (NO)/cGMP signalling is observed in age-related vascular disease. We hypothesize that this disturbed signalling involves effects of genomic instability, a primary causal factor in aging, on vascular smooth muscle cells (VSMCs) and that the underlying mechanism plays a role.......0061, P=2.89×10(-5)). In summary, these results show that genomic instability and cellular senescence in VSMCs increase PDE1 expression. This might play a role in aging-related loss of vasodilator function, VSMC senescence, increased blood pressure and vascular hypertrophy....

  7. Key factors affecting mechanical behavior of metallic glass nanowires

    Science.gov (United States)

    Zhang, Qi; Li, Qi-Kai; Li, Mo

    2017-01-01

    Both strengthening and weakening trends with decreasing diameter have been observed for metallic glass nanowires, sometimes even in the samples with the same chemical composition. How to reconcile the results has reminded a puzzle. Since the detailed stress state and microstructure of metallic glass nanowires may differ from each other significantly depending on preparation, to discover the intrinsic size effect it is necessary to study metallic glass nanowires fabricated differently. Here we show the complex size effects from one such class of metallic glass nanowires prepared by casting using molecular dynamics simulations. As compared with the nanowires of the same composition prepared by other methods, the cast nanowires deform nearly homogeneously with much lower strength but better ductility; and also show strengthening in tension but weakening in compression with decreasing wire diameter. The subtle size dependence is shown to be related to the key factors including internal and surface stress state, atomic structure variation, and presence of various gradients. The complex interplay of these factors at decreasing size leads to the different deformation behaviors.

  8. DNA microarray revealed and RNAi plants confirmed key genes conferring low Cd accumulation in barley grains

    DEFF Research Database (Denmark)

    Sun, Hongyan; Chen, Zhong-Hua; Chen, Fei

    2015-01-01

    accumulation and tolerance between the two contrasting barley genotypes: W6nk2 (a low-grain-Cd-accumulating and Cd-sensitive genotype) and Zhenong8 (a high-grain-Cd-accumulating and tolerant genotype). A DNA microarray analysis detected large-scale changes of gene expression in response to Cd stress...... with a substantial difference between the two genotypes. Cd stress led to higher expression of genes involved in transport, carbohydrate metabolism and signal transduction in the low-grain-Cd-accumulating genotype. Novel transporter genes such as zinc transporter genes were identified as being associated with low Cd......Background Understanding the mechanism of low Cd accumulation in crops is crucial for sustainable safe food production in Cd-contaminated soils. Results Confocal microscopy, atomic absorption spectrometry, gas exchange and chlorophyll fluorescence analyses revealed a distinct difference in Cd...

  9. Network topology reveal high connectance levels and few key microbial genera within soils

    Directory of Open Access Journals (Sweden)

    Manoeli eLupatini

    2014-05-01

    Full Text Available Microbes have a central role in soil global biogeochemical process, yet specific microbe-microbe relationships are largely unknown. Analytical approaches as network analysis may shed new lights in understanding of microbial ecology and environmental microbiology. We investigated the soil bacterial community interactions through cultivation-independent methods in several land uses common in two Brazilian biomes. Using correlation network analysis we identified bacterial genera that presented important microbial associations within the soil community. The associations revealed nonrandomly structured microbial communities and clusters of operational taxonomic units (OTUs that reflected relevant bacterial relationships. Possible keystone genera were found in each soil. Irrespective of the biome or land use studied only a small portion of OTUs showed positive or negative interaction with other members of the soil bacterial community. The more interactive genera were also more abundant however, within those genera, the abundance was not related to taxon importance as measured by the Betweenness Centrality. Most of the soil bacterial genera were important to the overall connectance of the network, whereas only few genera play a key role as connectors, mainly belonged to phyla Proteobacteria and Actinobacteria. Finally it was observed that each land use presented a different set of keystone genera and that no keystone genus presented a generalized distribution. Taking into account that species interactions could be more important to soil processes than species richness and abundance, especially in complex ecosystems, this approach might represent a step forward in microbial ecology beyond the conventional studies of microbial richness and abundance.

  10. Many-objective optimization and visual analytics reveal key trade-offs for London's water supply

    Science.gov (United States)

    Matrosov, Evgenii S.; Huskova, Ivana; Kasprzyk, Joseph R.; Harou, Julien J.; Lambert, Chris; Reed, Patrick M.

    2015-12-01

    In this study, we link a water resource management simulator to multi-objective search to reveal the key trade-offs inherent in planning a real-world water resource system. We consider new supplies and demand management (conservation) options while seeking to elucidate the trade-offs between the best portfolios of schemes to satisfy projected water demands. Alternative system designs are evaluated using performance measures that minimize capital and operating costs and energy use while maximizing resilience, engineering and environmental metrics, subject to supply reliability constraints. Our analysis shows many-objective evolutionary optimization coupled with state-of-the art visual analytics can help planners discover more diverse water supply system designs and better understand their inherent trade-offs. The approach is used to explore future water supply options for the Thames water resource system (including London's water supply). New supply options include a new reservoir, water transfers, artificial recharge, wastewater reuse and brackish groundwater desalination. Demand management options include leakage reduction, compulsory metering and seasonal tariffs. The Thames system's Pareto approximate portfolios cluster into distinct groups of water supply options; for example implementing a pipe refurbishment program leads to higher capital costs but greater reliability. This study highlights that traditional least-cost reliability constrained design of water supply systems masks asset combinations whose benefits only become apparent when more planning objectives are considered.

  11. Surface N-glycoproteome patterns reveal key proteins of neuronal differentiation.

    Science.gov (United States)

    Tyleckova, Jirina; Valekova, Ivona; Zizkova, Martina; Rakocyova, Michaela; Marsala, Silvia; Marsala, Martin; Gadher, Suresh Jivan; Kovarova, Hana

    2016-01-30

    Pluripotent stem cell-derived committed neural precursors are an important source of cells to treat neurodegenerative diseases including spinal cord injury. There remains an urgency to identify markers for monitoring of neural progenitor specificity, estimation of neural fate and follow-up correlation with therapeutic effect in preclinical studies using animal disease models. Cell surface capture technology was used to uncover the cell surface exposed N-glycoproteome of neural precursor cells upon neuronal differentiation as well as post-mitotic mature hNT neurons. The data presented depict an extensive study of surfaceome during neuronal differentiation, confirming glycosylation at a particular predicted site of many of the identified proteins. Quantitative changes detected in cell surface protein levels reveal a set of proteins that highlight the complexity of the neuronal differentiation process. Several of these proteins including the cell adhesion molecules ICAM1, CHL1, and astrotactin1 as well as LAMP1 were validated by SRM. Combination of immunofluorescence staining of ICAM1 and flow cytometry indicated a possible direction for future scrutiny of such proteins as targets for enrichment of the neuronal subpopulation from mixed cultures after differentiation of neural precursor cells. These surface proteins hold an important key for development of safe strategies in cell-replacement therapies of neuronal disorders. Neural stem and/or precursor cells have a great potential for cell-replacement therapies of neuronal diseases. Availability of well characterised and expandable neural cell lineage specific populations is critical for addressing such a challenge. In our study we identified and relatively quantified several hundred surface N-glycoproteins in the course of neuronal differentiation. We further confirmed the abundant changes for several cell adhesion proteins by SRM and outlined a strategy for utilisation of such N-glycoproteins in antibody based cell

  12. Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn.

    Directory of Open Access Journals (Sweden)

    Yongbin Dong

    Full Text Available The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize.

  13. Long-term monitoring reveals carbon-nitrogen metabolism key to microcystin production in eutrophic lakes

    Directory of Open Access Journals (Sweden)

    Lucas J Beversdorf

    2015-05-01

    Full Text Available The environmental drivers contributing to cyanobacterial dominance in aquatic systems have been extensively studied. However, understanding of toxic versus non-toxic cyanobacterial population dynamics and the mechanisms regulating cyanotoxin production remain elusive, both physiologically and ecologically. One reason is the disconnect between laboratory and field-based studies. Here, we combined three years of temporal data, including microcystin (MC concentrations, 16 years of long-term ecological research, and 10 years of molecular data to investigate the potential factors leading to the selection of toxic Microcystis and MC production. Our analysis revealed that nitrogen (N speciation and inorganic carbon (C availability might be important drivers of Microcystis population dynamics and that an imbalance in cellular C: N ratios may trigger MC production. More specifically, precipitous declines in ammonium concentrations lead to a transitional period of N stress, even in the presence of high nitrate concentrations, that we call the toxic phase. Following the toxic phase, temperature and cyanobacterial abundance remained elevated but MC concentrations drastically declined. Increases in ammonium due to lake turnover may have led to down regulation of MC synthesis or a shift in the community from toxic to non-toxic species. While total phosphorus (P to total N ratios were relatively low over the time-series, MC concentrations were highest when total N to total P ratios were also highest. Similarly, high C: N ratios were also strongly correlated to the toxic phase. We propose a metabolic model that corroborates molecular studies and reflects our ecological observations that C and N metabolism may regulate MC production physiologically and ecologically. In particular, we hypothesize that an imbalance between 2-oxoglutarate and ammonium in the cell regulates MC synthesis in the environment.

  14. The Mechanism about Key and Credential on Trusted Computing Platform and the Application Study

    Institute of Scientific and Technical Information of China (English)

    SHEN Zhidong; ZHANG Huanguo; ZHANG Miao; YAN Fei; ZHANG Liqiang

    2006-01-01

    Trusted Computing technology is quickly developing in recent years. This technology manages to improve the computer security and archive a trusted computing environment. The core of trusted computing technology is cryptology. In this paper, we analyze the key and credential mechanism which is two basic aspects in the cryptology application of trusted computing. We give an example application to illustrate that the TPM enabled key and credential mechanism can improve the security of computer system.

  15. Small molecules reveal an alternative mechanism of Bax activation.

    Science.gov (United States)

    Brahmbhatt, Hetal; Uehling, David; Al-Awar, Rima; Leber, Brian; Andrews, David

    2016-04-15

    The pro-apoptotic protein Bax commits a cell to death by permeabilizing the mitochondrial outer membrane (MOM). To obtain small-molecule probes for elucidating the molecular mechanism(s) of Bax activation, we screened for compounds that induced Bax-mediated liposome permeabilization. We identified five structurally different small molecules that promoted both Bax targeting to and oligomerization at membranes. All five compounds initiated Bax oligomerization in the absence of membranes by a mechanism unlike Bax activation by Bcl-2 homology 3 domain (BH3) proteins. Some of the compounds induced Bax/Bak-dependent apoptosis in cells. Activation of Bax by the most active compound was poorly inhibited by the anti-apoptotic protein Bcl-XL and requires a cysteine residue at position 126 of Bax that is not required for activation by BH3 proteins. Our results reveal a novel pathway for Bax activation independent of pro-apoptotic BH3 proteins that may have important implications for the regulation of Bax activity in cells. © 2016 The Author(s).

  16. Geometric Mechanics Reveals Optimal Complex Terrestrial Undulation Patterns

    Science.gov (United States)

    Gong, Chaohui; Astley, Henry; Schiebel, Perrin; Dai, Jin; Travers, Matthew; Goldman, Daniel; Choset, Howie; CMU Team; GT Team

    Geometric mechanics offers useful tools for intuitively analyzing biological and robotic locomotion. However, utility of these tools were previously restricted to systems that have only two internal degrees of freedom and in uniform media. We show kinematics of complex locomotors that make intermittent contacts with substrates can be approximated as a linear combination of two shape bases, and can be represented using two variables. Therefore, the tools of geometric mechanics can be used to analyze motions of locomotors with many degrees of freedom. To demonstrate the proposed technique, we present studies on two different types of snake gaits which utilize combinations of waves in the horizontal and vertical planes: sidewinding (in the sidewinder rattlesnake C. cerastes) and lateral undulation (in the desert specialist snake C. occipitalis). C. cerastes moves by generating posteriorly traveling body waves in the horizontal and vertical directions, with a relative phase offset equal to +/-π/2 while C. occipitalismaintains a π/2 offset of a frequency doubled vertical wave. Geometric analysis reveals these coordination patterns enable optimal movement in the two different styles of undulatory terrestrial locomotion. More broadly, these examples demonstrate the utility of geometric mechanics in analyzing realistic biological and robotic locomotion.

  17. Analysis of replication profiles reveals key role of RFC-Ctf18 in yeast replication stress response.

    Science.gov (United States)

    Crabbé, Laure; Thomas, Aubin; Pantesco, Véronique; De Vos, John; Pasero, Philippe; Lengronne, Armelle

    2010-11-01

    Maintenance of genome integrity relies on surveillance mechanisms that detect and signal arrested replication forks. Although evidence from budding yeast indicates that the DNA replication checkpoint (DRC) is primarily activated by single-stranded DNA (ssDNA), studies in higher eukaryotes have implicated primer ends in this process. To identify factors that signal primed ssDNA in Saccharomyces cerevisiae, we have screened a collection of checkpoint mutants for their ability to activate the DRC, using the repression of late origins as readout for checkpoint activity. This quantitative analysis reveals that neither RFC(Rad24) and the 9-1-1 clamp nor the alternative clamp loader RFC(Elg1) is required to signal paused forks. In contrast, we found that RFC(Ctf18) is essential for the Mrc1-dependent activation of Rad53 and for the maintenance of paused forks. These data identify RFC(Ctf18) as a key DRC mediator, potentially bridging Mrc1 and primed ssDNA to signal paused forks.

  18. Ternary structure reveals mechanism of a membrane diacylglycerol kinase

    Science.gov (United States)

    Li, Dianfan; Stansfeld, Phillip J.; Sansom, Mark S. P.; Keogh, Aaron; Vogeley, Lutz; Howe, Nicole; Lyons, Joseph A.; Aragao, David; Fromme, Petra; Fromme, Raimund; Basu, Shibom; Grotjohann, Ingo; Kupitz, Christopher; Rendek, Kimberley; Weierstall, Uwe; Zatsepin, Nadia A.; Cherezov, Vadim; Liu, Wei; Bandaru, Sateesh; English, Niall J.; Gati, Cornelius; Barty, Anton; Yefanov, Oleksandr; Chapman, Henry N.; Diederichs, Kay; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Marvin Seibert, M.; Caffrey, Martin

    2015-12-01

    Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.

  19. Integrative modelling reveals mechanisms linking productivity and plant species richness.

    Science.gov (United States)

    Grace, James B; Anderson, T Michael; Seabloom, Eric W; Borer, Elizabeth T; Adler, Peter B; Harpole, W Stanley; Hautier, Yann; Hillebrand, Helmut; Lind, Eric M; Pärtel, Meelis; Bakker, Jonathan D; Buckley, Yvonne M; Crawley, Michael J; Damschen, Ellen I; Davies, Kendi F; Fay, Philip A; Firn, Jennifer; Gruner, Daniel S; Hector, Andy; Knops, Johannes M H; MacDougall, Andrew S; Melbourne, Brett A; Morgan, John W; Orrock, John L; Prober, Suzanne M; Smith, Melinda D

    2016-01-21

    How ecosystem productivity and species richness are interrelated is one of the most debated subjects in the history of ecology. Decades of intensive study have yet to discern the actual mechanisms behind observed global patterns. Here, by integrating the predictions from multiple theories into a single model and using data from 1,126 grassland plots spanning five continents, we detect the clear signals of numerous underlying mechanisms linking productivity and richness. We find that an integrative model has substantially higher explanatory power than traditional bivariate analyses. In addition, the specific results unveil several surprising findings that conflict with classical models. These include the isolation of a strong and consistent enhancement of productivity by richness, an effect in striking contrast with superficial data patterns. Also revealed is a consistent importance of competition across the full range of productivity values, in direct conflict with some (but not all) proposed models. The promotion of local richness by macroecological gradients in climatic favourability, generally seen as a competing hypothesis, is also found to be important in our analysis. The results demonstrate that an integrative modelling approach leads to a major advance in our ability to discern the underlying processes operating in ecological systems.

  20. Comparative "-omics" in mycoplasma pneumoniae clinical isolates reveals key virulence factors

    OpenAIRE

    Maria Lluch-Senar; Luca Cozzuto; Jaime Cano; Javier Delgado; Verónica Llórens-Rico; Sabine Pereyre; Cécile Bebear; Luis Serrano

    2015-01-01

    The human respiratory tract pathogen M. pneumoniae is one of the best characterized minimal bacterium. Until now, two main groups of clinical isolates of this bacterium have been described (types 1 and 2), differing in the sequence of the P1 adhesin gene. Here, we have sequenced the genomes of 23 clinical isolates of M. pneumoniae. Studying SNPs, non-synonymous mutations, indels and genome rearrangements of these 23 strains and 4 previously sequenced ones, has revealed new subclasses in the t...

  1. Comparative "-omics" in Mycoplasma pneumoniae Clinical Isolates Reveals Key Virulence Factors.

    Directory of Open Access Journals (Sweden)

    Maria Lluch-Senar

    Full Text Available The human respiratory tract pathogen M. pneumoniae is one of the best characterized minimal bacterium. Until now, two main groups of clinical isolates of this bacterium have been described (types 1 and 2, differing in the sequence of the P1 adhesin gene. Here, we have sequenced the genomes of 23 clinical isolates of M. pneumoniae. Studying SNPs, non-synonymous mutations, indels and genome rearrangements of these 23 strains and 4 previously sequenced ones, has revealed new subclasses in the two main groups, some of them being associated with the country of isolation. Integrative analysis of in vitro gene essentiality and mutation rates enabled the identification of several putative virulence factors and antigenic proteins; revealing recombination machinery, glycerol metabolism and peroxide production as possible factors in the genetics and physiology of these pathogenic strains. Additionally, the transcriptomes and proteomes of two representative strains, one from each of the two main groups, have been characterized to evaluate the impact of mutations on RNA and proteins levels. This study has revealed that type 2 strains show higher expression levels of CARDS toxin, a protein recently shown to be one of the major factors of inflammation. Thus, we propose that type 2 strains could be more toxigenic than type 1 strains of M. pneumoniae.

  2. Foundations of mechanism design: A tutorial Part 1 – Key concepts and classical results

    Indian Academy of Sciences (India)

    Dinesh Garg; Y Narahari; Sujit Gujar

    2008-04-01

    Mechanism design, an important tool in microeconomics, has found widespread applications in modelling and solving decentralized design problems in many branches of engineering, notably computer science, electronic commerce, and network economics. Mechanism design is concerned with settings where a social planner faces the problem of aggregating the announced preferences of multiple agents into a collective decision when the agents exhibit strategic behaviour. The objective of this paper is to provide a tutorial introduction to the foundations and key results in mechanism design theory. The paper is in two parts. Part 1 focuses on basic concepts and classical results which form the foundation of mechanism design theory. Part 2 presents key advanced concepts and deeper results in mechanism design

  3. Dynamic Zebrafish Interactome Reveals Transcriptional Mechanisms of Dioxin Toxicity

    Science.gov (United States)

    Alexeyenko, Andrey; Wassenberg, Deena M.; Lobenhofer, Edward K.; Yen, Jerry; Linney, Elwood; Sonnhammer, Erik L. L.; Meyer, Joel N.

    2010-01-01

    Background In order to generate hypotheses regarding the mechanisms by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) causes toxicity, we analyzed global gene expression changes in developing zebrafish embryos exposed to this potent toxicant in the context of a dynamic gene network. For this purpose, we also computationally inferred a zebrafish (Danio rerio) interactome based on orthologs and interaction data from other eukaryotes. Methodology/Principal Findings Using novel computational tools to analyze this interactome, we distinguished between dioxin-dependent and dioxin-independent interactions between proteins, and tracked the temporal propagation of dioxin-dependent transcriptional changes from a few genes that were altered initially, to large groups of biologically coherent genes at later times. The most notable processes altered at later developmental stages were calcium and iron metabolism, embryonic morphogenesis including neuronal and retinal development, a variety of mitochondria-related functions, and generalized stress response (not including induction of antioxidant genes). Within the interactome, many of these responses were connected to cytochrome P4501A (cyp1a) as well as other genes that were dioxin-regulated one day after exposure. This suggests that cyp1a may play a key role initiating the toxic dysregulation of those processes, rather than serving simply as a passive marker of dioxin exposure, as suggested by earlier research. Conclusions/Significance Thus, a powerful microarray experiment coupled with a flexible interactome and multi-pronged interactome tools (which are now made publicly available for microarray analysis and related work) suggest the hypothesis that dioxin, best known in fish as a potent cardioteratogen, has many other targets. Many of these types of toxicity have been observed in mammalian species and are potentially caused by alterations to cyp1a. PMID:20463971

  4. Dynamic zebrafish interactome reveals transcriptional mechanisms of dioxin toxicity.

    Directory of Open Access Journals (Sweden)

    Andrey Alexeyenko

    Full Text Available BACKGROUND: In order to generate hypotheses regarding the mechanisms by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin causes toxicity, we analyzed global gene expression changes in developing zebrafish embryos exposed to this potent toxicant in the context of a dynamic gene network. For this purpose, we also computationally inferred a zebrafish (Danio rerio interactome based on orthologs and interaction data from other eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS: Using novel computational tools to analyze this interactome, we distinguished between dioxin-dependent and dioxin-independent interactions between proteins, and tracked the temporal propagation of dioxin-dependent transcriptional changes from a few genes that were altered initially, to large groups of biologically coherent genes at later times. The most notable processes altered at later developmental stages were calcium and iron metabolism, embryonic morphogenesis including neuronal and retinal development, a variety of mitochondria-related functions, and generalized stress response (not including induction of antioxidant genes. Within the interactome, many of these responses were connected to cytochrome P4501A (cyp1a as well as other genes that were dioxin-regulated one day after exposure. This suggests that cyp1a may play a key role initiating the toxic dysregulation of those processes, rather than serving simply as a passive marker of dioxin exposure, as suggested by earlier research. CONCLUSIONS/SIGNIFICANCE: Thus, a powerful microarray experiment coupled with a flexible interactome and multi-pronged interactome tools (which are now made publicly available for microarray analysis and related work suggest the hypothesis that dioxin, best known in fish as a potent cardioteratogen, has many other targets. Many of these types of toxicity have been observed in mammalian species and are potentially caused by alterations to cyp1a.

  5. Endogenous Purification Reveals GREB1 as a Key Estrogen Receptor Regulatory Factor

    Directory of Open Access Journals (Sweden)

    Hisham Mohammed

    2013-02-01

    Full Text Available Estrogen receptor-α (ER is the driving transcription factor in most breast cancers, and its associated proteins can influence drug response, but direct methods for identifying interacting proteins have been limited. We purified endogenous ER using an approach termed RIME (rapid immunoprecipitation mass spectrometry of endogenous proteins and discovered the interactome under agonist- and antagonist-liganded conditions in breast cancer cells, revealing transcriptional networks in breast cancer. The most estrogen-enriched ER interactor is GREB1, a potential clinical biomarker with no known function. GREB1 is shown to be a chromatin-bound ER coactivator and is essential for ER-mediated transcription, because it stabilizes interactions between ER and additional cofactors. We show a GREB1-ER interaction in three xenograft tumors, and using a directed protein-protein approach, we find GREB1-ER interactions in half of ER+ primary breast cancers. This finding is supported by histological expression of GREB1, which shows that GREB1 is expressed in half of ER+ cancers, and predicts good clinical outcome. These findings reveal an unexpected role for GREB1 as an estrogen-specific ER cofactor that is expressed in drug-sensitive contexts.

  6. Metaproteomic Analysis of a Chemosynthetic Hydrothermal Vent Community Reveals Insights into Key-Metabolic Processes

    Science.gov (United States)

    Steen, I.; Stokke, R.; Lanzen, A.; Pedersen, R.; Øvreås, L.; Urich, T.

    2010-12-01

    In 2005 researchers at the Centre for Geobiology, University of Bergen, Norway, discovered two active vent fields at the southwestern Mohns Ridge in the Norwegian-Greenland Sea. The fields harbours both low-temperature iron deposits and high-temperature white smoker vents. Distinct microbial mats were abundantly present and located in close vicinity to the hydrothermal vent sites. Characteristics of the mat environment were steep physical and chemical gradients with temperatures ranging from 10°C in the top layer to 90°C at 10 cm bsf and high concentrations of hydrogen sulfide and methane. The work presented here focus on the In situ community activities, and is part of an integrated strategy combining metagenomics, metatranscriptomics and metaproteomics to in-depth characterise these newly discovered hydrothermal vent communities. Extracted proteins were separated via SDS-PAGE. Peptides extracted after In-gel tryptic digest was injected into an Ultimate 3000 nanoLC system connected to a linear quadropole ion trap-orbitrap (LTQ-Orbitrap XL) mass spectrometer equipped with a nanoelectrospray ion source. A custom database of open reading frames (ORFs) from the combined metatranscriptome and metagenome datasets was implemented and searched against using Mascot 2.2; the IRMa tool box [1] was used in peptide validation. Validated ORFs were subjected to a Blastp search against Refseq with an E-value cut-off of 0.001. A total of 1097 proteins with ≥ 2 peptides were identified of which 921 gave a hit against Refseq, containing 519 unique proteins. Key enzymes of the sulfur oxidation pathway (sox) were found, which were taxonomically affiliated to Epsilonproteobacteria. In addition, this group actively expressed hydrogenases and membrane proteins involved in aerobic and anaerobic respiratory chains. Enzymes of dissimilatory sulfate-reduction (APS-reductase, AprAB and DsrA2) were found with closest hit to members of the Deltaproteobacteria. These findings indicate an

  7. Quantitative transcription dynamic analysis reveals candidate genes and key regulators for ethanol tolerance in Saccharomyces cerevisiae.

    Science.gov (United States)

    Ma, Menggen; Liu, Lewis Z

    2010-06-10

    Derived from our lignocellulosic conversion inhibitor-tolerant yeast, we generated an ethanol-tolerant strain Saccharomyces cerevisiae NRRL Y-50316 by enforced evolutionary adaptation. Using a newly developed robust mRNA reference and a master equation unifying gene expression data analyses, we investigated comparative quantitative transcription dynamics of 175 genes selected from previous studies for an ethanol-tolerant yeast and its closely related parental strain. A highly fitted master equation was established and applied for quantitative gene expression analyses using pathway-based qRT-PCR array assays. The ethanol-tolerant Y-50316 displayed significantly enriched background of mRNA abundance for at least 35 genes without ethanol challenge compared with its parental strain Y-50049. Under the ethanol challenge, the tolerant Y-50316 responded in consistent expressions over time for numerous genes belonging to groups of heat shock proteins, trehalose metabolism, glycolysis, pentose phosphate pathway, fatty acid metabolism, amino acid biosynthesis, pleiotropic drug resistance gene family and transcription factors. The parental strain showed repressed expressions for many genes and was unable to withstand the ethanol stress and establish a viable culture and fermentation. The distinct expression dynamics between the two strains and their close association with cell growth, viability and ethanol fermentation profiles distinguished the tolerance-response from the stress-response in yeast under the ethanol challenge. At least 82 genes were identified as candidate and key genes for ethanol-tolerance and subsequent fermentation under the stress. Among which, 36 genes were newly recognized by the present study. Most of the ethanol-tolerance candidate genes were found to share protein binding motifs of transcription factors Msn4p/Msn2p, Yap1p, Hsf1p and Pdr1p/Pdr3p. Enriched background of transcription abundance and enhanced expressions of ethanol-tolerance genes

  8. Quantitative transcription dynamic analysis reveals candidate genes and key regulators for ethanol tolerance in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Ma Menggen

    2010-06-01

    Full Text Available Abstract Background Derived from our lignocellulosic conversion inhibitor-tolerant yeast, we generated an ethanol-tolerant strain Saccharomyces cerevisiae NRRL Y-50316 by enforced evolutionary adaptation. Using a newly developed robust mRNA reference and a master equation unifying gene expression data analyses, we investigated comparative quantitative transcription dynamics of 175 genes selected from previous studies for an ethanol-tolerant yeast and its closely related parental strain. Results A highly fitted master equation was established and applied for quantitative gene expression analyses using pathway-based qRT-PCR array assays. The ethanol-tolerant Y-50316 displayed significantly enriched background of mRNA abundance for at least 35 genes without ethanol challenge compared with its parental strain Y-50049. Under the ethanol challenge, the tolerant Y-50316 responded in consistent expressions over time for numerous genes belonging to groups of heat shock proteins, trehalose metabolism, glycolysis, pentose phosphate pathway, fatty acid metabolism, amino acid biosynthesis, pleiotropic drug resistance gene family and transcription factors. The parental strain showed repressed expressions for many genes and was unable to withstand the ethanol stress and establish a viable culture and fermentation. The distinct expression dynamics between the two strains and their close association with cell growth, viability and ethanol fermentation profiles distinguished the tolerance-response from the stress-response in yeast under the ethanol challenge. At least 82 genes were identified as candidate and key genes for ethanol-tolerance and subsequent fermentation under the stress. Among which, 36 genes were newly recognized by the present study. Most of the ethanol-tolerance candidate genes were found to share protein binding motifs of transcription factors Msn4p/Msn2p, Yap1p, Hsf1p and Pdr1p/Pdr3p. Conclusion Enriched background of transcription abundance

  9. Multiscale computational analysis of Xenopus laevis morphogenesis reveals key insights of systems-level behavior

    Directory of Open Access Journals (Sweden)

    DeSimone Douglas W

    2007-10-01

    Full Text Available Abstract Background Tissue morphogenesis is a complex process whereby tissue structures self-assemble by the aggregate behaviors of independently acting cells responding to both intracellular and extracellular cues in their environment. During embryonic development, morphogenesis is particularly important for organizing cells into tissues, and although key regulatory events of this process are well studied in isolation, a number of important systems-level questions remain unanswered. This is due, in part, to a lack of integrative tools that enable the coupling of biological phenomena across spatial and temporal scales. Here, we present a new computational framework that integrates intracellular signaling information with multi-cell behaviors in the context of a spatially heterogeneous tissue environment. Results We have developed a computational simulation of mesendoderm migration in the Xenopus laevis explant model, which is a well studied biological model of tissue morphogenesis that recapitulates many features of this process during development in humans. The simulation couples, via a JAVA interface, an ordinary differential equation-based mass action kinetics model to compute intracellular Wnt/β-catenin signaling with an agent-based model of mesendoderm migration across a fibronectin extracellular matrix substrate. The emergent cell behaviors in the simulation suggest the following properties of the system: maintaining the integrity of cell-to-cell contact signals is necessary for preventing fractionation of cells as they move, contact with the Fn substrate and the existence of a Fn gradient provides an extracellular feedback loop that governs migration speed, the incorporation of polarity signals is required for cells to migrate in the same direction, and a delicate balance of integrin and cadherin interactions is needed to reproduce experimentally observed migratory behaviors. Conclusion Our computational framework couples two different

  10. Stress, deformation, conservation, and rheology: a survey of key concepts in continuum mechanics

    Science.gov (United States)

    Major, J.J.

    2013-01-01

    This chapter provides a brief survey of key concepts in continuum mechanics. It focuses on the fundamental physical concepts that underlie derivations of the mathematical formulations of stress, strain, hydraulic head, pore-fluid pressure, and conservation equations. It then shows how stresses are linked to strain and rates of distortion through some special cases of idealized material behaviors. The goal is to equip the reader with a physical understanding of key mathematical formulations that anchor continuum mechanics in order to better understand theoretical studies published in geomorphology.

  11. Transcriptomic analysis reveals key genes related to betalain biosynthesis in pulp coloration of Hylocereus polyrhizus

    Directory of Open Access Journals (Sweden)

    Hua eQingzhu

    2016-01-01

    Full Text Available Betalains have high nutritional value and bioactivities. Red pulp pitaya (Hylocereus polyrhizus is the only fruit containing abundant betalains for consumer. However, no information is available about genes involved in betalain biosynthesis in H. polyrhizus. Herein, two cDNA libraries of pitaya pulps with two different coloration stages (white and red pulp stages of Guanhuahong (H. polyrhizus were constructed. A total of about 12 Gb raw RNA-Seq data was generated and was de novo assembled into 122,677 transcripts with an average length of 1,183 bp and an N50 value of 2008. Approximately 99.99% of all transcripts were annotated based on seven public databases. A total of 8,871 transcripts were significantly regulated. Thirty-three candidate transcripts related to betalain biosynthesis were obtained from the transcriptome data. Transcripts encoding enzymes involved in betalain biosynthesis were analyzed using RT-qPCR at the whole pulp coloration stages of H. Polyrhizus (7-1 and H. Undatus (132-4. Nine key transcripts of betalain biosynthesis were identified. They were assigned to four kinds of genes in betalain biosynthetic pathway, including tyrosinase, 4, 5-DOPA dioxygenase extradiol, cytochrome P450 and glucosyltransferase. Ultimately, a preliminary betalain biosynthetic pathway for pitaya was proposed based on betalain analyses and gene expression profiles.

  12. Transcriptomic Analysis Reveals Key Genes Related to Betalain Biosynthesis in Pulp Coloration of Hylocereus polyrhizus.

    Science.gov (United States)

    Qingzhu, Hua; Chengjie, Chen; Zhe, Chen; Pengkun, Chen; Yuewen, Ma; Jingyu, Wu; Jian, Zheng; Guibing, Hu; Jietang, Zhao; Yonghua, Qin

    2015-01-01

    Betalains have high nutritional value and bioactivities. Red pulp pitaya (Hylocereus polyrhizus) is the only fruit containing abundant betalains for consumer. However, no information is available about genes involved in betalain biosynthesis in H. polyrhizus. Herein, two cDNA libraries of pitaya pulps with two different coloration stages (white and red pulp stages) of Guanhuahong (H. polyrhizus) were constructed. A total of about 12 Gb raw RNA-Seq data was generated and was de novo assembled into 122,677 transcripts with an average length of 1183 bp and an N50 value of 2008. Approximately 99.99% of all transcripts were annotated based on seven public databases. A total of 8871 transcripts were significantly regulated. Thirty-three candidate transcripts related to betalain biosynthesis were obtained from the transcriptome data. Transcripts encoding enzymes involved in betalain biosynthesis were analyzed using RT-qPCR at the whole pulp coloration stages of H. polyrhizus (7-1) and H. undatus (132-4). Nine key transcripts of betalain biosynthesis were identified. They were assigned to four kinds of genes in betalain biosynthetic pathway, including tyrosinase, 4, 5-DOPA dioxygenase extradiol, cytochrome P450 and glucosyltransferase. Ultimately, a preliminary betalain biosynthetic pathway for pitaya was proposed based on betalain analyses, gene expression profiles and published documents.

  13. The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions

    Energy Technology Data Exchange (ETDEWEB)

    Merchant, Sabeeha S

    2007-04-09

    Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

  14. Novel personalized pathway-based metabolomics models reveal key metabolic pathways for breast cancer diagnosis

    DEFF Research Database (Denmark)

    Huang, Sijia; Chong, Nicole; Lewis, Nathan

    2016-01-01

    . Methods: We propose that higher-order functional representation of metabolomics data, such as pathway-based metabolomic features, can be used as robust biomarkers for breast cancer. Towards this, we have developed a new computational method that uses personalized pathway dysregulation scores for disease...... the Curve, a receiver operating characteristic curve) of 0.968 and 0.934, sensitivities of 0.946 and 0.954, and specificities of 0.934 and 0.918. These two metabolomics-based pathway models are further validated by RNA-Seq-based TCGA (The Cancer Genome Atlas) breast cancer data, with AUCs of 0.995 and 0.......993. Moreover, important metabolic pathways, such as taurine and hypotaurine metabolism and the alanine, aspartate, and glutamate pathway, are revealed as critical biological pathways for early diagnosis of breast cancer. Conclusions: We have successfully developed a new type of pathway-based model to study...

  15. Genomes of cryptic chimpanzee Plasmodium species reveal key evolutionary events leading to human malaria.

    Science.gov (United States)

    Sundararaman, Sesh A; Plenderleith, Lindsey J; Liu, Weimin; Loy, Dorothy E; Learn, Gerald H; Li, Yingying; Shaw, Katharina S; Ayouba, Ahidjo; Peeters, Martine; Speede, Sheri; Shaw, George M; Bushman, Frederic D; Brisson, Dustin; Rayner, Julian C; Sharp, Paul M; Hahn, Beatrice H

    2016-03-22

    African apes harbour at least six Plasmodium species of the subgenus Laverania, one of which gave rise to human Plasmodium falciparum. Here we use a selective amplification strategy to sequence the genome of chimpanzee parasites classified as Plasmodium reichenowi and Plasmodium gaboni based on the subgenomic fragments. Genome-wide analyses show that these parasites indeed represent distinct species, with no evidence of cross-species mating. Both P. reichenowi and P. gaboni are 10-fold more diverse than P. falciparum, indicating a very recent origin of the human parasite. We also find a remarkable Laverania-specific expansion of a multigene family involved in erythrocyte remodelling, and show that a short region on chromosome 4, which encodes two essential invasion genes, was horizontally transferred into a recent P. falciparum ancestor. Our results validate the selective amplification strategy for characterizing cryptic pathogen species, and reveal evolutionary events that likely predisposed the precursor of P. falciparum to colonize humans.

  16. Virus and dsRNA-triggered transcriptional responses reveal key components of honey bee antiviral defense.

    Science.gov (United States)

    Brutscher, Laura M; Daughenbaugh, Katie F; Flenniken, Michelle L

    2017-07-25

    Recent high annual losses of honey bee colonies are associated with many factors, including RNA virus infections. Honey bee antiviral responses include RNA interference and immune pathway activation, but their relative roles in antiviral defense are not well understood. To better characterize the mechanism(s) of honey bee antiviral defense, bees were infected with a model virus in the presence or absence of dsRNA, a virus associated molecular pattern. Regardless of sequence specificity, dsRNA reduced virus abundance. We utilized next generation sequencing to examine transcriptional responses triggered by virus and dsRNA at three time-points post-infection. Hundreds of genes exhibited differential expression in response to co-treatment of dsRNA and virus. Virus-infected bees had greater expression of genes involved in RNAi, Toll, Imd, and JAK-STAT pathways, but the majority of differentially expressed genes are not well characterized. To confirm the virus limiting role of two genes, including the well-characterized gene, dicer, and a probable uncharacterized cyclin dependent kinase in honey bees, we utilized RNAi to reduce their expression in vivo and determined that virus abundance increased, supporting their involvement in antiviral defense. Together, these results further our understanding of honey bee antiviral defense, particularly the role of a non-sequence specific dsRNA-mediated antiviral pathway.

  17. Mutations in GDF5 reveal a key residue mediating BMP inhibition by NOGGIN.

    Directory of Open Access Journals (Sweden)

    Petra Seemann

    2009-11-01

    Full Text Available Signaling output of bone morphogenetic proteins (BMPs is determined by two sets of opposing interactions, one with heterotetrameric complexes of cell surface receptors, the other with secreted antagonists that act as ligand traps. We identified two mutations (N445K,T in patients with multiple synostosis syndrome (SYM1 in the BMP-related ligand GDF5. Functional studies of both mutants in chicken micromass culture demonstrated a gain of function caused by a resistance to the BMP-inhibitor NOGGIN and an altered signaling effect. Residue N445, situated within overlapping receptor and antagonist interfaces, is highly conserved among the BMP family with the exception of BMP9 and BMP10, in which it is substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to NOGGIN and show a high chondrogenic activity. Ectopic expression of BMP9 or the GDF5 mutants resulted in massive induction of cartilage in an in vivo chick model presumably by bypassing the feedback inhibition imposed by endogenous NOGGIN. Swapping residues at the mutation site alone was not sufficient to render Bmp9 NOG-sensitive; however, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. In conclusion, we show a new mechanism for abnormal joint development that interferes with a naturally occurring regulatory mechanism of BMP signaling.

  18. Molecular modifiers reveal a mechanism of pathological crystal growth inhibition

    Science.gov (United States)

    Chung, Jihae; Granja, Ignacio; Taylor, Michael G.; Mpourmpakis, Giannis; Asplin, John R.; Rimer, Jeffrey D.

    2016-08-01

    Crystalline materials are crucial to the function of living organisms, in the shells of molluscs, the matrix of bone, the teeth of sea urchins, and the exoskeletons of coccoliths. However, pathological biomineralization can be an undesirable crystallization process associated with human diseases. The crystal growth of biogenic, natural and synthetic materials may be regulated by the action of modifiers, most commonly inhibitors, which range from small ions and molecules to large macromolecules. Inhibitors adsorb on crystal surfaces and impede the addition of solute, thereby reducing the rate of growth. Complex inhibitor-crystal interactions in biomineralization are often not well elucidated. Here we show that two molecular inhibitors of calcium oxalate monohydrate crystallization—citrate and hydroxycitrate—exhibit a mechanism that differs from classical theory in that inhibitor adsorption on crystal surfaces induces dissolution of the crystal under specific conditions rather than a reduced rate of crystal growth. This phenomenon occurs even in supersaturated solutions where inhibitor concentration is three orders of magnitude less than that of the solute. The results of bulk crystallization, in situ atomic force microscopy, and density functional theory studies are qualitatively consistent with a hypothesis that inhibitor-crystal interactions impart localized strain to the crystal lattice and that oxalate and calcium ions are released into solution to alleviate this strain. Calcium oxalate monohydrate is the principal component of human kidney stones and citrate is an often-used therapy, but hydroxycitrate is not. For hydroxycitrate to function as a kidney stone treatment, it must be excreted in urine. We report that hydroxycitrate ingested by non-stone-forming humans at an often-recommended dose leads to substantial urinary excretion. In vitro assays using human urine reveal that the molecular modifier hydroxycitrate is as effective an inhibitor of nucleation

  19. Low-Temperature Biodiesel Research Reveals Potential Key to Successful Blend Performance (Fact Sheet)

    Energy Technology Data Exchange (ETDEWEB)

    2012-02-01

    Relatively low-cost solutions could improve reliability while making biodiesel blends an affordable option. While biodiesel has very low production costs and the potential to displace up to 10% of petroleum diesel, until now, issues with cold weather performance have prevented biodiesel blends from being widely adopted. Some biodiesel blends have exhibited unexplained low-temperature performance problems even at blend levels as low as 2% by volume. The most common low-temperature performance issue is vehicle stalling caused by fuel filter clogging, which prevents fuel from reaching the engine. Research at the National Renewable Energy Laboratory (NREL) reveals the properties responsible for these problems, clearing a path for the development of solutions and expanded use of energy-conserving and low-emissions alternative fuel. NREL researchers set out to study the unpredictable nature of biodiesel crystallization, the condition that impedes the flow of fuel in cold weather. Their research revealed for the first time that saturated monoglyceride impurities common to the biodiesel manufacturing process create crystals that can cause fuel filter clogging and other problems when cooling at slow rates. Biodiesel low-temperature operational problems are commonly referred to as 'precipitates above the cloud point (CP).' NREL's Advanced Biofuels team spiked distilled soy and animal fat-derived B100, as well as B20, B10, and B5 biodiesel blends with three saturated monoglycerides (SMGs) at concentration levels comparable to those of real-world fuels. Above a threshold or eutectic concentration, the SMGs (monomyristin, monopalmitin, and monostearin) were shown to significantly raise the biodiesel CP, and had an even greater impact on the final melting temperature. Researchers discovered that upon cooling, monoglyceride initially precipitates as a metastable crystal, but it transforms over time or upon slight heating into a more stable crystal with a much lower

  20. Topological robustness analysis of protein interaction networks reveals key targets for overcoming chemotherapy resistance in glioma

    Science.gov (United States)

    Azevedo, Hátylas; Moreira-Filho, Carlos Alberto

    2015-11-01

    Biological networks display high robustness against random failures but are vulnerable to targeted attacks on central nodes. Thus, network topology analysis represents a powerful tool for investigating network susceptibility against targeted node removal. Here, we built protein interaction networks associated with chemoresistance to temozolomide, an alkylating agent used in glioma therapy, and analyzed their modular structure and robustness against intentional attack. These networks showed functional modules related to DNA repair, immunity, apoptosis, cell stress, proliferation and migration. Subsequently, network vulnerability was assessed by means of centrality-based attacks based on the removal of node fractions in descending orders of degree, betweenness, or the product of degree and betweenness. This analysis revealed that removing nodes with high degree and high betweenness was more effective in altering networks’ robustness parameters, suggesting that their corresponding proteins may be particularly relevant to target temozolomide resistance. In silico data was used for validation and confirmed that central nodes are more relevant for altering proliferation rates in temozolomide-resistant glioma cell lines and for predicting survival in glioma patients. Altogether, these results demonstrate how the analysis of network vulnerability to topological attack facilitates target prioritization for overcoming cancer chemoresistance.

  1. A study revealing the key aroma compounds of steamed bread made by Chinese traditional sourdough.

    Science.gov (United States)

    Zhang, Guo-Hua; Wu, Tao; Sadiq, Faizan A; Yang, Huan-Yi; Liu, Tong-Jie; Ruan, Hui; He, Guo-Qing

    Aroma of Chinese steamed bread (CSB) is one of the important parameters that determines the overall quality attributes and consumer acceptance. However, the aroma profile of CSB still remains poorly understood, mainly because of relying on only a single method for aroma extraction in previous studies. Therefore, the objective of this study was to determine the volatile aroma compounds of five different samples of CSB using three different aroma extraction methods, namely solid-phase microextraction (SPME), simultaneous distillation-extraction (SDE), and purge and trap (P&T). All samples showed a unique aroma profile, which could be attributed to their unique microbial consortia. (E)-2-Nonenal and (E,E)-2,4-decadienal were the most prevalent aromatic compounds revealed by SDE, which have not been reported previously, while ethanol and acetic acid proved to be the most dominant compounds by both SPME and P&T. Our approach of combining three different aroma extraction methods provided better insights into the aroma profile of CSB, which had remained largely unknown in previous studies.

  2. A study revealing the key aroma compounds of steamed bread made by Chinese traditional sourdough*

    Science.gov (United States)

    Zhang, Guo-hua; Wu, Tao; Sadiq, Faizan A.; Yang, Huan-yi; Liu, Tong-jie; Ruan, Hui; He, Guo-qing

    2016-01-01

    Aroma of Chinese steamed bread (CSB) is one of the important parameters that determines the overall quality attributes and consumer acceptance. However, the aroma profile of CSB still remains poorly understood, mainly because of relying on only a single method for aroma extraction in previous studies. Therefore, the objective of this study was to determine the volatile aroma compounds of five different samples of CSB using three different aroma extraction methods, namely solid-phase microextraction (SPME), simultaneous distillation–extraction (SDE), and purge and trap (P&T). All samples showed a unique aroma profile, which could be attributed to their unique microbial consortia. (E)-2-Nonenal and (E,E)-2,4-decadienal were the most prevalent aromatic compounds revealed by SDE, which have not been reported previously, while ethanol and acetic acid proved to be the most dominant compounds by both SPME and P&T. Our approach of combining three different aroma extraction methods provided better insights into the aroma profile of CSB, which had remained largely unknown in previous studies. PMID:27704748

  3. Characterization of a C3 Deoxygenation Pathway Reveals a Key Branch Point in Aminoglycoside Biosynthesis.

    Science.gov (United States)

    Lv, Meinan; Ji, Xinjian; Zhao, Junfeng; Li, Yongzhen; Zhang, Chen; Su, Li; Ding, Wei; Deng, Zixin; Yu, Yi; Zhang, Qi

    2016-05-25

    Apramycin is a clinically interesting aminoglycoside antibiotic (AGA) containing a highly unique bicyclic octose moiety, and this octose is deoxygenated at the C3 position. Although the biosynthetic pathways for most 2-deoxystreptamine-containing AGAs have been well characterized, the pathway for apramycin biosynthesis, including the C3 deoxygenation process, has long remained unknown. Here we report detailed investigation of apramycin biosynthesis by a series of genetic, biochemical and bioinformatical studies. We show that AprD4 is a novel radical S-adenosyl-l-methionine (SAM) enzyme, which uses a noncanonical CX3CX3C motif for binding of a [4Fe-4S] cluster and catalyzes the dehydration of paromamine, a pseudodisaccharide intermediate in apramycin biosynthesis. We also show that AprD3 is an NADPH-dependent reductase that catalyzes the reduction of the dehydrated product from AprD4-catalyzed reaction to generate lividamine, a C3' deoxygenated product of paromamine. AprD4 and AprD3 do not form a tight catalytic complex, as shown by protein complex immunoprecipitation and other assays. The AprD4/AprD3 enzyme system acts on different pseudodisaccharide substrates but does not catalyze the deoxygenation of oxyapramycin, an apramycin analogue containing a C3 hydroxyl group on the octose moiety, suggesting that oxyapramycin and apramycin are partitioned into two parallel pathways at an early biosynthetic stage. Functional dissection of the C6 dehydrogenase AprQ shows the crosstalk between different AGA biosynthetic gene clusters from the apramycin producer Streptomyces tenebrarius, and reveals the remarkable catalytic versatility of AprQ. Our study highlights the intriguing chemistry in apramycin biosynthesis and nature's ingenuity in combinatorial biosynthesis of natural products.

  4. Physiological, biochemical, and proteome profiling reveals key pathways underlying the drought stress responses of Hippophae rhamnoides.

    Science.gov (United States)

    He, Cai Y; Zhang, Guo Y; Zhang, Jian G; Duan, Ai G; Luo, Hong M

    2016-10-01

    The effects of drought on plant growth and development are occurring as a result of climate change and the growing scarcity of water resources. Hippophae rhamnoides has been exploited for soil and water conservation for many years. However, the outstanding drought-resistance mechanisms possessed by this species remain unclear. The protein, physiological, and biochemical responses to medium and severe drought stresses in H. rhamnoides seedlings are analyzed. Linear decreases in photosynthesis rate, transpiration rate, and the content of indole acetic acid in roots, as well as a linear increase in the contents of abscisic acid, superoxide dismutase, glutathione reductase, and zeatin riboside in leaves are observed as water potential decreased. At the same time, cell membrane permeability, malondialdehyde, stomatal conductance, water use efficiency, and contents of zeatin riboside in roots and indole acetic acid in leaves showed nonconsistent changes. DIGE and MS/MS analysis identified 51 differently expressed protein spots in leaves with functions related to epigenetic modification and PTM in addition to normal metabolism, photosynthesis, signal transduction, antioxidative systems, and responses to stimuli. This study provides new insights into the responses and adaptations in this drought-resistant species and may benefit future agricultural production. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Transcriptomic Analysis of Musca domestica to Reveal Key Genes of the Prophenoloxidase-Activating System.

    Science.gov (United States)

    Li, Dianxiang; Liang, Yongli; Wang, Xianwei; Wang, Lei; Qi, Mei; Yu, Yang; Luan, Yuanyuan

    2015-09-01

    The proPO system regulates melanization in arthropods. However, the genes that are involved in the proPO system in housefly Musca domestica remain unclear. Thus, this study analyzed the combined transcriptome obtained from M. domestica larvae, pupae, and adults that were either normal or bacteria-challenged by an Escherichia coli and Staphylococcus aureus mixture. A total of 54,821,138 clean reads (4.93 Gb) were yielded by Illumina sequencing, which were de novo assembled into 89,842 unigenes. Of the 89,842 unigenes, based on a similarity search with known genes in other insects, 24 putative genes related to the proPO system were identified. Eight of the identified genes encoded for peptidoglycan recognition receptors, two encoded for prophenoloxidases, three encoded for prophenoloxidase-activating enzymes, and 11 encoded for serine proteinase inhibitors. The expression levels of these identified genes were investigated by qRT-PCR assay, which were consistent with expected activation process of the proPO system, and their activation functions were confirmed by the measurement of phenoloxidase activity in bacteria-infected larvae after proPO antibody blockage, suggesting these candidate genes might have potentially different roles in the activation of proPO system. Collectively, this study has provided the comprehensive transcriptomic data of an insect and some fundamental basis toward achieving understanding of the activation mechanisms and immune functions of the proPO system in M. domestica.

  6. Gene expression profiling reveals potential key pathways involved in pyrazinamide-mediated hepatotoxicity in Wistar rats.

    Science.gov (United States)

    Zhang, Yun; Jiang, Zhenzhou; Su, Yijing; Chen, Mi; Li, Fu; Liu, Li; Sun, Lixin; Wang, Yun; Zhang, Shuang; Zhang, Luyong

    2013-08-01

    Pyrazinamide (PZA) is an important sterilizing prodrug that shortens the duration of tuberculosis therapy. However, hepatotoxicity has been reported during clinical trials investigating PZA. To determine the hepatotoxic effects of PZA in vivo and to further investigate the underlying cellular mechanism, we profiled the gene expression patterns of PZA-treated rat livers by microarray analysis. Wistar rats of both sexes were orally administered PZA at doses of 0.5, 1.0 and 2.0 g kg(-1) for 28 days. Body weight, absolute and relative liver weight, biochemical analysis, histopathology, oxidative stress parameters in liver homogenates and changes in global transcriptomic expression were evaluated to study the hepatotoxic effects of PZA. Our results confirm the dose-dependent and sex-related hepatotoxicity of PZA. Female rats were more sensitive to PZA-induced hepatotoxicity than males. Furthermore, changes in the activity of major antioxidant enzymes and nonenzymatic antioxidants (superoxide dismutase, total antioxidant capacity, glutathione and malondialdehyde), indicating the development of oxidative stress, were more significant in the PZA-treated group. PZA-induced gene expression changes were related to pathways involved in drug metabolism, peroxisome proliferator-activated receptor (PPAR) signaling, oxidative stress and apoptosis. Real-time polymerase chain reaction confirmed the regulation of selected genes involved in PZA-hepatotoxicity (Ephx1, Cyp2b1, Gstm1, Gstp1, Fabp7, Acaa1, Cpt-1b, Cyp8b1, Hmox1 and Ntrk1). We observed for the first time that these genes have effects on PZA-induced hepatotoxicity. In addition, drug metabolism and PPAR signaling pathways may play an important role in PZA hepatotoxicity. Taken together, these findings will be useful for future PZA hepatotoxicity studies.

  7. A study revealing the key aroma compounds of steamed bread made by Chinese traditional sourdough

    Institute of Scientific and Technical Information of China (English)

    Guo-hua ZHANG; Tao WU; Faizan A SADIQ; Huan-yi YANG; Tong-jie LIU; Hui RUAN; Guo-qing HE

    2016-01-01

    Aroma of Chinese steamed bread (CSB) is one of the important parameters that determines the overall quality attributes and consumer acceptance. However, the aroma profile of CSB stil remains poorly understood, mainly because of relying on only a single method for aroma extraction in previous studies. Therefore, the objective of this study was to determine the volatile aroma compounds of five different samples of CSB using three different aroma extraction methods, namely solid-phase microextraction (SPME), simultaneous distilation–extraction (SDE), and purge and trap (P&T). Al samples showed a unique aroma profile, which could be attributed to their unique microbial consortia. (E)-2-Nonenal and (E,E)-2,4-decadienal were the most prevalent aromatic compounds revealed by SDE, which have not been reported previously, while ethanol and acetic acid proved to be the most dominant compounds by both SPME and P&T. Our approach of combining three different aroma extraction methods provided better insights into the aroma profile of CSB, which had remained largely unknown in previous studies.%中文概要题目:传统酸面团制备的馒头特征风味物质的研究目的:通过同时蒸馏萃取(SDE)、顶空固相微萃取(SPME)及吹扫捕集(P&T)技术结合气相色谱-嗅闻-质谱(GC-O-MS)技术对传统酸面团制备的馒头特征风味成分进行分析,并初步探讨特征风味形成机理。创新点:首次采用不同风味物质分析技术探索传统酸面团制备的馒头关键风味成分。方法:采用同时SDE、SPME及P&T方法,对不同传统酸面团制备的馒头特征风味物质进行分析。结论:SDE结果显示,反式-2-壬烯醛和反式-2,4-癸二烯醛为传统酸面团馒头中关键风味成分。SPME和P&T 方法表明乙醇和醋酸是关键风味物质。因此,需要结合不同技术方法,全面分析传统酸面团制备的馒头中特征风味成分。

  8. The extraordinary joint material of an articulated coralline alga. I. Mechanical characterization of a key adaptation.

    Science.gov (United States)

    Denny, Mark W; King, Felicia A

    2016-06-15

    Flexibility is key to survival for seaweeds exposed to the extreme hydrodynamic environment of wave-washed rocky shores. This poses a problem for coralline algae, whose calcified cell walls make them rigid. Through the course of evolution, erect coralline algae have solved this problem by incorporating joints (genicula) into their morphology, allowing their fronds to be as flexible as those of uncalcified seaweeds. To provide the flexibility required by this structural innovation, the joint material of Calliarthron cheilosporioides, a representative articulated coralline alga, relies on an extraordinary tissue that is stronger, more extensible and more fatigue resistant than the tissue of other algal fronds. Here, we report on experiments that reveal the viscoelastic properties of this material. On the one hand, its compliance is independent of the rate of deformation across a wide range of deformation rates, a characteristic of elastic solids. This deformation rate independence allows joints to maintain their flexibility when loaded by the unpredictable - and often rapidly imposed - hydrodynamic force of breaking waves. On the other hand, the genicular material has viscous characteristics that similarly augment its function. The genicular material dissipates much of the energy absorbed as a joint is deformed during cyclic wave loading, which potentially reduces the chance of failure by fatigue, and the material accrues a limited amount of deformation through time. This limited creep increases the flexibility of the joints while preventing them from gradually stretching to the point of failure. These new findings provide the basis for understanding how the microscale architecture of genicular cell walls results in the adaptive mechanical properties of coralline algal joints.

  9. CMOS-MEMS Test-Key for Extracting Wafer-Level Mechanical Properties

    Directory of Open Access Journals (Sweden)

    Pei-Zen Chang

    2012-12-01

    Full Text Available This paper develops the technologies of mechanical characterization of CMOS-MEMS devices, and presents a robust algorithm for extracting mechanical properties, such as Young’s modulus, and mean stress, through the external electrical circuit behavior of the micro test-key. An approximate analytical solution for the pull-in voltage of bridge-type test-key subjected to electrostatic load and initial stress is derived based on Euler’s beam model and the minimum energy method. Then one can use the aforesaid closed form solution of the pull-in voltage to extract the Young’s modulus and mean stress of the test structures. The test cases include the test-key fabricated by a TSMC 0.18 μm standard CMOS process, and the experimental results refer to Osterberg’s work on the pull-in voltage of single crystal silicone microbridges. The extracted material properties calculated by the present algorithm are valid. Besides, this paper also analyzes the robustness of this algorithm regarding the dimension effects of test-keys. This mechanical properties extracting method is expected to be applicable to the wafer-level testing in micro-device manufacture and compatible with the wafer-level testing in IC industry since the test process is non-destructive.

  10. Revealing the Differences Between Free and Complexed Enzyme Mechanisms and Factors Contributing to Cell Wall Recalcitrance

    Energy Technology Data Exchange (ETDEWEB)

    Resch, M.

    2014-09-08

    Enzymatic depolymerization of polysaccharides is a key step in the production of fuels and chemicals from lignocellulosic biomass, and discovery of synergistic biomass-degrading enzyme paradigms will enable improved conversion processes. Historically, revealing insights into enzymatic saccharification mechanisms on plant cell walls has been hindered by uncharacterized substrates and low resolution imaging techniques. Also, translating findings between model substrates to intact biomass is critical for evaluating enzyme performance. Here we employ a fungal free enzyme cocktail, a complexed cellulosomal system, and a combination of the two to investigate saccharification mechanisms on cellulose I, II and III along with corn stover from Clean Fractionation (CF), which is an Organosolv pretreatment. The insoluble Cellulose Enriched Fraction (CEF) from CF contains mainly cellulose with minor amounts of residual hemicellulose and lignin, the amount of which depends on the CF pretreatment severity. Enzymatic digestions at both low and high-solids loadings demonstrate that CF reduces the amount of enzyme required to depolymerize polysaccharides relative to deacetylated, dilute acid pretreated corn stover. Transmission and scanning electron microscopy of the biomass provides evidence for the different mechanisms of enzymatic deconstruction between free and complexed enzyme systems, and reveals the basis for the synergistic relationship between the two enzyme paradigms on a process-relevant substrate for the first time. These results also demonstrate that the presence of lignin, rather than cellulose morphology, is more detrimental to cellulosome action than to free cellulases. As enzyme costs are a major economic driver for biorefineries, this study provides key inputs for the evaluation of CF as a pretreatment method for biomass conversion.

  11. Asynchronous Group Key Distribution on top of the CC2420 Security Mechanisms for Sensor Networks

    DEFF Research Database (Denmark)

    Hansen, Morten Tranberg

    2009-01-01

    scheme with no time synchronization requirements. The scheme decreases the number of key updates by providing them on an as needed basis according to the amount of network traffic. We evaluate the CC2420 radio security mechanism and show how to use it as a basis to implement secure group communication......A sensor network is a network consisting of small, inexpensive, low-powered sensor nodes that communicate to complete a common task. Sensor nodes are characterized by having limited communication and computation capabilities, energy, and storage. They often are deployed in hostile environments...... creating a demand for encryption and authentication of the messages sent between them. Due to severe resource constraints on the sensor nodes, efficient key distribution schemes and secure communication protocols with low overhead are desired. In this paper we present an asynchronous group key distribution...

  12. Pedigree-based analysis of derivation of genome segments of an elite rice reveals key regions during its breeding.

    Science.gov (United States)

    Zhou, Degui; Chen, Wei; Lin, Zechuan; Chen, Haodong; Wang, Chongrong; Li, Hong; Yu, Renbo; Zhang, Fengyun; Zhen, Gang; Yi, Junliang; Li, Kanghuo; Liu, Yaoguang; Terzaghi, William; Tang, Xiaoyan; He, Hang; Zhou, Shaochuan; Deng, Xing Wang

    2016-02-01

    Analyses of genome variations with high-throughput assays have improved our understanding of genetic basis of crop domestication and identified the selected genome regions, but little is known about that of modern breeding, which has limited the usefulness of massive elite cultivars in further breeding. Here we deploy pedigree-based analysis of an elite rice, Huanghuazhan, to exploit key genome regions during its breeding. The cultivars in the pedigree were resequenced with 7.6× depth on average, and 2.1 million high-quality single nucleotide polymorphisms (SNPs) were obtained. Tracing the derivation of genome blocks with pedigree and information on SNPs revealed the chromosomal recombination during breeding, which showed that 26.22% of Huanghuazhan genome are strictly conserved key regions. These major effect regions were further supported by a QTL mapping of 260 recombinant inbred lines derived from the cross of Huanghuazhan and a very dissimilar cultivar, Shuanggui 36, and by the genome profile of eight cultivars and 36 elite lines derived from Huanghuazhan. Hitting these regions with the cloned genes revealed they include numbers of key genes, which were then applied to demonstrate how Huanghuazhan were bred after 30 years of effort and to dissect the deficiency of artificial selection. We concluded the regions are helpful to the further breeding based on this pedigree and performing breeding by design. Our study provides genetic dissection of modern rice breeding and sheds new light on how to perform genomewide breeding by design. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  13. The autoregulatory loop: A common mechanism of regulation of key sex determining genes in insects

    Indian Academy of Sciences (India)

    Suresh Kumar Kumar; Gajula Gopinath; Nagraj Sambrani; Kallare P Arunkumar

    2016-06-01

    Sex determination in most insects is structured as a gene cascade, wherein a primary signal is passed through a series of sex-determining genes, culminating in a downstream double-switch known as doublesex that decides the sexual fate of the embryo. From the literature available on sex determination cascades, it becomes apparent that sex determination mechanisms have evolved rapidly. The primary signal that provides the cue to determine the sex of the embryo varies remarkably, not only among taxa, but also within taxa. Furthermore, the upstream key gene in the cascade also varies between species and even among closely related species. The order Insecta alone provides examples of astoundingly complex diversity of upstream key genes in sex determination mechanisms. Besides, unlike key upstream genes, the downstream double-switch gene is alternatively spliced to form functional sex-specific isoforms. This sex-specific splicing is conserved across insect taxa. The genes involved in the sex determination cascade such as Sex-lethal (Sxl) in Drosophila melanogaster, transformer (tra) in many other dipterans, coleopterans and hymenopterans, Feminizer (fem) in Apis mellifera, and IGF-II mRNA-binding protein (Bmimp) in Bombyx mori are reported to be regulated by an autoregulatory positive feedback loop. In this review, by taking examples from various insects, we propose the hypothesis that autoregulatory loop mechanisms of sex determination might be a general strategy. We also discuss the possible reasons for the evolution of autoregulatory loops in sex determination cascades and their impact on binary developmental choices.

  14. The autoregulatory loop: A common mechanism of regulation of key sex determining genes in insects.

    Science.gov (United States)

    Sawanth, Suresh Kumar; Gopinath, Gajula; Sambrani, Nagraj; Arunkumar, Kallare P

    2016-06-01

    Sex determination in most insects is structured as a gene cascade, wherein a primary signal is passed through a series of sex-determining genes, culminating in a downstream double-switch known as doublesex that decides the sexual fate of the embryo. From the literature available on sex determination cascades, it becomes apparent that sex determination mechanisms have evolved rapidly. The primary signal that provides the cue to determine the sex of the embryo varies remarkably, not only among taxa, but also within taxa. Furthermore, the upstream key gene in the cascade also varies between species and even among closely related species. The order Insecta alone provides examples of astoundingly complex diversity of upstream key genes in sex determination mechanisms. Besides, unlike key upstream genes, the downstream double-switch gene is alternatively spliced to form functional sex-specific isoforms. This sex-specific splicing is conserved across insect taxa. The genes involved in the sex determination cascade such as Sex-lethal (Sxl) in Drosophila melanogaster, transformer (tra) in many other dipterans, coleopterans and hymenopterans, Feminizer (fem) in Apis mellifera, and IGF-II mRNA-binding protein (Bmimp) in Bombyx mori are reported to be regulated by an autoregulatory positive feedback loop. In this review, by taking examples from various insects, we propose the hypothesis that autoregulatory loop mechanisms of sex determination might be a general strategy. We also discuss the possible reasons for the evolution of autoregulatory loops in sex determination cascades and their impact on binary developmental choices.

  15. Electron flow in reaction mechanisms--revealed from first principles.

    Science.gov (United States)

    Knizia, Gerald; Klein, Johannes E M N

    2015-04-27

    The "curly arrow" of Robinson and Ingold is the primary tool for describing and rationalizing reaction mechanisms. Despite this approach's ubiquity and stellar success, its physical basis has never been clarified and a direct connection to quantum chemistry has never been found. Here we report that the bond rearrangements expressed by curly arrows can be directly observed in ab initio computations, as transformations of intrinsic bond orbitals (IBOs) along the reaction coordinate. Our results clarify that curly arrows are rooted in physical reality-a notion which has been challenged before-and show how quantum chemistry can directly establish reaction mechanisms in intuitive terms and unprecedented detail.

  16. Revealing the complex conduction heat transfer mechanism of nanofluids.

    Science.gov (United States)

    Sergis, A; Hardalupas, Y

    2015-12-01

    Nanofluids are two-phase mixtures consisting of small percentages of nanoparticles (sub 1-10 %vol) inside a carrier fluid. The typical size of nanoparticles is less than 100 nm. These fluids have been exhibiting experimentally a significant increase of thermal performance compared to the corresponding carrier fluids, which cannot be explained using the classical thermodynamic theory. This study deciphers the thermal heat transfer mechanism for the conductive heat transfer mode via a molecular dynamics simulation code. The current findings are the first of their kind and conflict with the proposed theories for heat transfer propagation through micron-sized slurries and pure matter. The authors provide evidence of a complex new type of heat transfer mechanism, which explains the observed abnormal heat transfer augmentation. The new mechanism appears to unite a number of popular speculations for the thermal heat transfer mechanism employed by nanofluids as predicted by the majority of the researchers of the field into a single one. The constituents of the increased diffusivity of the nanoparticle can be attributed to mismatching of the local temperature profiles between parts of the surface of the solid and the fluid resulting in increased local thermophoretic effects. These effects affect the region surrounding the solid manifesting interfacial layer phenomena (Kapitza resistance). In this region, the activity of the fluid and the interactions between the fluid and the nanoparticle are elevated. Isotropic increased nanoparticle mobility is manifested as enhanced Brownian motion and diffusion effects.

  17. Untargeted metabolomics analysis reveals key pathways responsible for the synergistic killing of colistin and doripenem combination against Acinetobacter baumannii

    Science.gov (United States)

    Maifiah, Mohd Hafidz Mahamad; Creek, Darren J.; Nation, Roger L.; Forrest, Alan; Tsuji, Brian T.; Velkov, Tony; Li, Jian

    2017-01-01

    Combination therapy is deployed for the treatment of multidrug-resistant Acinetobacter baumannii, as it can rapidly develop resistance to current antibiotics. This is the first study to investigate the synergistic effect of colistin/doripenem combination on the metabolome of A. baumannii. The metabolite levels were measured using LC-MS following treatment with colistin (2 mg/L) or doripenem (25 mg/L) alone, and their combination at 15 min, 1 hr and 4 hr (n = 4). Colistin caused early (15 min and 1 hr) disruption of the bacterial outer membrane and cell wall, as demonstrated by perturbation of glycerophospholipids and fatty acids. Concentrations of peptidoglycan biosynthesis metabolites decreased at 4 hr by doripenem alone, reflecting its mechanism of action. The combination induced significant changes to more key metabolic pathways relative to either monotherapy. Down-regulation of cell wall biosynthesis (via D-sedoheptulose 7-phosphate) and nucleotide metabolism (via D-ribose 5-phosphate) was associated with perturbations in the pentose phosphate pathway induced initially by colistin (15 min and 1 hr) and later by doripenem (4 hr). We discovered that the combination synergistically killed A. baumannii via time-dependent inhibition of different key metabolic pathways. Our study highlights the significant potential of systems pharmacology in elucidating the mechanism of synergy and optimizing antibiotic pharmacokinetics/pharmacodynamics. PMID:28358014

  18. Dynamic Symmetric Key Mobile Commerce Scheme Based on Self-Verified Mechanism

    Directory of Open Access Journals (Sweden)

    Jiachen Yang

    2014-01-01

    Full Text Available In terms of the security and efficiency of mobile e-commerce, the authors summarized the advantages and disadvantages of several related schemes, especially the self-verified mobile payment scheme based on the elliptic curve cryptosystem (ECC and then proposed a new type of dynamic symmetric key mobile commerce scheme based on self-verified mechanism. The authors analyzed the basic algorithm based on self-verified mechanisms and detailed the complete transaction process of the proposed scheme. The authors analyzed the payment scheme based on the security and high efficiency index. The analysis shows that the proposed scheme not only meets the high efficiency of mobile electronic payment premise, but also takes the security into account. The user confirmation mechanism at the end of the proposed scheme further strengthens the security of the proposed scheme. In brief, the proposed scheme is more efficient and practical than most of the existing schemes.

  19. Key Mechanisms for Employee-Driven Innovation in Governmental Client Organisations

    DEFF Research Database (Denmark)

    Sørensen, Henrik; Wandahl, Søren; Ussing, Lene Faber;

    2016-01-01

    the mechanisms of organisational trust, innovation safety, organisational practices, cultural settings and the recognition and rewards structure to motivate employees. The decision-making procedures should be transparent especially to the idea-owner and the employees involved, and the implementation and putting......In order to ensure that knowledge and ideas are transformed into innovative contributions, a systematic approach is important. The aim of this research was to identify the specific mechanisms that are key to implementing an innovation process model when conducting Employee-driven Innovation...... in a governmental client organisation. The methodological approach was an exploratory case study of a governmental client organisation within the Danish construction industry. The units of analysis were groups of employees and managers within the case organisation. The primary mechanisms identified were...

  20. Multiscale simulations reveal key features of the proton-pumping mechanism in cytochrome c oxidase.

    Science.gov (United States)

    Liang, Ruibin; Swanson, Jessica M J; Peng, Yuxing; Wikström, Mårten; Voth, Gregory A

    2016-07-05

    Cytochrome c oxidase (CcO) reduces oxygen to water and uses the released free energy to pump protons across the membrane. We have used multiscale reactive molecular dynamics simulations to explicitly characterize (with free-energy profiles and calculated rates) the internal proton transport events that enable proton pumping during first steps of oxidation of the fully reduced enzyme. Our results show that proton transport from amino acid residue E286 to both the pump loading site (PLS) and to the binuclear center (BNC) are thermodynamically driven by electron transfer from heme a to the BNC, but that the former (i.e., pumping) is kinetically favored whereas the latter (i.e., transfer of the chemical proton) is rate-limiting. The calculated rates agree with experimental measurements. The backflow of the pumped proton from the PLS to E286 and from E286 to the inside of the membrane is prevented by large free-energy barriers for the backflow reactions. Proton transport from E286 to the PLS through the hydrophobic cavity and from D132 to E286 through the D-channel are found to be strongly coupled to dynamical hydration changes in the corresponding pathways and, importantly, vice versa.

  1. Mechanical Properties of Single Collagen Fibrils Revealed by Force Spectroscopy

    Science.gov (United States)

    Graham, John; Phillips, Charlotte; Grandbois, Michel

    2004-03-01

    In the field of biomechanics, collagen fibrils are believed to be robust mechanical structures characterized by a low extensibility. Until very recently, information on the mechanical properties of collagen fibrils could only be derived from ensemble measurements performed on complete tissues such as bone, skin and tendon. Here we measure force-elongation/relaxation profiles of single collagen fibrils using atomic force microscopy-based force spectroscopy. The elongation profiles indicate that in vitro assembled heterotrimeric type I collagen fibrils are characterized by a large extensibility. Numerous discontinuities and a plateau in the force profile indicate major reorganization occurs within the fibrils in the 1.5 -- 4.5 nN range. Our study demonstrates that newly assembled collagen fibrils are robust structures with a significant reserve of elasticity that could play a determinant role in cellular motion in the context of tissue growth and morphogenesis. In contrast, homotrimeric collagen fibrils corresponding to osteogenesis imperfecta pathology exhibit a marked difference in their elasticity profile.

  2. Striking Plasticity of CRISPR-Cas9 and Key Role of Non-target DNA, as Revealed by Molecular Simulations

    Science.gov (United States)

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system recently emerged as a transformative genome-editing technology that is innovating basic bioscience and applied medicine and biotechnology. The endonuclease Cas9 associates with a guide RNA to match and cleave complementary sequences in double stranded DNA, forming an RNA:DNA hybrid and a displaced non-target DNA strand. Although extensive structural studies are ongoing, the conformational dynamics of Cas9 and its interplay with the nucleic acids during association and DNA cleavage are largely unclear. Here, by employing multi-microsecond time scale molecular dynamics, we reveal the conformational plasticity of Cas9 and identify key determinants that allow its large-scale conformational changes during nucleic acid binding and processing. We show how the “closure” of the protein, which accompanies nucleic acid binding, fundamentally relies on highly coupled and specific motions of the protein domains, collectively initiating the prominent conformational changes needed for nucleic acid association. We further reveal a key role of the non-target DNA during the process of activation of the nuclease HNH domain, showing how the nontarget DNA positioning triggers local conformational changes that favor the formation of a catalytically competent Cas9. Finally, a remarkable conformational plasticity is identified as an intrinsic property of the HNH domain, constituting a necessary element that allows for the HNH repositioning. These novel findings constitute a reference for future experimental studies aimed at a full characterization of the dynamic features of the CRISPR-Cas9 system, and—more importantly—call for novel structure engineering efforts that are of fundamental importance for the rational design of new genome-engineering applications. PMID:27800559

  3. The key microorganisms for anaerobic degradation of pentachlorophenol in paddy soil as revealed by stable isotope probing.

    Science.gov (United States)

    Tong, Hui; Liu, Chengshuai; Li, Fangbai; Luo, Chunling; Chen, Manjia; Hu, Min

    2015-11-15

    Pentachlorophenol (PCP) is a common residual persistent pesticide in paddy soil and has resulted in harmful effect on soil ecosystem. The anaerobic microbial transformation of PCP, therefore, has been received much attentions, especially the functional microbial communities for the reductive transformation. However, the key functional microorganisms for PCP mineralization in the paddy soil still remain unknown. In this work, DNA-based stable isotope probing (SIP) was applied to explore the key microorganisms responsible for PCP mineralization in paddy soil. The SIP results indicated that the dominant bacteria responsible for PCP biodegradation belonged to the genus Dechloromonas of the class β-Proteobacteria. In addition, the increased production of (13)CH4 and (13)CO2 indicated that the addition of lactate enhanced the rate of biodegradation and mineralization of PCP. Two archaea classified as the genera of Methanosaeta and Methanocella of class Methanobacteria were enriched in the heavy fraction when with lactate, whereas no archaea was detected in the absence of lactate. These findings provide direct evidence for the species of bacteria and archaea responsible for anaerobic PCP or its breakdown products mineralization and reveal a new insight into the microorganisms linked with PCP degradation in paddy soil. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. The key microorganisms for anaerobic degradation of pentachlorophenol in paddy soil as revealed by stable isotope probing

    Energy Technology Data Exchange (ETDEWEB)

    Tong, Hui [Guangdong Key Laboratory of Agricultural Environment Pollution Integrated Control, Guangdong Institute of Eco-Environmental and Soil Sciences, Guangzhou 510650 (China); Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Liu, Chengshuai [State Key Laboratory of Environmental Geochemistry, Institute of Geochemistry, Chinese Academy of Sciences, Guiyang 550009 (China); Li, Fangbai, E-mail: cefbli@soil.gd.cn [Guangdong Key Laboratory of Agricultural Environment Pollution Integrated Control, Guangdong Institute of Eco-Environmental and Soil Sciences, Guangzhou 510650 (China); Luo, Chunling [Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640 (China); Chen, Manjia; Hu, Min [Guangdong Key Laboratory of Agricultural Environment Pollution Integrated Control, Guangdong Institute of Eco-Environmental and Soil Sciences, Guangzhou 510650 (China)

    2015-11-15

    Highlights: • SIP suggested that Dechloromonas can mineralize PCP in soil. • Methanosaeta and Methanocella acquired PCP-derived carbon. • Lactate enhanced microbial degradation of PCP in soil. - Abstract: Pentachlorophenol (PCP) is a common residual persistent pesticide in paddy soil and has resulted in harmful effect on soil ecosystem. The anaerobic microbial transformation of PCP, therefore, has been received much attentions, especially the functional microbial communities for the reductive transformation. However, the key functional microorganisms for PCP mineralization in the paddy soil still remain unknown. In this work, DNA-based stable isotope probing (SIP) was applied to explore the key microorganisms responsible for PCP mineralization in paddy soil. The SIP results indicated that the dominant bacteria responsible for PCP biodegradation belonged to the genus Dechloromonas of the class β-Proteobacteria. In addition, the increased production of {sup 13}CH{sub 4} and {sup 13}CO{sub 2} indicated that the addition of lactate enhanced the rate of biodegradation and mineralization of PCP. Two archaea classified as the genera of Methanosaeta and Methanocella of class Methanobacteria were enriched in the heavy fraction when with lactate, whereas no archaea was detected in the absence of lactate. These findings provide direct evidence for the species of bacteria and archaea responsible for anaerobic PCP or its breakdown products mineralization and reveal a new insight into the microorganisms linked with PCP degradation in paddy soil.

  5. Truth-Bonding and Other Truth-Revealing Mechanisms for Courts

    OpenAIRE

    Cooter, Robert D.; Emons, Winand

    2000-01-01

    In trials witnesses often gain by slanting their testimony. The law tries to elicit the truth from witnesses by cross-examination under threat of criminal prosecution for perjury. As a truth-revealing mechanism, perjury law is crude and ineffective. We develop the mathematical form of a perfect truth-revealing mechanism, which exactly offsets the gain from slanted testimony by the risk of a possible sanction. Implementing an effective truth-revealing mechanism requires a witness to certify...

  6. Probing protein flexibility reveals a mechanism for selective promiscuity

    Science.gov (United States)

    Pabon, Nicolas A; Camacho, Carlos J

    2017-01-01

    Many eukaryotic regulatory proteins adopt distinct bound and unbound conformations, and use this structural flexibility to bind specifically to multiple partners. However, we lack an understanding of how an interface can select some ligands, but not others. Here, we present a molecular dynamics approach to identify and quantitatively evaluate the interactions responsible for this selective promiscuity. We apply this approach to the anticancer target PD-1 and its ligands PD-L1 and PD-L2. We discover that while unbound PD-1 exhibits a hard-to-drug hydrophilic interface, conserved specific triggers encoded in the cognate ligands activate a promiscuous binding pathway that reveals a flexible hydrophobic binding cavity. Specificity is then established by additional contacts that stabilize the PD-1 cavity into distinct bound-like modes. Collectively, our studies provide insight into the structural basis and evolution of multiple binding partners, and also suggest a biophysical approach to exploit innate binding pathways to drug seemingly undruggable targets. DOI: http://dx.doi.org/10.7554/eLife.22889.001 PMID:28432789

  7. Molecular Mechanisms Regulating Hepcidin Revealed by Hepcidin Disorders

    Directory of Open Access Journals (Sweden)

    Clara Camaschella

    2011-01-01

    Full Text Available Iron is essential for human life, but toxic if present in excess. To avoid iron overload and maintain iron homeostasis, all cells are able to regulate their iron content through the post-transcriptional control of iron genes operated by the cytosolic iron regulatory proteins that interact with iron responsive elements on iron gene mRNA. At the systemic level, iron homeostasis is regulated by the liver peptide hepcidin. Disruption of these regulatory loops leads to genetic diseases characterized by iron deficiency (iron-refractory iron-deficiency anemia or iron overload (hemochromatosis. Alterations of the same systems are also found in acquired disorders, such as iron-loading anemias characterized by ineffective erythropoiesis and anemia of chronic diseases (ACD associated with common inflammatory conditions. In ACD, iron is present in the body, but maldistributed, being deficient for erythropoiesis, but sequestered in macrophages. Studies of the hepcidin regulation by iron and inflammatory cytokines are revealing new pathways that might become targets of new therapeutic intervention in iron disorders.

  8. Hydroxyapatite as a key biomaterial: quantum-mechanical simulation of its surfaces in interaction with biomolecules.

    Science.gov (United States)

    Corno, Marta; Rimola, Albert; Bolis, Vera; Ugliengo, Piero

    2010-06-28

    Hydroxyapatite is the mineral component of human bones and teeth enamel and is used as synthetic biomaterial. It also grows outside bioglasses as a response of their incorporation in body fluids. The focus is then on understanding the microscopic steps occurring at its surfaces as this allows researchers to understand the key features of biomolecular adhesion. This perspective article deals with in silico simulations of these processes by quantum-mechanical methods based on density functional theory using the hybrid B3LYP functional and Gaussian basis functions.

  9. Dual mechanism of ion permeation through VDAC revealed with inorganic phosphate ions and phosphate metabolites.

    Science.gov (United States)

    Krammer, Eva-Maria; Vu, Giang Thi; Homblé, Fabrice; Prévost, Martine

    2015-01-01

    In the exchange of metabolites and ions between the mitochondrion and the cytosol, the voltage-dependent anion channel (VDAC) is a key element, as it forms the major transport pathway for these compounds through the mitochondrial outer membrane. Numerous experimental studies have promoted the idea that VDAC acts as a regulator of essential mitochondrial functions. In this study, using a combination of molecular dynamics simulations, free-energy calculations, and electrophysiological measurements, we investigated the transport of ions through VDAC, with a focus on phosphate ions and metabolites. We showed that selectivity of VDAC towards small anions including monovalent phosphates arises from short-lived interactions with positively charged residues scattered throughout the pore. In dramatic contrast, permeation of divalent phosphate ions and phosphate metabolites (AMP and ATP) involves binding sites along a specific translocation pathway. This permeation mechanism offers an explanation for the decrease in VDAC conductance measured in the presence of ATP or AMP at physiological salt concentration. The binding sites occur at similar locations for the divalent phosphate ions, AMP and ATP, and contain identical basic residues. ATP features a marked affinity for a central region of the pore lined by two lysines and one arginine of the N-terminal helix. This cluster of residues together with a few other basic amino acids forms a "charged brush" which facilitates the passage of the anionic metabolites through the pore. All of this reveals that VDAC controls the transport of the inorganic phosphates and phosphate metabolites studied here through two different mechanisms.

  10. A molecular phylogeny of Acronychia, Euodia, Melicope and relatives (Rutaceae) reveals polyphyletic genera and key innovations for species richness.

    Science.gov (United States)

    Appelhans, Marc S; Wen, Jun; Wagner, Warren L

    2014-10-01

    We present the first detailed phylogenetic study of the genus Melicope, the largest genus of the Citrus family (Rutaceae). The phylogenetic analysis sampled about 50% of the 235 accepted species of Melicope as well as representatives of 26 related genera, most notably Acronychia and Euodia. The results based on five plastid and nuclear markers have revealed that Acronychia, Euodia and Melicope are each not monophyletic in their current circumscriptions and that several small genera mainly from Australia and New Caledonia need to be merged with one of the three genera to ensure monophyly at the generic level. The phylogenetic position of the drupaceous Acronychia in relation to Melicope, which has capsular or follicular fruits, remains unclear and Acronychia might be a separate genus or a part of Melicope. The seed coats of Melicope, Acronychia and related genera show adaptations to bird-dispersal, which might be regarded as key innovations for species radiations. Euodia and its relatives, which lack these adaptations, include only about 20 species while the Melicope-Acronychia group consists of about 340 species. The drupaceous genera Comptonella, Dutaillyea, Picrella and Sarcomelicope are nested within Melicope and need to be merged with Melicope. The expanded genus is a prime example of the artificial classification system of Engler, who defined Rutaceous subfamilies mainly based on gynoecial and fruit characters.

  11. Genome Alignment Spanning Major Poaceae Lineages Reveals Heterogeneous Evolutionary Rates and Alters Inferred Dates for Key Evolutionary Events.

    Science.gov (United States)

    Wang, Xiyin; Wang, Jingpeng; Jin, Dianchuan; Guo, Hui; Lee, Tae-Ho; Liu, Tao; Paterson, Andrew H

    2015-06-01

    Multiple comparisons among genomes can clarify their evolution, speciation, and functional innovations. To date, the genome sequences of eight grasses representing the most economically important Poaceae (grass) clades have been published, and their genomic-level comparison is an essential foundation for evolutionary, functional, and translational research. Using a formal and conservative approach, we aligned these genomes. Direct comparison of paralogous gene pairs all duplicated simultaneously reveal striking variation in evolutionary rates among whole genomes, with nucleotide substitution slowest in rice and up to 48% faster in other grasses, adding a new dimension to the value of rice as a grass model. We reconstructed ancestral genome contents for major evolutionary nodes, potentially contributing to understanding the divergence and speciation of grasses. Recent fossil evidence suggests revisions of the estimated dates of key evolutionary events, implying that the pan-grass polyploidization occurred ∼96 million years ago and could not be related to the Cretaceous-Tertiary mass extinction as previously inferred. Adjusted dating to reflect both updated fossil evidence and lineage-specific evolutionary rates suggested that maize subgenome divergence and maize-sorghum divergence were virtually simultaneous, a coincidence that would be explained if polyploidization directly contributed to speciation. This work lays a solid foundation for Poaceae translational genomics. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

  12. Deep sequencing of Lotus corniculatus L. reveals key enzymes and potential transcription factors related to the flavonoid biosynthesis pathway.

    Science.gov (United States)

    Wang, Ying; Hua, Wenping; Wang, Jian; Hannoufa, Abdelali; Xu, Ziqin; Wang, Zhezhi

    2013-04-01

    Lotus corniculatus L. is used worldwide as a forage crop due to its abundance of secondary metabolites and its ability to grow in severe environments. Although the entire genome of L. corniculatus var. japonicus R. is being sequenced, the differences in morphology and production of secondary metabolites between these two related species have led us to investigate this variability at the genetic level, in particular the differences in flavonoid biosynthesis. Our goal is to use the resulting information to develop more valuable forage crops and medicinal materials. Here, we conducted Illumina/Solexa sequencing to profile the transcriptome of L. corniculatus. We produced 26,492,952 short reads that corresponded to 2.38 gigabytes of total nucleotides. These reads were then assembled into 45,698 unigenes, of which a large number associated with secondary metabolism were annotated. In addition, we identified 2,998 unigenes based on homology with L. japonicus transcription factors (TFs) and grouped them into 55 families. Meanwhile, a comparison of four tag-based digital gene expression libraries, built from the flowers, pods, leaves, and roots, revealed distinct patterns of spatial expression of candidate unigenes in flavonoid biosynthesis. Based on these results, we identified many key enzymes from L. corniculatus which were different from reference genes of L. japonicus, and five TFs that are potential enhancers in flavonoid biosynthesis. Our results provide initial genetics resources that will be valuable in efforts to manipulate the flavonoid metabolic pathway in plants.

  13. Key Based Mutual Authentication (KBMA Mechanism for Secured Access in MobiCloud Environment

    Directory of Open Access Journals (Sweden)

    Donald A. Cecil

    2016-01-01

    Full Text Available Mobile Cloud Computing (MCC fuels innovation in Mobile Computing and opens new pathways between mobile devices and infrastructures. There are several issues in MCC environment as it integrates various technologies. Among all issues, security lies on the top where many users are not willing to adopt the cloud services. This paper focuses on the authentication. The objective of this paper is to provide a mechanism for authenticating all the entities involved in accessing the cloud services. A mechanism called Key Based Mutual Authentication (KBMA is proposed which is divided into two processes namely registration and authentication. Registration is a one-time process where the users are registered for accessing the cloud services by giving the desired unique information. Authentication process is carried out mutually to verify the identities of Device and Cloud Service Provider (CSP. Scyther tool is used for analysing the vulnerability in terms of attacks. The result claims show that the proposed mechanism is resilient against various attacks.

  14. Key Characteristics of Carcinogens as a Basis for Organizing Data on Mechanisms of Carcinogenesis.

    Science.gov (United States)

    Smith, Martyn T; Guyton, Kathryn Z; Gibbons, Catherine F; Fritz, Jason M; Portier, Christopher J; Rusyn, Ivan; DeMarini, David M; Caldwell, Jane C; Kavlock, Robert J; Lambert, Paul F; Hecht, Stephen S; Bucher, John R; Stewart, Bernard W; Baan, Robert A; Cogliano, Vincent J; Straif, Kurt

    2016-06-01

    A recent review by the International Agency for Research on Cancer (IARC) updated the assessments of the > 100 agents classified as Group 1, carcinogenic to humans (IARC Monographs Volume 100, parts A-F). This exercise was complicated by the absence of a broadly accepted, systematic method for evaluating mechanistic data to support conclusions regarding human hazard from exposure to carcinogens. IARC therefore convened two workshops in which an international Working Group of experts identified 10 key characteristics, one or more of which are commonly exhibited by established human carcinogens. These characteristics provide the basis for an objective approach to identifying and organizing results from pertinent mechanistic studies. The 10 characteristics are the abilities of an agent to 1) act as an electrophile either directly or after metabolic activation; 2) be genotoxic; 3) alter DNA repair or cause genomic instability; 4) induce epigenetic alterations; 5) induce oxidative stress; 6) induce chronic inflammation; 7) be immunosuppressive; 8) modulate receptor-mediated effects; 9) cause immortalization; and 10) alter cell proliferation, cell death, or nutrient supply. We describe the use of the 10 key characteristics to conduct a systematic literature search focused on relevant end points and construct a graphical representation of the identified mechanistic information. Next, we use benzene and polychlorinated biphenyls as examples to illustrate how this approach may work in practice. The approach described is similar in many respects to those currently being implemented by the U.S. EPA's Integrated Risk Information System Program and the U.S. National Toxicology Program. Smith MT, Guyton KZ, Gibbons CF, Fritz JM, Portier CJ, Rusyn I, DeMarini DM, Caldwell JC, Kavlock RJ, Lambert P, Hecht SS, Bucher JR, Stewart BW, Baan R, Cogliano VJ, Straif K. 2016. Key characteristics of carcinogens as a basis for organizing data on mechanisms of carcinogenesis. Environ Health

  15. Key Characteristics of Carcinogens as a Basis for Organizing Data on Mechanisms of Carcinogenesis

    Science.gov (United States)

    Smith, Martyn T.; Guyton, Kathryn Z.; Gibbons, Catherine F.; Fritz, Jason M.; Portier, Christopher J.; Rusyn, Ivan; DeMarini, David M.; Caldwell, Jane C.; Kavlock, Robert J.; Lambert, Paul F.; Hecht, Stephen S.; Bucher, John R.; Stewart, Bernard W.; Baan, Robert A.; Cogliano, Vincent J.; Straif, Kurt

    2015-01-01

    Background: A recent review by the International Agency for Research on Cancer (IARC) updated the assessments of the > 100 agents classified as Group 1, carcinogenic to humans (IARC Monographs Volume 100, parts A–F). This exercise was complicated by the absence of a broadly accepted, systematic method for evaluating mechanistic data to support conclusions regarding human hazard from exposure to carcinogens. Objectives and Methods: IARC therefore convened two workshops in which an international Working Group of experts identified 10 key characteristics, one or more of which are commonly exhibited by established human carcinogens. Discussion: These characteristics provide the basis for an objective approach to identifying and organizing results from pertinent mechanistic studies. The 10 characteristics are the abilities of an agent to 1) act as an electrophile either directly or after metabolic activation; 2) be genotoxic; 3) alter DNA repair or cause genomic instability; 4) induce epigenetic alterations; 5) induce oxidative stress; 6) induce chronic inflammation; 7) be immunosuppressive; 8) modulate receptor-mediated effects; 9) cause immortalization; and 10) alter cell proliferation, cell death, or nutrient supply. Conclusion: We describe the use of the 10 key characteristics to conduct a systematic literature search focused on relevant end points and construct a graphical representation of the identified mechanistic information. Next, we use benzene and polychlorinated biphenyls as examples to illustrate how this approach may work in practice. The approach described is similar in many respects to those currently being implemented by the U.S. EPA’s Integrated Risk Information System Program and the U.S. National Toxicology Program. Citation: Smith MT, Guyton KZ, Gibbons CF, Fritz JM, Portier CJ, Rusyn I, DeMarini DM, Caldwell JC, Kavlock RJ, Lambert P, Hecht SS, Bucher JR, Stewart BW, Baan R, Cogliano VJ, Straif K. 2016. Key characteristics of carcinogens as a

  16. Constant-net-time headway as a key mechanism behind pedestrian flow dynamics.

    Science.gov (United States)

    Johansson, Anders

    2009-08-01

    We show that keeping a constant lower limit on the net-time headway is the key mechanism behind the dynamics of pedestrian streams. There is a large variety in flow and speed as functions of density for empirical data of pedestrian streams obtained from studies in different countries. The net-time headway, however, stays approximately constant over all these different data sets. By using this fact, we demonstrate how the underlying dynamics of pedestrian crowds, naturally follows from local interactions. This means that there is no need to come up with an arbitrary fit function (with arbitrary fit parameters) as has traditionally been done. Further, by using not only the average density values but the variance as well, we show how the recently reported stop-and-go waves [Helbing, Phys. Rev. E 75, 046109 (2007)] emerge when local density variations take values exceeding a certain maximum global (average) density, which makes pedestrians stop.

  17. A Face Centered Cubic Key Agreement Mechanism for Mobile Ad Hoc Networks

    Science.gov (United States)

    Askoxylakis, Ioannis G.; Markantonakis, Konstantinos; Tryfonas, Theo; May, John; Traganitis, Apostolos

    Mobile ad hoc networking is an operating mode for rapid mobile node networking. Each node relies on adjacent nodes in order to achieve and maintain connectivity and functionality. Security is considered among the main issues for the successful deployment of mobile ad hoc networks (MANETs). In this paper we introduce a weak to strong authentication mechanism associated with a multiparty contributory key establishment method. The latter is designed for MANETs with dynamic changing topologies, due to continuous flow of incoming and departing nodes. We introduce a new cube algorithm based on the face-centered cubic (FCC) structure. The proposed architecture employs elliptic curve cryptography, which is considered more efficient for thin clients where processing power and energy consumption are significant constraints.

  18. Transcriptome analysis of tomato flower pedicel tissues reveals abscission zone-specific modulation of key meristem activity genes.

    Directory of Open Access Journals (Sweden)

    Xiang Wang

    Full Text Available Tomato flower abscises at the anatomically distinct abscission zone that separates the pedicel into basal and apical portions. During abscission, cell separation occurs only at the abscission zone indicating distinctive molecular regulation in its cells. We conducted a transcriptome analysis of tomato pedicel tissues during ethylene promoted abscission. We found that the abscission zone was the most active site with the largest set of differentially expressed genes when compared with basal and apical portions. Gene Ontology analyses revealed enriched transcription regulation and hydrolase activities in the abscission zone. We also demonstrate coordinated responses of hormone and cell wall related genes. Besides, a number of ESTs representing homologs of key Arabidopsis shoot apical meristem activity genes were found to be preferentially expressed in the abscission zone, including WUSCHEL (WUS, KNAT6, LATERAL ORGAN BOUNDARIES DOMAIN PROTEIN 1(LBD1, and BELL-like homeodomain protein 1 (BLH1, as well as tomato axillary meristem genes BLIND (Bl and LATERAL SUPPRESSOR (Ls. More interestingly, the homologs of WUS and the potential functional partner OVATE FAMILIY PROTEIN (OFP were subsequently down regulated during abscission while Bl and AGL12 were continuously and specifically induced in the abscission zone. The expression patterns of meristem activity genes corroborate the idea that cells of the abscission zone confer meristem-like nature and coincide with the course of abscission and post-abscission cell differentiation. Our data therefore propose a possible regulatory scheme in tomato involving meristem genes that may be required not only for the abscission zone development, but also for abscission.

  19. Representing Microbial Dormancy in Soil Decomposition Models Improves Model Performance and Reveals Key Ecosystem Controls on Microbial Activity

    Science.gov (United States)

    He, Y.; Yang, J.; Zhuang, Q.; Wang, G.; Liu, Y.

    2014-12-01

    Climate feedbacks from soils can result from environmental change and subsequent responses of plant and microbial communities and nutrient cycling. Explicit consideration of microbial life history traits and strategy may be necessary to predict climate feedbacks due to microbial physiology and community changes and their associated effect on carbon cycling. In this study, we developed an explicit microbial-enzyme decomposition model and examined model performance with and without representation of dormancy at six temperate forest sites with observed soil efflux ranged from 4 to 10 years across different forest types. We then extrapolated the model to all temperate forests in the Northern Hemisphere (25-50°N) to investigate spatial controls on microbial and soil C dynamics. Both models captured the observed soil heterotrophic respiration (RH), yet no-dormancy model consistently exhibited large seasonal amplitude and overestimation in microbial biomass. Spatially, the total RH from temperate forests based on dormancy model amounts to 6.88PgC/yr, and 7.99PgC/yr based on no-dormancy model. However, no-dormancy model notably overestimated the ratio of microbial biomass to SOC. Spatial correlation analysis revealed key controls of soil C:N ratio on the active proportion of microbial biomass, whereas local dormancy is primarily controlled by soil moisture and temperature, indicating scale-dependent environmental and biotic controls on microbial and SOC dynamics. These developments should provide essential support to modeling future soil carbon dynamics and enhance the avenue for collaboration between empirical soil experiment and modeling in the sense that more microbial physiological measurements are needed to better constrain and evaluate the models.

  20. Transcriptome analysis of tomato flower pedicel tissues reveals abscission zone-specific modulation of key meristem activity genes.

    Science.gov (United States)

    Wang, Xiang; Liu, Danmei; Li, Aili; Sun, Xiuli; Zhang, Rongzhi; Wu, Liang; Liang, Yanchun; Mao, Long

    2013-01-01

    Tomato flower abscises at the anatomically distinct abscission zone that separates the pedicel into basal and apical portions. During abscission, cell separation occurs only at the abscission zone indicating distinctive molecular regulation in its cells. We conducted a transcriptome analysis of tomato pedicel tissues during ethylene promoted abscission. We found that the abscission zone was the most active site with the largest set of differentially expressed genes when compared with basal and apical portions. Gene Ontology analyses revealed enriched transcription regulation and hydrolase activities in the abscission zone. We also demonstrate coordinated responses of hormone and cell wall related genes. Besides, a number of ESTs representing homologs of key Arabidopsis shoot apical meristem activity genes were found to be preferentially expressed in the abscission zone, including WUSCHEL (WUS), KNAT6, LATERAL ORGAN BOUNDARIES DOMAIN PROTEIN 1(LBD1), and BELL-like homeodomain protein 1 (BLH1), as well as tomato axillary meristem genes BLIND (Bl) and LATERAL SUPPRESSOR (Ls). More interestingly, the homologs of WUS and the potential functional partner OVATE FAMILIY PROTEIN (OFP) were subsequently down regulated during abscission while Bl and AGL12 were continuously and specifically induced in the abscission zone. The expression patterns of meristem activity genes corroborate the idea that cells of the abscission zone confer meristem-like nature and coincide with the course of abscission and post-abscission cell differentiation. Our data therefore propose a possible regulatory scheme in tomato involving meristem genes that may be required not only for the abscission zone development, but also for abscission.

  1. Structure of the trehalose-6-phosphate phosphatase from Brugia malayi reveals key design principles for anthelmintic drugs.

    Directory of Open Access Journals (Sweden)

    Jeremiah D Farelli

    2014-07-01

    Full Text Available Parasitic nematodes are responsible for devastating illnesses that plague many of the world's poorest populations indigenous to the tropical areas of developing nations. Among these diseases is lymphatic filariasis, a major cause of permanent and long-term disability. Proteins essential to nematodes that do not have mammalian counterparts represent targets for therapeutic inhibitor discovery. One promising target is trehalose-6-phosphate phosphatase (T6PP from Brugia malayi. In the model nematode Caenorhabditis elegans, T6PP is essential for survival due to the toxic effect(s of the accumulation of trehalose 6-phosphate. T6PP has also been shown to be essential in Mycobacterium tuberculosis. We determined the X-ray crystal structure of T6PP from B. malayi. The protein structure revealed a stabilizing N-terminal MIT-like domain and a catalytic C-terminal C2B-type HAD phosphatase fold. Structure-guided mutagenesis, combined with kinetic analyses using a designed competitive inhibitor, trehalose 6-sulfate, identified five residues important for binding and catalysis. This structure-function analysis along with computational mapping provided the basis for the proposed model of the T6PP-trehalose 6-phosphate complex. The model indicates a substrate-binding mode wherein shape complementarity and van der Waals interactions drive recognition. The mode of binding is in sharp contrast to the homolog sucrose-6-phosphate phosphatase where extensive hydrogen-bond interactions are made to the substrate. Together these results suggest that high-affinity inhibitors will be bi-dentate, taking advantage of substrate-like binding to the phosphoryl-binding pocket while simultaneously utilizing non-native binding to the trehalose pocket. The conservation of the key residues that enforce the shape of the substrate pocket in T6PP enzymes suggest that development of broad-range anthelmintic and antibacterial therapeutics employing this platform may be possible.

  2. Target Organ Metabolism, Toxicity, and Mechanisms of Trichloroethylene and Perchloroethylene: Key Similarities, Differences, and Data Gaps.

    Science.gov (United States)

    Cichocki, Joseph A; Guyton, Kathryn Z; Guha, Neela; Chiu, Weihsueh A; Rusyn, Ivan; Lash, Lawrence H

    2016-10-01

    Trichloroethylene (TCE) and perchloroethylene or tetrachloroethylene (PCE) are high-production volume chemicals with numerous industrial applications. As a consequence of their widespread use, these chemicals are ubiquitous environmental contaminants to which the general population is commonly exposed. It is widely assumed that TCE and PCE are toxicologically similar; both are simple olefins with three (TCE) or four (PCE) chlorines. Nonetheless, despite decades of research on the adverse health effects of TCE or PCE, few studies have directly compared these two toxicants. Although the metabolic pathways are qualitatively similar, quantitative differences in the flux and yield of metabolites exist. Recent human health assessments have uncovered some overlap in target organs that are affected by exposure to TCE or PCE, and divergent species- and sex-specificity with regard to cancer and noncancer hazards. The objective of this minireview is to highlight key similarities, differences, and data gaps in target organ metabolism and mechanism of toxicity. The main anticipated outcome of this review is to encourage research to 1) directly compare the responses to TCE and PCE using more sensitive biochemical techniques and robust statistical comparisons; 2) more closely examine interindividual variability in the relationship between toxicokinetics and toxicodynamics for TCE and PCE; 3) elucidate the effect of coexposure to these two toxicants; and 4) explore new mechanisms for target organ toxicity associated with TCE and/or PCE exposure. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  3. Steady streaming: A key mixing mechanism in low-Reynolds-number acinar flows

    Science.gov (United States)

    Kumar, Haribalan; Tawhai, Merryn H.; Hoffman, Eric A.; Lin, Ching-Long

    2011-01-01

    Study of mixing is important in understanding transport of submicron sized particles in the acinar region of the lung. In this article, we investigate transport in view of advective mixing utilizing Lagrangian particle tracking techniques: tracer advection, stretch rate and dispersion analysis. The phenomenon of steady streaming in an oscillatory flow is found to hold the key to the origin of kinematic mixing in the alveolus, the alveolar mouth and the alveolated duct. This mechanism provides the common route to folding of material lines and surfaces in any region of the acinar flow, and has no bearing on whether the geometry is expanding or if flow separates within the cavity or not. All analyses consistently indicate a significant decrease in mixing with decreasing Reynolds number (Re). For a given Re, dispersion is found to increase with degree of alveolation, indicating that geometry effects are important. These effects of Re and geometry can also be explained by the streaming mechanism. Based on flow conditions and resultant convective mixing measures, we conclude that significant convective mixing in the duct and within an alveolus could originate only in the first few generations of the acinar tree as a result of nonzero inertia, flow asymmetry, and large Keulegan–Carpenter (KC) number. PMID:21580803

  4. Actin polymerization as a key innate immune effector mechanism to control Salmonella infection.

    Science.gov (United States)

    Man, Si Ming; Ekpenyong, Andrew; Tourlomousis, Panagiotis; Achouri, Sarra; Cammarota, Eugenia; Hughes, Katherine; Rizzo, Alessandro; Ng, Gilbert; Wright, John A; Cicuta, Pietro; Guck, Jochen R; Bryant, Clare E

    2014-12-09

    Salmonellosis is one of the leading causes of food poisoning worldwide. Controlling bacterial burden is essential to surviving infection. Nucleotide-binding oligomerization domain-like receptors (NLRs), such as NLRC4, induce inflammasome effector functions and play a crucial role in controlling Salmonella infection. Inflammasome-dependent production of IL-1β recruits additional immune cells to the site of infection, whereas inflammasome-mediated pyroptosis of macrophages releases bacteria for uptake by neutrophils. Neither of these functions is known to directly kill intracellular salmonellae within macrophages. The mechanism, therefore, governing how inflammasomes mediate intracellular bacterial-killing and clearance in host macrophages remains unknown. Here, we show that actin polymerization is required for NLRC4-dependent regulation of intracellular bacterial burden, inflammasome assembly, pyroptosis, and IL-1β production. NLRC4-induced changes in actin polymerization are physically manifested as increased cellular stiffness, and leads to reduced bacterial uptake, production of antimicrobial molecules, and arrested cellular migration. These processes act in concert to limit bacterial replication in the cell and dissemination in tissues. We show, therefore, a functional link between innate immunity and actin turnover in macrophages that underpins a key host defense mechanism for the control of salmonellosis.

  5. Domain Motions and Functionally-Key Residues of l-Alanine Dehydrogenase Revealed by an Elastic Network Model

    Directory of Open Access Journals (Sweden)

    Xing-Yuan Li

    2015-12-01

    Full Text Available Mycobacterium tuberculosis l-alanine dehydrogenase (l-MtAlaDH plays an important role in catalyzing l-alanine to ammonia and pyruvate, which has been considered to be a potential target for tuberculosis treatment. In the present work, the functional domain motions encoded in the structure of l-MtAlaDH were investigated by using the Gaussian network model (GNM and the anisotropy network model (ANM. The slowest modes for the open-apo and closed-holo structures of the enzyme show that the domain motions have a common hinge axis centered in residues Met133 and Met301. Accompanying the conformational transition, both the 1,4-dihydronicotinamide adenine dinucleotide (NAD-binding domain (NBD and the substrate-binding domain (SBD move in a highly coupled way. The first three slowest modes of ANM exhibit the open-closed, rotation and twist motions of l-MtAlaDH, respectively. The calculation of the fast modes reveals the residues responsible for the stability of the protein, and some of them are involved in the interaction with the ligand. Then, the functionally-important residues relevant to the binding of the ligand were identified by using a thermodynamic method. Our computational results are consistent with the experimental data, which will help us to understand the physical mechanism for the function of l-MtAlaDH.

  6. Metatranscriptomics reveals the molecular mechanism of large granule formation in granular anammox reactor

    KAUST Repository

    Bagchi, Samik

    2016-06-20

    Granules enriched with anammox bacteria are essential in enhancing the treatment of ammonia-rich wastewater, but little is known about how anammox bacteria grow and multiply inside granules. Here, we combined metatranscriptomics, quantitative PCR and 16S rRNA gene sequencing to study the changes in community composition, metabolic gene content and gene expression in a granular anammox reactor with the objective of understanding the molecular mechanism of anammox growth and multiplication that led to formation of large granules. Size distribution analysis revealed the spatial distribution of granules in which large granules having higher abundance of anammox bacteria (genus Brocadia) dominated the bottom biomass. Metatranscriptomics analysis detected all the essential transcripts for anammox metabolism. During the later stage of reactor operation, higher expression of ammonia and nitrite transport proteins and key metabolic enzymes mainly in the bottom large granules facilitated anammox bacteria activity. The high activity resulted in higher growth and multiplication of anammox bacteria and expanded the size of the granules. This conceptual model for large granule formation proposed here may assist in the future design of anammox processes for mainstream wastewater treatment.

  7. Metatranscriptomics reveals the molecular mechanism of large granule formation in granular anammox reactor

    Science.gov (United States)

    Bagchi, Samik; Lamendella, Regina; Strutt, Steven; van Loosdrecht, Mark C. M.; Saikaly, Pascal E.

    2016-06-01

    Granules enriched with anammox bacteria are essential in enhancing the treatment of ammonia-rich wastewater, but little is known about how anammox bacteria grow and multiply inside granules. Here, we combined metatranscriptomics, quantitative PCR and 16S rRNA gene sequencing to study the changes in community composition, metabolic gene content and gene expression in a granular anammox reactor with the objective of understanding the molecular mechanism of anammox growth and multiplication that led to formation of large granules. Size distribution analysis revealed the spatial distribution of granules in which large granules having higher abundance of anammox bacteria (genus Brocadia) dominated the bottom biomass. Metatranscriptomics analysis detected all the essential transcripts for anammox metabolism. During the later stage of reactor operation, higher expression of ammonia and nitrite transport proteins and key metabolic enzymes mainly in the bottom large granules facilitated anammox bacteria activity. The high activity resulted in higher growth and multiplication of anammox bacteria and expanded the size of the granules. This conceptual model for large granule formation proposed here may assist in the future design of anammox processes for mainstream wastewater treatment.

  8. Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography.

    Science.gov (United States)

    Fukuda, Yohta; Tse, Ka Man; Nakane, Takanori; Nakatsu, Toru; Suzuki, Mamoru; Sugahara, Michihiro; Inoue, Shigeyuki; Masuda, Tetsuya; Yumoto, Fumiaki; Matsugaki, Naohiro; Nango, Eriko; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Song, Changyong; Hatsui, Takaki; Yabashi, Makina; Nureki, Osamu; Murphy, Michael E P; Inoue, Tsuyoshi; Iwata, So; Mizohata, Eiichi

    2016-03-15

    Proton-coupled electron transfer (PCET), a ubiquitous phenomenon in biological systems, plays an essential role in copper nitrite reductase (CuNiR), the key metalloenzyme in microbial denitrification of the global nitrogen cycle. Analyses of the nitrite reduction mechanism in CuNiR with conventional synchrotron radiation crystallography (SRX) have been faced with difficulties, because X-ray photoreduction changes the native structures of metal centers and the enzyme-substrate complex. Using serial femtosecond crystallography (SFX), we determined the intact structures of CuNiR in the resting state and the nitrite complex (NC) state at 2.03- and 1.60-Å resolution, respectively. Furthermore, the SRX NC structure representing a transient state in the catalytic cycle was determined at 1.30-Å resolution. Comparison between SRX and SFX structures revealed that photoreduction changes the coordination manner of the substrate and that catalytically important His255 can switch hydrogen bond partners between the backbone carbonyl oxygen of nearby Glu279 and the side-chain hydroxyl group of Thr280. These findings, which SRX has failed to uncover, propose a redox-coupled proton switch for PCET. This concept can explain how proton transfer to the substrate is involved in intramolecular electron transfer and why substrate binding accelerates PCET. Our study demonstrates the potential of SFX as a powerful tool to study redox processes in metalloenzymes.

  9. Edge-melting: nanoscale key-mechanism to explain nanoparticle formation from heated TEM grids

    Energy Technology Data Exchange (ETDEWEB)

    Cesaria, Maura, E-mail: maura.cesaria@le.infn.it [Department of Mathematics and Physics “E. De Giorgi”, University of Salento, Via Arnesano, 73100 Lecce (Italy); Taurino, Antonietta; Catalano, Massimo [Institute for Microelectronics and Microsystems, IMM-CNR, Via Monteroni, 73100 Lecce (Italy); Caricato, Anna Paola; Martino, Maurizio [Department of Mathematics and Physics “E. De Giorgi”, University of Salento, Via Arnesano, 73100 Lecce (Italy)

    2016-03-01

    Graphical abstract: - Highlights: • Nanoparticle formation from metal grids explained by edge melting as key mechanism. • The inconsistency of bulk phenomenology invoking the vapor pressure is discussed. • Surface-melting and size-dependent evaporation are questioned as unsatisfactory. • Edge-melting: edges, corners, facets invoked as highly thermally unstable surfaces. • The polycrystalline nature of the really occurring metal grids is accounted for. - Abstract: In this study, we examine at both experimental and fundamental levels, the experimental evidence of nanoparticle formation in transmission electron microscopy (TEM) metal grids annealed at temperatures lower than the melting point of the corresponding metal bulk material. Our experimental investigation considers the most thermally unstable TEM grids (i.e. Cu-grids) and inspects the possible sources and mechanisms of contamination of thin films, conventionally deposited on carbon-coated Cu-grids. The investigations are supported by morphological–compositional analyses performed in different regions of the TEM sample. Then, a general model is formulated and discussed in order to explain the grid thermal instability, based on the critical role of edge-melting (i.e. melting initiated at edges and corners of the grid bars), the enhanced rate of evaporation from a liquid surface and the polycristallinity of the grid bars. Hence, we totally disregard conventional arguments such as bulk evaporation and metal vapor pressure and, in order to emphasize and clarify the alternative point of view of our model, we also overview the nano-scale melting phenomenology relevant to our discussion and survey the discrepancies reported in the literature.

  10. Inhibition of VEGF: a novel mechanism to control angiogenesis by Withania somnifera's key metabolite Withaferin A.

    Science.gov (United States)

    Saha, Sanjib; Islam, Md Khirul; Shilpi, Jamil A; Hasan, Shihab

    2013-01-01

    Angiogenesis, or new blood vessel formation from existing one, plays both beneficial and detrimental roles in living organisms in different aspects. Vascular endothelial growth factor (VEGF), a signal protein, well established as key regulator of vasculogenesis and angiogenesis. VEGF ensures oxygen supply to the tissues when blood supply is not adequate, or tissue environment is in hypoxic condition. Limited expression of VEGF is necessary, but if it is over expressed, then it can lead to serious disease like cancer. Cancers that have ability to express VEGF are more efficient to grow and metastasize because solid cancers cannot grow larger than a limited size without adequate blood and oxygen supply. Anti-VEGF drugs are already available in the market to control angiogenesis, but they are often associated with severe side-effects like fetal bleeding and proteinuria in the large number of patients. To avoid such side-effects, new insight is required to find potential compounds as anti-VEGF from natural sources. In the present investigation, molecular docking studies were carried out to find the potentiality of Withaferin A, a key metabolite of Withania somnifera, as an inhibitor of VEGF. Molecular Docking studies were performed in DockingServer and SwissDock. Bevacizumab, a commercial anti-VEGF drug, was used as reference to compare the activity of Withaferin A. X-ray crystallographic structure of VEGF, was retrieved from Protein Data Bank (PDB), and used as drug target protein. Structure of Withaferin A and Bevacizumab was obtained from PubChem and ZINC databases. Molecular visualization was performed using UCSF Chimera. Withaferin A showed favorable binding with VEGF with low binding energy in comparison to Bevacizumab. Molecular Docking studies also revealed potential protein-ligand interactions for both Withaferin A and Bevacizumab. Conclusively our results strongly suggest that Withaferin A is a potent anti-VEGF agent as ascertained by its potential

  11. Mechanism of the superior mechanical strength of nanometer-sized metal single crystals revealed

    KAUST Repository

    Afify, N. D.

    2013-10-01

    Clear understanding of the superior mechanical strength of nanometer-sized metal single crystals is required to derive advanced mechanical components retaining such superiority. Although high quality studies have been reported on nano-crystalline metals, the superiority of small single crystals has neither been fundamentally explained nor quantified to this date. Here we present a molecular dynamics study of aluminum single crystals in the size range from 4.1 nm to 40.5 nm. We show that the ultimate mechanical strength deteriorates exponentially as the single crystal size increases. The small crystals superiority is explained by their ability to continuously form vacancies and to recover them. © 2013 Published by Elsevier B.V.

  12. Severe inflammatory reaction induced by peritoneal trauma is the key driving mechanism of postoperative adhesion formation

    Directory of Open Access Journals (Sweden)

    Pismensky Sergei V

    2011-11-01

    Full Text Available Abstract Background Many factors have been put forward as a driving mechanism of surgery-triggered adhesion formation (AF. In this study, we underline the key role of specific surgical trauma related with open surgery (OS and laparoscopic (LS conditions in postoperative AF and we aimed to study peritoneal tissue inflammatory reaction (TIR, remodelling specific complications of open surgery (OS versus LS and subsequently evaluating AF induced by these conditions. Methods A prospective randomized study was done in 80 anaesthetised female Wistar rats divided equally into 2 groups. Specific traumatic OS conditions were induced by midline incision line (MIL extension and tissue drying and specific LS conditions were remodelled by intraperitoneal CO2 insufflation at the 10 cm of water. TIR was evaluated at the 24th, 72nd, 120th and 168th hour by scoring scale. Statistical analysis was performed by the non-parametric t test and two-way ANOVA using Bonferroni post-tests. Results More pronounced residual TIR was registered after OS than after LS. There were no significant TIR interactions though highly significant differences were observed between the OS and LS groups (p th and 72nd; p th and p th hrs. Adhesion free wounds were observed in 20.0 and 31.0% of cases after creation of OS and LS conditions respectively; with no significant differences between these values (p > 0.05. However larger adhesion size (41.67 ± 33.63 was observed after OS in comparison with LS (20.31 ± 16.38. The upper-lower 95% confidential limits ranged from 60.29 to 23.04 and from 29.04 to 11.59 respectively after OS and LS groups with significant differences (p = 0.03. Analogous changes were observed in adhesion severity values. Subsequently, severe TIR parameters were followed by larger sizes of severe postoperative adhesions in the OS group than those observed in the LS group. Conclusions MIL extension and tissue drying seem to be the key factors in the pathogenesis of

  13. The transcriptional programme of Salmonella enterica serovar Typhimurium reveals a key role for tryptophan metabolism in biofilms

    Directory of Open Access Journals (Sweden)

    Cochrane Brett

    2009-12-01

    Full Text Available Abstract Background Biofilm formation enhances the capacity of pathogenic Salmonella bacteria to survive stresses that are commonly encountered within food processing and during host infection. The persistence of Salmonella within the food chain has become a major health concern, as biofilms can serve as a reservoir for the contamination of food products. While the molecular mechanisms required for the survival of bacteria on surfaces are not fully understood, transcriptional studies of other bacteria have demonstrated that biofilm growth triggers the expression of specific sets of genes, compared with planktonic cells. Until now, most gene expression studies of Salmonella have focused on the effect of infection-relevant stressors on virulence or the comparison of mutant and wild-type bacteria. However little is known about the physiological responses taking place inside a Salmonella biofilm. Results We have determined the transcriptomic and proteomic profiles of biofilms of Salmonella enterica serovar Typhimurium. We discovered that 124 detectable proteins were differentially expressed in the biofilm compared with planktonic cells, and that 10% of the S. Typhimurium genome (433 genes showed a 2-fold or more change in the biofilm compared with planktonic cells. The genes that were significantly up-regulated implicated certain cellular processes in biofilm development including amino acid metabolism, cell motility, global regulation and tolerance to stress. We found that the most highly down-regulated genes in the biofilm were located on Salmonella Pathogenicity Island 2 (SPI2, and that a functional SPI2 secretion system regulator (ssrA was required for S. Typhimurium biofilm formation. We identified STM0341 as a gene of unknown function that was needed for biofilm growth. Genes involved in tryptophan (trp biosynthesis and transport were up-regulated in the biofilm. Deletion of trpE led to decreased bacterial attachment and this biofilm defect

  14. The transcriptional programme of Salmonella enterica serovar Typhimurium reveals a key role for tryptophan metabolism in biofilms.

    LENUS (Irish Health Repository)

    Hamilton, Shea

    2009-12-11

    Abstract Background Biofilm formation enhances the capacity of pathogenic Salmonella bacteria to survive stresses that are commonly encountered within food processing and during host infection. The persistence of Salmonella within the food chain has become a major health concern, as biofilms can serve as a reservoir for the contamination of food products. While the molecular mechanisms required for the survival of bacteria on surfaces are not fully understood, transcriptional studies of other bacteria have demonstrated that biofilm growth triggers the expression of specific sets of genes, compared with planktonic cells. Until now, most gene expression studies of Salmonella have focused on the effect of infection-relevant stressors on virulence or the comparison of mutant and wild-type bacteria. However little is known about the physiological responses taking place inside a Salmonella biofilm. Results We have determined the transcriptomic and proteomic profiles of biofilms of Salmonella enterica serovar Typhimurium. We discovered that 124 detectable proteins were differentially expressed in the biofilm compared with planktonic cells, and that 10% of the S. Typhimurium genome (433 genes) showed a 2-fold or more change in the biofilm compared with planktonic cells. The genes that were significantly up-regulated implicated certain cellular processes in biofilm development including amino acid metabolism, cell motility, global regulation and tolerance to stress. We found that the most highly down-regulated genes in the biofilm were located on Salmonella Pathogenicity Island 2 (SPI2), and that a functional SPI2 secretion system regulator (ssrA) was required for S. Typhimurium biofilm formation. We identified STM0341 as a gene of unknown function that was needed for biofilm growth. Genes involved in tryptophan (trp) biosynthesis and transport were up-regulated in the biofilm. Deletion of trpE led to decreased bacterial attachment and this biofilm defect was restored by

  15. Acquisition of docetaxel resistance in breast cancer cells reveals upregulation of ABCB1 expression as a key mediator of resistance accompanied by discrete upregulation of other specific genes and pathways

    DEFF Research Database (Denmark)

    Ninel Hansen, Stine; Westergaard, David; Borg Houlberg Thomsen, Mathilde

    2015-01-01

    to be prominent at higher docetaxel concentrations (second-phase response). Additional resistance mechanisms were indicated by gene expression profiling, including genes in the interferon-inducible protein family in MCF7RES and cancer testis antigen family in MDARES. Also, upregulated expression of various ABC...... resistance and thereby identify key molecular mechanisms and predictive molecular characteristics to docetaxel resistance. Two docetaxel-resistant cell lines, MCF7RES and MDARES, were generated from their respective parental cell lines MCF-7 and MDA-MB-231 by stepwise selection in docetaxel dose increments...... analysis singled out ABCB1, which encodes permeability glycoprotein (Pgp), as the top upregulated gene in both MCF7RES and MDARES. Functional validation revealed Pgp as a key resistance mediator at low docetaxel concentrations (first-phase response), whereas additional resistance mechanisms appeared...

  16. Mitochondrial glutathione depletion reveals a novel role for the pyruvate dehydrogenase complex as a key H2O2 emitting source under conditions of nutrient overload

    Science.gov (United States)

    Fisher-Wellman, Kelsey H.; Gilliam, Laura A. A.; Lin, Chien-Te; Cathey, Brook L.; Lark, Daniel S.; Neufer, P. Darrell

    2014-01-01

    Once regarded as “byproducts” of aerobic metabolism, the production of superoxide/H2O2 is now understood to be a highly specialized and extensively regulated process responsible for exerting control over a vast number of thiol-containing proteins, collectively referred to as the redox-sensitive proteome. Although disruptions within this process, secondary to elevated peroxide exposure, have been linked to disease, delineation of the sources and mechanisms regulating increased peroxide burden remain poorly defined and as such difficult to target using pharmacotherapy. Here we identify the pyruvate dehydrogenase complex (PDC) as a key source of H2O2 within skeletal muscle mitochondria under conditions of depressed glutathione redox buffering integrity. Treatment of permeabilized myofibers with varying concentrations of the glutathione depleting agent 1-chloro-2,4-dinitrobenzene (CDNB) led to a dose-dependent increase in pyruvate-supported JH2O2 emission, with emission rates eventually rising to exceed those of all substrate combinations tested. This striking sensitivity to glutathione depletion was observed in permeabilized fibers prepared from multiple species and was specific to PDC. Physiological oxidation of the cellular glutathione pool following high fat feeding in rodents was found to elevate PDC JH2O2 emission, as well as increase the sensitivity of the complex to GSH depletion. These findings reveal PDC as a potential major site of H2O2 production that is extremely sensitive to mitochondrial glutathione redox status. PMID:24056031

  17. Revealing of photon-number splitting attack on quantum key distribution system by photon-number resolving devices

    Science.gov (United States)

    Gaidash, A. A.; Egorov, V. I.; Gleim, A. V.

    2016-08-01

    Quantum cryptography allows distributing secure keys between two users so that any performed eavesdropping attempt would be immediately discovered. However, in practice an eavesdropper can obtain key information from multi-photon states when attenuated laser radiation is used as a source of quantum states. In order to prevent actions of an eavesdropper, it is generally suggested to implement special cryptographic protocols, like decoy states or SARG04. In this paper, we describe an alternative method based on monitoring photon number statistics after detection. We provide a useful rule of thumb to estimate approximate order of difference of expected distribution and distribution in case of attack. Formula for calculating a minimum value of total pulses or time-gaps to resolve attack is shown. Also formulas for actual fraction of raw key known to Eve were derived. This method can therefore be used with any system and even combining with mentioned special protocols.

  18. Instrumented Indentation of Lung Reveals Significant Short Term Alteration in Mechanical Behavior with 100% Oxygen

    Directory of Open Access Journals (Sweden)

    Maricris R. Silva

    2010-01-01

    Full Text Available In critical care, trauma, or other situations involving reduced lung function, oxygen is given to avoid hypoxia. It is known that under certain conditions and long time (several hours exposure, oxygen is toxic to the lungs, the possible mechanisms being direct cellular damage or surfactant dysfunction. Our key objective was to investigate possible changes in lung function when exposed to 100% oxygen in the short term (several tidal volumes. We performed mechanical tests on lobar surfaces of excised mammalian lungs inflated with air or 100% oxygen, examining (i stiffness, (ii non-linear mechanical response and (iii induced alveolar deformation. Our results showed that within five tidal volumes of breathing 100% oxygen, lung mechanics are significantly altered. In addition, after five tidal volumes of laboratory air, lung mechanical behavior begins to return to pre-oxygen levels, indicating some reversibility. These significant and short-term mechanical effects of oxygen could be linked to oxygen toxicity.

  19. Museum specimens reveal loss of pollen host plants as key factor driving wild bee decline in The Netherlands

    NARCIS (Netherlands)

    Scheper, J.A.; Reemer, M.; Kats, van R.J.M.; Ozinga, W.A.; Linden, van der G.T.J.; Schaminee, J.H.J.; Siepel, H.; Kleijn, D.

    2014-01-01

    Evidence for declining populations of both wild and managed bees has raised concern about a potential global pollination crisis. Strategies to mitigate bee loss generally aim to enhance floral resources. However, we do not really know whether loss of preferred floral resources is the key driver of b

  20. Analysis of the Staphylococcus aureus DgkB Structure Reveals a Common Catalytic Mechanism for the Soluble Diacylglycerol Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Miller, Darcie J.; Jerga, Agoston; Rock, Charles O.; White, Stephen W. (SJCH)

    2008-08-11

    Soluble diacylglycerol (DAG) kinases function as regulators of diacylglycerol metabolism in cell signaling and intermediary metabolism. We report the structure of a DAG kinase, DgkB from Staphylococcus aureus, both as the free enzyme and in complex with ADP. The molecule is a tight homodimer, and each monomer comprises two domains with the catalytic center located within the interdomain cleft. Two distinctive features of DkgB are a structural Mg{sup 2+} site and an associated Asp{center_dot}water{center_dot}Mg{sup 2+} network that extends toward the active site locale. Site-directed mutagenesis revealed that these features play important roles in the catalytic mechanism. The key active site residues and the components of the Asp{center_dot}water{center_dot}Mg{sup 2+} network are conserved in the catalytic cores of the mammalian signaling DAG kinases, indicating that these enzymes use the same mechanism and have similar structures as DgkB.

  1. Analysis of the Staphylococcus aureus DgkB structure reveals a common catalytic mechanism for the soluble diacylglycerol kinases.

    Science.gov (United States)

    Miller, Darcie J; Jerga, Agoston; Rock, Charles O; White, Stephen W

    2008-07-01

    Soluble diacylglycerol (DAG) kinases function as regulators of diacylglycerol metabolism in cell signaling and intermediary metabolism. We report the structure of a DAG kinase, DgkB from Staphylococcus aureus, both as the free enzyme and in complex with ADP. The molecule is a tight homodimer, and each monomer comprises two domains with the catalytic center located within the interdomain cleft. Two distinctive features of DkgB are a structural Mg2+ site and an associated Asp*water*Mg2+ network that extends toward the active site locale. Site-directed mutagenesis revealed that these features play important roles in the catalytic mechanism. The key active site residues and the components of the Asp*water*Mg2+ network are conserved in the catalytic cores of the mammalian signaling DAG kinases, indicating that these enzymes use the same mechanism and have similar structures as DgkB.

  2. Crystal Structure of the ERp44-Peroxiredoxin 4 Complex Reveals the Molecular Mechanisms of Thiol-Mediated Protein Retention.

    Science.gov (United States)

    Yang, Kai; Li, De-Feng; Wang, Xi'e; Liang, Jinzhao; Sitia, Roberto; Wang, Chih-Chen; Wang, Xi

    2016-10-04

    ERp44 controls the localization and transport of diverse proteins in the early secretory pathway. The mechanisms that allow client recognition and the source of the oxidative power for forming intermolecular disulfides are as yet unknown. Here we present the structure of ERp44 bound to a client, peroxiredoxin 4. Our data reveal that ERp44 binds the oxidized form of peroxiredoxin 4 via thiol-disulfide interchange reactions. The structure explains the redox-dependent recognition and characterizes the essential non-covalent interactions at the interface. The ERp44-Prx4 covalent complexes can be reduced by glutathione and protein disulfide isomerase family members in the ER, allowing the two components to recycle. This work provides insights into the mechanisms of thiol-mediated protein retention and indicates the key roles of ERp44 in this biochemical cycle to optimize oxidative folding and redox homeostasis.

  3. Same pattern, different mechanism: Locking onto the role of key species in seafloor ecosystem process

    OpenAIRE

    2016-01-01

    Seafloor biodiversity is a key mediator of ecosystem functioning, but its role is often excluded from global budgets or simplified to black boxes in models. New techniques allow quantification of the behavior of animals living below the sediment surface and assessment of the ecosystem consequences of complex interactions, yielding a better understanding of the role of seafloor animals in affecting key processes like primary productivity. Combining predictions based on natural history, behavio...

  4. Crystal structure and site-directed mutational analysis reveals key residues involved in Escherichia coli ZapA function.

    Science.gov (United States)

    Roach, Elyse J; Kimber, Matthew S; Khursigara, Cezar M

    2014-08-22

    FtsZ is an essential cell division protein in Escherichia coli, and its localization, filamentation, and bundling at the mid-cell are required for Z-ring stability. Once assembled, the Z-ring recruits a series of proteins that comprise the bacterial divisome. Zaps (FtsZ-associated proteins) stabilize the Z-ring by increasing lateral interactions between individual filaments, bundling FtsZ to provide a scaffold for divisome assembly. The x-ray crystallographic structure of E. coli ZapA was determined, identifying key structural differences from the existing ZapA structure from Pseudomonas aeruginosa, including a charged α-helix on the globular domains of the ZapA tetramer. Key helix residues in E. coli ZapA were modified using site-directed mutagenesis. These ZapA variants significantly decreased FtsZ bundling in protein sedimentation assays when compared with WT ZapA proteins. Electron micrographs of ZapA-bundled FtsZ filaments showed the modified ZapA variants altered the number of FtsZ filaments per bundle. These in vitro results were corroborated in vivo by expressing the ZapA variants in an E. coli ΔzapA strain. In vivo, ZapA variants that altered FtsZ bundling showed an elongated phenotype, indicating improper cell division. Our findings highlight the importance of key ZapA residues that influence the extent of FtsZ bundling and that ultimately affect Z-ring formation in dividing cells.

  5. Comparison of the aggregation of homologous β2-microglobulin variants reveals protein solubility as a key determinant of amyloid formation.

    Science.gov (United States)

    Pashley, Clare L; Hewitt, Eric W; Radford, Sheena E

    2016-02-13

    The mouse and human β2-microglobulin protein orthologs are 70% identical in sequence and share 88% sequence similarity. These proteins are predicted by various algorithms to have similar aggregation and amyloid propensities. However, whilst human β2m (hβ2m) forms amyloid-like fibrils in denaturing conditions (e.g. pH2.5) in the absence of NaCl, mouse β2m (mβ2m) requires the addition of 0.3M NaCl to cause fibrillation. Here, the factors which give rise to this difference in amyloid propensity are investigated. We utilise structural and mutational analyses, fibril growth kinetics and solubility measurements under a range of pH and salt conditions, to determine why these two proteins have different amyloid propensities. The results show that, although other factors influence the fibril growth kinetics, a striking difference in the solubility of the proteins is a key determinant of the different amyloidogenicity of hβ2m and mβ2m. The relationship between protein solubility and lag time of amyloid formation is not captured by current aggregation or amyloid prediction algorithms, indicating a need to better understand the role of solubility on the lag time of amyloid formation. The results demonstrate the key contribution of protein solubility in determining amyloid propensity and lag time of amyloid formation, highlighting how small differences in protein sequence can have dramatic effects on amyloid formation.

  6. Combining experimental and mathematical modeling to reveal mechanisms of macrophage-dependent left ventricular remodeling

    Directory of Open Access Journals (Sweden)

    Dai Qiuxia

    2011-05-01

    Full Text Available Abstract Background Progressive remodeling of the left ventricle (LV following myocardial infarction (MI can lead to congestive heart failure, but the underlying initiation factors remain poorly defined. The objective of this study, accordingly, was to determine the key factors and elucidate the regulatory mechanisms of LV remodeling using integrated computational and experimental approaches. Results By examining the extracellular matrix (ECM gene expression and plasma analyte levels in C57/BL6J mice LV post-MI and ECM gene responses to transforming growth factor (TGF-β1 in cultured cardiac fibroblasts, we found that key factors in LV remodeling included macrophages, fibroblasts, transforming growth factor-β1, matrix metalloproteinase-9 (MMP-9, and specific collagen subtypes. We established a mathematical model to study LV remodeling post-MI by quantifying the dynamic balance between ECM construction and destruction. The mathematical model incorporated the key factors and demonstrated that TGF-β1 stimuli and MMP-9 interventions with different strengths and intervention times lead to different LV remodeling outcomes. The predictions of the mathematical model fell within the range of experimental measurements for these interventions, providing validation for the model. Conclusions In conclusion, our results demonstrated that the balance between ECM synthesis and degradation, controlled by interactions of specific key factors, determines the LV remodeling outcomes. Our mathematical model, based on the balance between ECM construction and destruction, provides a useful tool for studying the regulatory mechanisms and for predicting LV remodeling outcomes.

  7. Novel Model of Tendon Regeneration Reveals Distinct Cell Mechanisms Underlying Regenerative and Fibrotic Tendon Healing

    Science.gov (United States)

    Howell, Kristen; Chien, Chun; Bell, Rebecca; Laudier, Damien; Tufa, Sara F.; Keene, Douglas R.; Andarawis-Puri, Nelly; Huang, Alice H.

    2017-01-01

    To date, the cell and molecular mechanisms regulating tendon healing are poorly understood. Here, we establish a novel model of tendon regeneration using neonatal mice and show that neonates heal via formation of a ‘neo-tendon’ that differentiates along the tendon specific lineage with functional restoration of gait and mechanical properties. In contrast, adults heal via fibrovascular scar, aberrant differentiation toward cartilage and bone, with persistently impaired function. Lineage tracing identified intrinsic recruitment of Scx-lineage cells as a key cellular mechanism of neonatal healing that is absent in adults. Instead, adult Scx-lineage tenocytes are not recruited into the defect but transdifferentiate into ectopic cartilage; in the absence of tenogenic cells, extrinsic αSMA-expressing cells persist to form a permanent scar. Collectively, these results establish an exciting model of tendon regeneration and uncover a novel cellular mechanism underlying regenerative vs non-regenerative tendon healing. PMID:28332620

  8. Network theory inspired analysis of time-resolved expression data reveals key players guiding P. patens stem cell development.

    Science.gov (United States)

    Busch, Hauke; Boerries, Melanie; Bao, Jie; Hanke, Sebastian T; Hiss, Manuel; Tiko, Theodhor; Rensing, Stefan A

    2013-01-01

    Transcription factors (TFs) often trigger developmental decisions, yet, their transcripts are often only moderately regulated and thus not easily detected by conventional statistics on expression data. Here we present a method that allows to determine such genes based on trajectory analysis of time-resolved transcriptome data. As a proof of principle, we have analysed apical stem cells of filamentous moss (P. patens) protonemata that develop from leaflets upon their detachment from the plant. By our novel correlation analysis of the post detachment transcriptome kinetics we predict five out of 1,058 TFs to be involved in the signaling leading to the establishment of pluripotency. Among the predicted regulators is the basic helix loop helix TF PpRSL1, which we show to be involved in the establishment of apical stem cells in P. patens. Our methodology is expected to aid analysis of key players of developmental decisions in complex plant and animal systems.

  9. Mitochondrial glutathione depletion reveals a novel role for the pyruvate dehydrogenase complex as a key H2O2-emitting source under conditions of nutrient overload.

    Science.gov (United States)

    Fisher-Wellman, Kelsey H; Gilliam, Laura A A; Lin, Chien-Te; Cathey, Brook L; Lark, Daniel S; Neufer, P Darrell

    2013-12-01

    Once regarded as a "by-product" of aerobic metabolism, the production of superoxide/H2O2 is now understood to be a highly specialized and extensively regulated process responsible for exerting control over a vast number of thiol-containing proteins, collectively referred to as the redox-sensitive proteome. Although disruptions within this process, secondary to elevated peroxide exposure, have been linked to disease, the sources and mechanisms regulating increased peroxide burden remain poorly defined and as such are difficult to target using pharmacotherapy. Here we identify the pyruvate dehydrogenase complex (PDC) as a key source of H2O2 within skeletal muscle mitochondria under conditions of depressed glutathione redox buffering integrity. Treatment of permeabilized myofibers with varying concentrations of the glutathione-depleting agent 1-chloro-2,4-dinitrobenzene led to a dose-dependent increase in pyruvate-supported JH2O2 emission (the flux of H2O2 diffusing out of the mitochondrial matrix into the surrounding assay medium), with emission rates eventually rising to exceed those of all substrate combinations tested. This striking sensitivity to glutathione depletion was observed in permeabilized fibers prepared from multiple species and was specific to PDC. Physiological oxidation of the cellular glutathione pool after high-fat feeding in rodents was found to elevate PDC JH2O2 emission, as well as increasing the sensitivity of the complex to GSH depletion. These findings reveal PDC as a potential major site of H2O2 production that is extremely sensitive to mitochondrial glutathione redox status. Published by Elsevier Inc.

  10. Structural characterization of native autoinducing peptides and abiotic analogues reveals key features essential for activation and inhibition of an AgrC quorum sensing receptor in Staphylococcus aureus.

    Science.gov (United States)

    Tal-Gan, Yftah; Ivancic, Monika; Cornilescu, Gabriel; Cornilescu, Claudia C; Blackwell, Helen E

    2013-12-11

    Staphylococcus aureus is a major human pathogen that uses quorum sensing (QS) to control virulence. Its QS system is regulated by macrocyclic peptide signals (or autoinducing peptides (AIPs)) and their cognate transmembrane receptors (AgrCs). Four different specificity groups of S. aureus have been identified to date (groups I-IV), each of which uses a different AIP:AgrC pair. Non-native ligands capable of intercepting AIP:AgrC binding, and thereby QS, in S. aureus have attracted considerable interest as chemical tools to study QS pathways and as possible antivirulence strategies for the treatment of infection. We recently reported a set of analogues of the group-III AIP that are capable of strongly modulating the activity of all four AgrC receptors. Critical to the further development of such ligands is a detailed understanding of the structural features of both native AIPs and non-native analogues that are essential for activity. Herein, we report the first three-dimensional structural analysis of the known native AIP signals (AIPs-I-IV) and several AIP-III analogues with varied biological activities using NMR spectroscopy. Integration of these NMR studies with the known agonism and antagonism profiles of these peptides in AgrC-III revealed two key structural elements that control AIP-III (and non-native peptide) activity: (1) a tri-residue hydrophobic "knob" essential for both activation and inhibition and (2) a fourth anchor point on the exocyclic tail needed for receptor activation. These results provide strong structural support for a mechanism of AIP-mediated AgrC activation and inhibition in S. aureus , and should facilitate the design of new AgrC ligands with enhanced activities (as agonists or antagonists) and simplified chemical structures.

  11. Transcriptomic analysis by RNA-seq reveals AP-1 pathway as key regulator that green tea may rely on to inhibit lung tumorigenesis.

    Science.gov (United States)

    Pan, Jing; Zhang, Qi; Xiong, Donghai; Vedell, Peter; Yan, Ying; Jiang, Hui; Cui, Peng; Ding, Feng; Tichelaar, Jay W; Wang, Yian; Lubet, Ronald A; You, Ming

    2014-01-01

    Green tea is a promising chemopreventive agent for lung cancer. Multiple signaling events have been reported, however, the relative importance of these mechanisms in mediating the chemopreventive function of green tea is unclear. In the present study, to examine the involvement of AP-1 in green tea polyphenols induced tumor inhibition, human NSCLC cell line H1299 and mouse SPON 10 cells were identified as AP-1 dependent, as these two lines exhibit high constitutive AP-1 activity, and when TAM67 expression was induced with doxycycline, cell growth was inhibited and correlated with suppressed AP-1 activity. RNA-seq was used to determine the global transcriptional effects of AP-1 inhibition and also uncover the possible involvement of AP-1 in tea polyphenols induced chemoprevention. TAM67 mediated changes in gene expression were identified, and within down-regulated genes, AP-1 was identified as a key transcription regulator. RNA-seq analysis revealed that Polyphenon E-treated cells shared 293 commonly down-regulated genes within TAM67 expressing H1299 cells, and by analysis of limited Chip-seq data, over 10% of the down-regulated genes contain a direct AP-1 binding site, indicating that Polyphenon E elicits chemopreventive activity by regulating AP-1 target genes. Conditional TAM67 expressing transgenic mice and NSCLC cell lines were used to further confirm that the chemopreventive activity of green tea is AP-1 dependent. Polyphenon E lost its chempreventive function both in vitro and in vivo when AP-1 was inhibited, indicating that AP-1 inhibition is a major pathway through which green tea exhibits chemopreventive effects.

  12. Expression profiling reveals Spot 42 small RNA as a key regulator in the central metabolism of Aliivibrio salmonicida

    Directory of Open Access Journals (Sweden)

    Hansen Geir Å

    2012-01-01

    Full Text Available Abstract Background Spot 42 was discovered in Escherichia coli nearly 40 years ago as an abundant, small and unstable RNA. Its biological role has remained obscure until recently, and is today implicated in having broader roles in the central and secondary metabolism. Spot 42 is encoded by the spf gene. The gene is ubiquitous in the Vibrionaceae family of gamma-proteobacteria. One member of this family, Aliivibrio salmonicida, causes cold-water vibriosis in farmed Atlantic salmon. Its genome encodes Spot 42 with 84% identity to E. coli Spot 42. Results We generated a A. salmonicida spf deletion mutant. We then used microarray and Northern blot analyses to monitor global effects on the transcriptome in order to provide insights into the biological roles of Spot 42 in this bacterium. In the presence of glucose, we found a surprisingly large number of ≥ 2X differentially expressed genes, and several major cellular processes were affected. A gene encoding a pirin-like protein showed an on/off expression pattern in the presence/absence of Spot 42, which suggests that Spot 42 plays a key regulatory role in the central metabolism by regulating the switch between fermentation and respiration. Interestingly, we discovered an sRNA named VSsrna24, which is encoded immediately downstream of spf. This new sRNA has an expression pattern opposite to that of Spot 42, and its expression is repressed by glucose. Conclusions We hypothesize that Spot 42 plays a key role in the central metabolism, in part by regulating the pyruvat dehydrogenase enzyme complex via pirin.

  13. Transcriptional profiling of human brain endothelial cells reveals key properties crucial for predictive in vitro blood-brain barrier models.

    Directory of Open Access Journals (Sweden)

    Eduard Urich

    Full Text Available Brain microvascular endothelial cells (BEC constitute the blood-brain barrier (BBB which forms a dynamic interface between the blood and the central nervous system (CNS. This highly specialized interface restricts paracellular diffusion of fluids and solutes including chemicals, toxins and drugs from entering the brain. In this study we compared the transcriptome profiles of the human immortalized brain endothelial cell line hCMEC/D3 and human primary BEC. We identified transcriptional differences in immune response genes which are directly related to the immortalization procedure of the hCMEC/D3 cells. Interestingly, astrocytic co-culturing reduced cell adhesion and migration molecules in both BECs, which possibly could be related to regulation of immune surveillance of the CNS controlled by astrocytic cells within the neurovascular unit. By matching the transcriptome data from these two cell lines with published transcriptional data from freshly isolated mouse BECs, we discovered striking differences that could explain some of the limitations of using cultured BECs to study BBB properties. Key protein classes such as tight junction proteins, transporters and cell surface receptors show differing expression profiles. For example, the claudin-5, occludin and JAM2 expression is dramatically reduced in the two human BEC lines, which likely explains their low transcellular electric resistance and paracellular leakiness. In addition, the human BEC lines express low levels of unique brain endothelial transporters such as Glut1 and Pgp. Cell surface receptors such as LRP1, RAGE and the insulin receptor that are involved in receptor-mediated transport are also expressed at very low levels. Taken together, these data illustrate that BECs lose their unique protein expression pattern outside of their native environment and display a more generic endothelial cell phenotype. A collection of key genes that seems to be highly regulated by the local

  14. Transcriptome Analysis of Ullrich Congenital Muscular Dystrophy Fibroblasts Reveals a Disease Extracellular Matrix Signature and Key Molecular Regulators.

    Directory of Open Access Journals (Sweden)

    Sonia Paco

    Full Text Available Collagen VI related myopathies encompass a range of phenotypes with involvement of skeletal muscle, skin and other connective tissues. They represent a severe and relatively common form of congenital disease for which there is no treatment. Collagen VI in skeletal muscle and skin is produced by fibroblasts.In order to gain insight into the consequences of collagen VI mutations and identify key disease pathways we performed global gene expression analysis of dermal fibroblasts from patients with Ullrich Congenital Muscular Dystrophy with and without vitamin C treatment. The expression data were integrated using a range of systems biology tools. Results were validated by real-time PCR, western blotting and functional assays.We found significant changes in the expression levels of almost 600 genes between collagen VI deficient and control fibroblasts. Highly regulated genes included extracellular matrix components and surface receptors, including integrins, indicating a shift in the interaction between the cell and its environment. This was accompanied by a significant increase in fibroblasts adhesion to laminin. The observed changes in gene expression profiling may be under the control of two miRNAs, miR-30c and miR-181a, which we found elevated in tissue and serum from patients and which could represent novel biomarkers for muscular dystrophy. Finally, the response to vitamin C of collagen VI mutated fibroblasts significantly differed from healthy fibroblasts. Vitamin C treatment was able to revert the expression of some key genes to levels found in control cells raising the possibility of a beneficial effect of vitamin C as a modulator of some of the pathological aspects of collagen VI related diseases.

  15. Aberrant splicing is a common mutational mechanism in MKS1, a key player in Meckel-Gruber syndrome.

    Science.gov (United States)

    Frank, Valeska; Ortiz Brüchle, Nadina; Mager, Silke; Frints, Susanna G M; Bohring, Axel; du Bois, Gabriele; Debatin, Irmgard; Seidel, Heide; Senderek, Jan; Besbas, Nesrin; Todt, Unda; Kubisch, Christian; Grimm, Tiemo; Teksen, Fulya; Balci, Sevim; Zerres, Klaus; Bergmann, Carsten

    2007-06-01

    Meckel-Gruber syndrome (MKS) is an autosomal recessive, usually lethal multisystemic disorder characterized by early developmental anomalies of the central nervous system, cystic kidney dysplasia, hepatobiliary ductal plate malformation, and postaxial polydactyly. Three MKS loci have been mapped and recently, two genes were identified: MKS1 on 17q22 in Caucasian kindreds and MKS3 on 8q22 in Omani and Pakistani families, putting MKS on the growing list of ciliary disorders ("ciliopathies"). We performed linkage analysis for MKS1-3 in 14 consanguineous and/or multiplex families of different ethnic origins with histologic diagnosis and at least three classic MKS manifestations in each kindred. Unexpectedly, only five families were linked to any of the known MKS loci, clearly indicating further locus heterogeneity. All five families showed homozygosity for MKS1 and, intriguingly, were of non-Caucasian origin. MKS1 sequencing revealed no mutation in two of these pedigrees, whereas different, novel splicing defects were identified in the three other families and an additional sporadic German patient. Given that all of our mutations and two of the in total four known MKS1 changes cause aberrant splicing (while the other two known mutations were frameshift mutations), we hypothesize that splicing defects are a crucial mutational mechanism in MKS1 which apparently is one of the main loci and key players in MKS. Our results indicate that MKS1 mutations are not restricted to the Caucasian gene pool and suggest further genetic heterogeneity for MKS. Overall, our data have immediate implications for genetic counselling and testing approaches in MKS. (c) 2007 Wiley-Liss, Inc.

  16. Understanding the pelvic pain mechanism is key to find an adequate therapeutic approach.

    Science.gov (United States)

    Van Kerrebroeck, Philip

    2016-06-25

    Pain is a natural mechanism to actual or potential tissue damage and involves both a sensory and an emotional experience. In chronic pelvic pain, localisation of pain can be widespread and can cause considerable distress. A multidisciplinary approach is needed in order to fully understand the pelvic pain mechanism and to identify an adequate therapeutic approach.

  17. Key role of local regulation in chemosensing revealed by a new molecular interaction-based modeling method.

    Directory of Open Access Journals (Sweden)

    Martin Meier-Schellersheim

    2006-07-01

    Full Text Available The signaling network underlying eukaryotic chemosensing is a complex combination of receptor-mediated transmembrane signals, lipid modifications, protein translocations, and differential activation/deactivation of membrane-bound and cytosolic components. As such, it provides particularly interesting challenges for a combined computational and experimental analysis. We developed a novel detailed molecular signaling model that, when used to simulate the response to the attractant cyclic adenosine monophosphate (cAMP, made nontrivial predictions about Dictyostelium chemosensing. These predictions, including the unexpected existence of spatially asymmetrical, multiphasic, cyclic adenosine monophosphate-induced PTEN translocation and phosphatidylinositol-(3,4,5P3 generation, were experimentally verified by quantitative single-cell microscopy leading us to propose significant modifications to the current standard model for chemoattractant-induced biochemical polarization in this organism. Key to this successful modeling effort was the use of "Simmune," a new software package that supports the facile development and testing of detailed computational representations of cellular behavior. An intuitive interface allows user definition of complex signaling networks based on the definition of specific molecular binding site interactions and the subcellular localization of molecules. It automatically translates such inputs into spatially resolved simulations and dynamic graphical representations of the resulting signaling network that can be explored in a manner that closely parallels wet lab experimental procedures. These features of Simmune were critical to the model development and analysis presented here and are likely to be useful in the computational investigation of many aspects of cell biology.

  18. The genetic potential for key biogeochemical processes in Arctic frost flowers and young sea ice revealed by metagenomic analysis.

    Science.gov (United States)

    Bowman, Jeff S; Berthiaume, Chris T; Armbrust, E Virginia; Deming, Jody W

    2014-08-01

    Newly formed sea ice is a vast and biogeochemically active environment. Recently, we reported an unusual microbial community dominated by members of the Rhizobiales in frost flowers at the surface of Arctic young sea ice based on the presence of 16S gene sequences related to these strains. Here, we use metagenomic analysis of two samples, from a field of frost flowers and the underlying young sea ice, to explore the metabolic potential of this surface ice community. The analysis links genes for key biogeochemical processes to the Rhizobiales, including dimethylsulfide uptake, betaine glycine turnover, and halocarbon production. Nodulation and nitrogen fixation genes characteristic of terrestrial root-nodulating Rhizobiales were generally lacking from these metagenomes. Non-Rhizobiales clades at the ice surface had genes that would enable additional biogeochemical processes, including mercury reduction and dimethylsulfoniopropionate catabolism. Although the ultimate source of the observed microbial community is not known, considerations of the possible role of eolian deposition or transport with particles entrained during ice formation favor a suspended particle source for this microbial community.

  19. Genome-wide functional analysis of plasmodium protein phosphatases reveals key regulators of parasite development and differentiation

    KAUST Repository

    Guttery, David S.

    2014-07-09

    Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria. © 2014 The Authors.

  20. Multiscale Homogenization Theory: An Analysis Tool for Revealing Mechanical Design Principles in Bone and Bone Replacement Materials

    Science.gov (United States)

    Hellmich, Christian; Fritsch, Andreas; Dormieux, Luc

    Biomimetics deals with the application of nature-made "design solutions" to the realm of engineering. In the quest to understand mechanical implications of structural hierarchies found in biological materials, multiscale mechanics may hold the key to understand "building plans" inherent to entire material classes, here bone and bone replacement materials. Analyzing a multitude of biophysical hierarchical and biomechanical experiments through homogenization theories for upscaling stiffness and strength properties reveals the following design principles: The elementary component "collagen" induces, right at the nanolevel, the mechanical anisotropy of bone materials, which is amplified by fibrillar collagen-based structures at the 100-nm scale, and by pores in the micrometer-to-millimeter regime. Hydroxyapatite minerals are poorly organized, and provide stiffness and strength in a quasi-brittle manner. Water layers between hydroxyapatite crystals govern the inelastic behavior of the nanocomposite, unless the "collagen reinforcement" breaks. Bone replacement materials should mimic these "microstructural mechanics" features as closely as possible if an imitation of the natural form of bone is desired (Gebeshuber et al., Adv Mater Res 74:265-268, 2009).

  1. Genomic and protein expression analysis reveals flap endonuclease 1 (FEN1) as a key biomarker in breast and ovarian cancer.

    Science.gov (United States)

    Abdel-Fatah, Tarek M A; Russell, Roslin; Albarakati, Nada; Maloney, David J; Dorjsuren, Dorjbal; Rueda, Oscar M; Moseley, Paul; Mohan, Vivek; Sun, Hongmao; Abbotts, Rachel; Mukherjee, Abhik; Agarwal, Devika; Illuzzi, Jennifer L; Jadhav, Ajit; Simeonov, Anton; Ball, Graham; Chan, Stephen; Caldas, Carlos; Ellis, Ian O; Wilson, David M; Madhusudan, Srinivasan

    2014-10-01

    FEN1 has key roles in Okazaki fragment maturation during replication, long patch base excision repair, rescue of stalled replication forks, maintenance of telomere stability and apoptosis. FEN1 may be dysregulated in breast and ovarian cancers and have clinicopathological significance in patients. We comprehensively investigated FEN1 mRNA expression in multiple cohorts of breast cancer [training set (128), test set (249), external validation (1952)]. FEN1 protein expression was evaluated in 568 oestrogen receptor (ER) negative breast cancers, 894 ER positive breast cancers and 156 ovarian epithelial cancers. FEN1 mRNA overexpression was highly significantly associated with high grade (p = 4.89 × 10(-57)), high mitotic index (p = 5.25 × 10(-28)), pleomorphism (p = 6.31 × 10(-19)), ER negative (p = 9.02 × 10(-35)), PR negative (p = 9.24 × 10(-24)), triple negative phenotype (p = 6.67 × 10(-21)), PAM50.Her2 (p = 5.19 × 10(-13)), PAM50. Basal (p = 2.7 × 10(-41)), PAM50.LumB (p = 1.56 × 10(-26)), integrative molecular cluster 1 (intClust.1) (p = 7.47 × 10(-12)), intClust.5 (p = 4.05 × 10(-12)) and intClust. 10 (p = 7.59 × 10(-38)) breast cancers. FEN1 mRNA overexpression is associated with poor breast cancer specific survival in univariate (p = 4.4 × 10(-16)) and multivariate analysis (p = 9.19 × 10(-7)). At the protein level, in ER positive tumours, FEN1 overexpression remains significantly linked to high grade, high mitotic index and pleomorphism (ps cancers, similarly, FEN1 overexpression is associated with high grade, high stage and poor survival (ps breast and ovarian epithelial cancer.

  2. An analysis of key issues in the clean development mechanism based on the UNEP Risoe clean development mechanism pipeline

    DEFF Research Database (Denmark)

    Fenhann, Jørgen Villy; Staun, Frederik

    2010-01-01

    This paper provides a description of the analysis contained in the UNEP Risoe Clean Development Mechanism (CDM) pipeline not available on the site, some of the impacts/problems of the CDM and a way forward post-2012. The successful development of the CDM since it started in December 2003 is docum......This paper provides a description of the analysis contained in the UNEP Risoe Clean Development Mechanism (CDM) pipeline not available on the site, some of the impacts/problems of the CDM and a way forward post-2012. The successful development of the CDM since it started in December 2003...

  3. Transcriptome analysis of sexually dimorphic Chinese white wax scale insects reveals key differences in developmental programs and transcription factor expression.

    Science.gov (United States)

    Yang, Pu; Chen, Xiao-Ming; Liu, Wei-Wei; Feng, Ying; Sun, Tao

    2015-01-01

    The Chinese white wax scale insect, Ericerus pela, represents one of the most dramatic examples of sexual dimorphism in any insect species. In this study, we showed that although E. pela males display complete metamorphosis similar to holometabolous insects, the species forms the sister group to Acyrthosiphon pisum and cluster with hemimetabolous insects. The gene expression profile and Gene Ontology (GO) analyses revealed that the two sexes engaged in distinct developmental programs. In particular, female development appeared to prioritize the expression of genes related to cellular, metabolic, and developmental processes and to anatomical structure formation in nymphs. By contrast, male nymphal development is characterized by the significant down-regulation of genes involved in chitin, the respiratory system, and neurons. The wing and appendage morphogenesis, anatomical and tissue structure morphogenesis programs activated after male nymphal development. Transcription factors (that convey juvenile hormone or ecdysone signals, and Hox genes) and DNA methyltransferase were also differentially expressed between females and males. These results may indicate the roles that these differentially expressed genes play in regulating sexual dimorphism through orchestrating complex genetic programs. This differential expression was particularly prominent for processes linked to female development and wing development in males.

  4. Hepatic gene expression profiling reveals key pathways involved in leptin-mediated weight loss in ob/ob mice.

    Directory of Open Access Journals (Sweden)

    Ashok Sharma

    Full Text Available BACKGROUND: Leptin, a cytokine-like protein, plays an important role in the regulation of body weight through inhibition of food intake and stimulation of energy expenditure. Leptin circulates in blood and acts on the brain, which sends downstream signals to regulate body weight. Leptin therapy has been successful in treating leptin deficient obese patients. However, high levels of leptin have been observed in more common forms of obesity indicating a state of leptin resistance which limits the application of leptin in the treatment of obesity. If the central effect of leptin could be by-passed and genes which respond to leptin treatment could be regulated directly, new therapeutic targets for the treatment of obesity may be possible. The purpose of this study was to identify genes and subsequent pathways correlated with leptin-mediated weight loss. METHODOLOGY/PRINCIPAL FINDINGS: WE UTILIZED MICROARRAY TECHNOLOGY TO COMPARE HEPATIC GENE EXPRESSION CHANGES AFTER TWO TYPES OF LEPTIN ADMINISTRATION: one involving a direct stimulatory effect when administered peripherally (subcutaneous: SQ and another that is indirect, involving a hypothalamic relay that suppresses food intake when leptin is administered centrally (intracerebroventricular: ICV. We identified 214 genes that correlate with leptin mediated weight loss. Several biological processes such as mitochondrial metabolic pathways, lipid metabolic and catabolic processes, lipid biosynthetic processes, carboxylic acid metabolic processes, iron ion binding and glutathione S-transferases were downregulated after leptin administration. In contrast, genes involved in the immune system inflammatory response and lysosomal activity were found to be upregulated. Among the cellular compartments mitochondrion (32 genes, endoplasmic reticulum (22 genes and vacuole (8 genes were significantly over represented. CONCLUSIONS/SIGNIFICANCE: In this study we have identified key molecular pathways and downstream

  5. Hepatic gene expression profiling reveals key pathways involved in leptin-mediated weight loss in ob/ob mice.

    Science.gov (United States)

    Sharma, Ashok; Bartell, Shoshana M; Baile, Clifton A; Chen, Bo; Podolsky, Robert H; McIndoe, Richard A; She, Jin-Xiong

    2010-08-16

    Leptin, a cytokine-like protein, plays an important role in the regulation of body weight through inhibition of food intake and stimulation of energy expenditure. Leptin circulates in blood and acts on the brain, which sends downstream signals to regulate body weight. Leptin therapy has been successful in treating leptin deficient obese patients. However, high levels of leptin have been observed in more common forms of obesity indicating a state of leptin resistance which limits the application of leptin in the treatment of obesity. If the central effect of leptin could be by-passed and genes which respond to leptin treatment could be regulated directly, new therapeutic targets for the treatment of obesity may be possible. The purpose of this study was to identify genes and subsequent pathways correlated with leptin-mediated weight loss. WE UTILIZED MICROARRAY TECHNOLOGY TO COMPARE HEPATIC GENE EXPRESSION CHANGES AFTER TWO TYPES OF LEPTIN ADMINISTRATION: one involving a direct stimulatory effect when administered peripherally (subcutaneous: SQ) and another that is indirect, involving a hypothalamic relay that suppresses food intake when leptin is administered centrally (intracerebroventricular: ICV). We identified 214 genes that correlate with leptin mediated weight loss. Several biological processes such as mitochondrial metabolic pathways, lipid metabolic and catabolic processes, lipid biosynthetic processes, carboxylic acid metabolic processes, iron ion binding and glutathione S-transferases were downregulated after leptin administration. In contrast, genes involved in the immune system inflammatory response and lysosomal activity were found to be upregulated. Among the cellular compartments mitochondrion (32 genes), endoplasmic reticulum (22 genes) and vacuole (8 genes) were significantly over represented. In this study we have identified key molecular pathways and downstream genes which respond to leptin treatment and are involved in leptin-mediated weight

  6. Comparative Transcriptome Analysis of Fetal Skin Reveals Key Genes Related to Hair Follicle Morphogenesis in Cashmere Goats.

    Directory of Open Access Journals (Sweden)

    Ye Gao

    Full Text Available Cashmere goat skin contains two types of hair follicles (HF: primary hair follicles (PHF and secondary hair follicles (SHF. Although multiple genetic determinants associated with HF formation have been identified, the molecules that determine the independent morphogenesis of HF in cashmere goats remain elusive. The growth and development of SHF directly influence the quantity and quality of cashmere production. Here, we report the transcriptome profiling analysis of nine skin samples from cashmere goats using 60- and 120-day-old embryos (E60 and E120, respectively, as well as newborns (NB, through RNA-sequencing (RNA-seq. HF morphological changes indicated that PHF were initiated at E60, with maturation from E120, while differentiation of SHF was identified at E120 until formation of cashmere occurred after birth (NB. The RNA-sequencing analysis generated over 20.6 million clean reads from each mRNA library. The number of differentially expressed genes (DEGs in E60 vs. E120, E120 vs. NB, and E60 vs. NB were 1,024, 0 and 1,801, respectively, indicating that no significant differences were found at transcriptomic levels between E120 and NB. Key genes including B4GALT4, TNC, a-integrin, and FGFR1, were up-regulated and expressed in HF initiation from E60 to E120, while regulatory genes such as GPRC5D, PAD3, HOXC13, PRR9, VSIG8, LRRC15, LHX2, MSX-2, and FOXN1 were up-regulated and expressed in HF keratinisation and hair shaft differentiation from E120 and NB to E60. Several genes belonging to the KRT and KRTAP gene families were detected throughout the three HF developmental stages. The transcriptional trajectory analyses of all DEGs indicated that immune privilege, glycosaminoglycan biosynthesis, extracellular matrix receptor interaction, and growth factor receptors all played dominant roles in the epithelial-mesenchymal interface and HF formation. We found that the Wnt, transforming growth factor-beta/bone morphogenetic protein, and Notch family

  7. Genomic and protein expression analysis reveals Flap endonuclease 1 (FEN1) as a key biomarker in breast and ovarian cancer

    Science.gov (United States)

    Abdel-Fatah, Tarek MA; Russell, Roslin; Albarakati, Nada; Maloney, David J; Dorjsuren, Dorjbal; Rueda, Oscar M; Moseley, Paul; Mohan, Vivek; Sun, Hongmao; Abbotts, Rachel; Mukherjee, Abhik; Agarwal, Devika; Illuzzi, Jennifer L.; Jadhav, Ajit; Simeonov, Anton; Ball, Graham; Chan, Stephen; Caldas, Carlos; Ellis, Ian O; Wilson, David M; Madhusudan, Srinivasan

    2015-01-01

    FEN1 has key roles in Okazaki fragment maturation during replication, long patch base excision repair, rescue of stalled replication forks, maintenance of telomere stability and apoptosis. FEN1 may be dysregulated in breast and ovarian cancers and have clinicopathological significance in patients. We comprehensively investigated FEN1 mRNA expression in multiple cohorts of breast cancer [training set (128), test set (249), external validation (1952)]. FEN1 protein expression was evaluated in 568 oestrogen receptor (ER) negative breast cancers, 894 ER positive breast cancers and 156 ovarian epithelial cancers. FEN1 mRNA overexpression was highly significantly associated with high grade (p=4.89 × 10−57), high mitotic index (p=5.25 × 10−28), pleomorphism (p=6.31 × 10−19), ER negative (p=9.02 × 10−35), PR negative (p=9.24 × 10−24), triple negative phenotype (p=6.67 × 10−21), PAM50.Her2 (p=5.19 × 10−13), PAM50.Basal (p=2.7 × 10−41), PAM50.LumB (p=1.56 × 10−26), integrative molecular cluster 1 (intClust.1) (p=7.47 × 10−12), intClust.5 (p=4.05 × 10−12) and intClust. 10 (p=7.59 × 10−38) breast cancers. FEN1 mRNA overexpression is associated with poor breast cancer specific survival in univariate (p= 4.4 × 10−16) and multivariate analysis (p= 9.19 × 10−7). At the protein level, in ER positive tumours, FEN1 overexpression remains significantly linked to high grade, high mitotic index and pleomorphism (ps<0.01). In ER negative tumours, high FEN1 is significantly associated with pleomorphism, tumour type, lymphovascular invasion, triple negative phenotype, EGFR and HER2 expression (ps<0.05). In ER positive as well as in ER negative tumours, FEN1 protein overexpression is associated with poor survival in univariate and multivariate analysis (ps<0.01). In ovarian epithelial cancers, similarly, FEN1 overexpression is associated with high grade, high stage and poor survival (ps<0.05). We conclude that FEN1 is a promising biomarker in breast

  8. Secondary structure of the rRNA ITS2 region reveals key evolutionary patterns in acroporid corals.

    Science.gov (United States)

    Coleman, Annette W; van Oppen, Madeleine J H

    2008-10-01

    This study investigates the ribosomal RNA transcript secondary structure in corals as confirmed by compensatory base changes in Isopora/Acropora species. These species are unique versus all other corals in the absence of a eukaryote-wide conserved structural component, the helix III in internal transcriber spacer (ITS) 2, and their variability in the 5.8S-LSU helix basal to ITS2, a helix with pairings identical among all other scleractinian corals. Furthermore, Isopora/Acropora individuals display at least two, and as many as three, ITS sequence isotypes in their genome which appear to be capable of function. From consideration of the conserved elements in ITS2 and flanking regions, it appears that there are three major groups within the IsoporaAcropora lineage: the Isopora + Acropora "longi" group, the large group including Caribbean Acropora + the Acropora "carib" types plus the bulk of the Indo-Pacific Acropora species, and the remaining enigmatic "pseudo" group found in the Pacific. Interbreeding is possible among Caribbean A. palmata and A. cervicornis and among some species of Indo-Pacific Acropora. Recombinant ITS sequences are obvious among these latter, such that morphology (as represented by species name) does not correlate with common ITS sequence. The combination of characters revealed by RNA secondary structure analyses suggests a recent past/current history of interbreeding among the Indo-Pacific Acropora species and a shared ancestry of some of these with the Caribbean Acropora. The unusual absence of helix III of ITS2 of Isopora/Acropora species may have some causative role in the equally unusual instability in the 5.8S-LSU helix basal to ITS2 of this species complex.

  9. Mechanism of intermediate filament recognition by plakin repeat domains revealed by envoplakin targeting of vimentin

    Science.gov (United States)

    Fogl, Claudia; Mohammed, Fiyaz; Al-Jassar, Caezar; Jeeves, Mark; Knowles, Timothy J.; Rodriguez-Zamora, Penelope; White, Scott A.; Odintsova, Elena; Overduin, Michael; Chidgey, Martyn

    2016-03-01

    Plakin proteins form critical connections between cell junctions and the cytoskeleton; their disruption within epithelial and cardiac muscle cells cause skin-blistering diseases and cardiomyopathies. Envoplakin has a single plakin repeat domain (PRD) which recognizes intermediate filaments through an unresolved mechanism. Herein we report the crystal structure of envoplakin's complete PRD fold, revealing binding determinants within its electropositive binding groove. Four of its five internal repeats recognize negatively charged patches within vimentin via five basic determinants that are identified by nuclear magnetic resonance spectroscopy. Mutations of the Lys1901 or Arg1914 binding determinants delocalize heterodimeric envoplakin from intracellular vimentin and keratin filaments in cultured cells. Recognition of vimentin is abolished when its residues Asp112 or Asp119 are mutated. The latter slot intermediate filament rods into basic PRD domain grooves through electrosteric complementarity in a widely applicable mechanism. Together this reveals how plakin family members form dynamic linkages with cytoskeletal frameworks.

  10. Pyrosequencing reveals the key microorganisms involved in sludge alkaline fermentation for efficient short-chain fatty acids production.

    Science.gov (United States)

    Zheng, Xiong; Su, Yinglong; Li, Xiang; Xiao, Naidong; Wang, Dongbo; Chen, Yinguang

    2013-05-07

    Short-chain fatty acids (SCFAs) have been regarded as the excellent carbon source of wastewater biological nutrient removal, and sludge alkaline (pH 10) fermentation has been reported to achieve highly efficient SCFAs production. In this study, the underlying mechanisms for the improved SCFAs production at pH 10 were investigated by using 454 pyrosequencing and fluorescent in situ hybridization (FISH) to analyze the microbial community structures in sludge fermentation reactors. It was found that sludge fermentation at pH 10 increased the abundances of Pseudomonas sp. and Alcaligenes sp., which were able to excrete extracellular proteases and depolymerases, and thus enhanced the hydrolysis of insoluble sludge protein and polyhydroxyalkanoates (PHA). Meanwhile, the abundance of acid-producing bacteria (such as Clostridium sp.) in the reactor of pH 10 was also higher than that of uncontrolled pH, which benefited the acidification of soluble organic substrates. Further study indicated that sludge fermentation at pH 10 significantly decreased the number of methanogenic archaea, resulting in lower SCFAs consumption and lower methane production. Therefore, anaerobic sludge fermentation under alkaline conditions increased the abundances of bacteria involved in sludge hydrolysis and acidification, and decreased the abundance of methanogenic archaea, which favored the competition of bacteria over methanogens and resulted in the efficient production of SCFAs.

  11. Evaluation of Secure Multi-Hop Node Authentication and Key Establishment Mechanisms for Wireless Sensor Networks

    Directory of Open Access Journals (Sweden)

    Ismail Mansour

    2014-09-01

    Full Text Available Designing secure authentication mechanisms in wireless sensor networks in order to associate a node to a secure network is not an easy task due to the limitations of this type of networks. In this paper, we propose different multi-hop node authentication protocols for wireless sensor networks. For each protocol, we provide a formal proof to verify the security of our proposals using Scyther, which is an automatic cryptographic protocols verification tool. We also provide implementation results in terms of execution time consumption obtained by real measurements on TelosB motes. These protocols offer different security mechanisms depending on the design of the protocol itself. Moreover, we evaluate the overhead of protection of each solution by studying the effect on execution time overhead of each protocol. Finally, we propose a mechanism to detect possible attack based on our evaluation results.

  12. An analysis of key issues in the clean development mechanism based on the UNEP Risoe clean development mechanism pipeline

    DEFF Research Database (Denmark)

    Fenhann, Jørgen Villy; Staun, Frederik

    2010-01-01

    This paper provides a description of the analysis contained in the UNEP Risoe Clean Development Mechanism (CDM) pipeline not available on the site, some of the impacts/problems of the CDM and a way forward post-2012. The successful development of the CDM since it started in December 2003 is docum......This paper provides a description of the analysis contained in the UNEP Risoe Clean Development Mechanism (CDM) pipeline not available on the site, some of the impacts/problems of the CDM and a way forward post-2012. The successful development of the CDM since it started in December 2003...... is documented. The CDM pipeline includes calculation of the investment in the CDM projects. The total investment in the 2171 registered CDM projects is approximately US$60 billion. It is shown that programmatic CDM gives hope for an increased share of projects in Africa, since 16% of the programmatic CDM...

  13. The firing patterns of spinal neurons: in situ patch-clamp recordings reveal a key role for potassium currents.

    Science.gov (United States)

    Winlove, Crawford I P; Roberts, Alan

    2012-10-01

    Neuron firing patterns underpin the detection and processing of stimuli, influence synaptic interactions, and contribute to the function of networks. To understand how intrinsic membrane properties determine firing patterns, we investigated the biophysical basis of single and repetitive firing in spinal neurons of hatchling Xenopus laevis tadpoles, a well-understood vertebrate model; experiments were conducted in situ. Primary sensory Rohon-Beard (RB) neurons fire singly in response to depolarising current, and dorsolateral (DL) interneurons fire repetitively. RB neurons exhibited a large tetrodotoxin-sensitive sodium current; in DL neurons, the sodium current density was significantly lower. High-voltage-activated calcium currents were similar in both neuron types. There was no evidence of persistent sodium currents, low-voltage-activated calcium currents, or hyperpolarisation-activated currents. In RB neurons, the potassium current was dominated by a tetraethylammonium-sensitive slow component (I(Ks) ); a fast component (I(Kf) ), sensitive to 4-aminopyridine, predominated in DL neurons. Sequential current-clamp and voltage-clamp recordings in individual neurons suggest that high densities of I(Ks) prevent repetitive firing; where I(Ks) is small, I(Kf) density determines the frequency of repetitive firing. Intermediate densities of I(Ks) and I(Kf) allow neurons to fire a few additional spikes on strong depolarisation; this property typifies a novel subset of RB neurons, and may activate escape responses. We discuss how this ensemble of currents and firing patterns underpins the operation of the Xenopus locomotor network, and suggest how simple mechanisms might underlie the similar firing patterns seen in the neurons of diverse species.

  14. Microstructures and Key Properties of Mechanically Deposited Zn-Al Coatings

    Institute of Scientific and Technical Information of China (English)

    WANG Shengmin; HE Mingyi; ZHAO Xiaojun; PENG Zenghua; LIU Li

    2009-01-01

    Zn-Al coatings can provide protection to exposed steel parts in most environments. For this reason, the investigation of Zn-Al coatings become very popular in recent years. In order to study the microstructures and properties of mechanically deposited Zn-Al coating, zinc powders and aluminum powders were used to deposit Zn-Al coating by mechanical plating. The microstructures, phase constitutes and compositions of the coating were observed and analyzed with optical microscopy (OM), scanning electron microscopy(SEM), X-ray diffraction(XRD) and X-ray energy-dispersive spectroscopy(EDS). The results of observation show that the coating consists of almost spherically shaped zinc particles point contacting with each other; the coatings are composed of zinc particles, aluminum particles, interstice, and tin; extra fine zinc powders and some smaller interspersed inclusions are positioned in the interstices. Porosity and thickness of the coating were tested by ferroxyl test and magnetic method. The corrosion resistance of coatings was studied by neutral salt spraying test(NSS) , immersion test and electrochemical polarization. It is found that the thickness of the coating dose lacks uniformity, with an uneven thickness distribution and an average variation of approximately 2-5μm; the coating can afford cathodic protection to the steel substrate; the corrosion resistance of Zn-Al coatings is better than that of the mechanically plated zinc coatings with same thickness. These conclusions can be applied to improve anti-corrosion performance by mechanically deposit Zn-Al coatings.

  15. Temperature Dependence of Laser-Induced Demagnetization in Ni: A Key for Identifying the Underlying Mechanism

    Science.gov (United States)

    Roth, T.; Schellekens, A. J.; Alebrand, S.; Schmitt, O.; Steil, D.; Koopmans, B.; Cinchetti, M.; Aeschlimann, M.

    2012-04-01

    The microscopic mechanisms responsible for the ultrafast loss of magnetic order triggered in ferromagnetic metals by optical excitation are still under debate. One of the ongoing controversies is about the thermal origin of ultrafast demagnetization. Although different theoretical investigations support a main driving mechanism of thermal origin, alternative descriptions in terms of coherent interaction between the laser and the spin system or superdiffusive spin transport have been proposed. Another important matter of debate originates from the experimental observation of two time scales in the demagnetization dynamics of the 4f ferromagnet gadolinium. Here, it is still unclear whether it is necessary to invoke two distinct microscopic mechanisms to explain such behavior, or if one single mechanism is indeed sufficient. To uncover the physics behind these two unsolved issues, we explore the dependence of ultrafast-demagnetization dynamics in nickel through a survey of different laser intensities and ambient temperatures. Measurements in a large range of these external parameters are performed by means of the time-resolved magneto-optical Kerr effect and display a pronounced change in the maximum loss of magnetization and in the temporal profile of the demagnetization traces. The most striking observation is that the same material system (nickel) can show a transition from a one-step (one time scale) to a two-step (two time scales) demagnetization, occurring on increasing the ambient temperature. We find that the fluence and the temperature dependence of ultrafast demagnetization—including the transition from one-step to two-step dynamics—are reproduced theoretically assuming only a single scattering mechanism coupling the spin system to the temperature of the electronic system. This finding means that the origin of ultrafast demagnetization is thermal and that only a single microscopic channel is sufficient to describe magnetization dynamics in the 3d

  16. Could the mechanisms of bariatric surgery hold the key for novel therapies?

    DEFF Research Database (Denmark)

    Tam, C S; Berthoud, H-R; Bueter, M;

    2011-01-01

    Bariatric surgery is the most effective method for promoting dramatic and durable weight loss in morbidly obese subjects. Furthermore, type 2 diabetes is resolved in over 80% of patients. The mechanisms behind the amelioration in metabolic abnormalities are largely unknown but may be due to chang...... of obesity and diabetes. Promising data on energy metabolism, gastrointestinal physiology, hedonic response and food intake were reviewed and discussed....

  17. Hydrolytic degradation of N,N‧-ethylenedimaleimide: Crystal structures of key intermediates and proposed mechanisms

    Science.gov (United States)

    Tan, Xue-Jie; Cheng, Shuang-Shuang; Shi, Yan; Xing, Dian-Xiang; Liu, Yun; Li, Hui; Feng, Wen-Quan; Yang, Jian-Bo

    2016-12-01

    Maleimide groups are used extensively in bioconjugation reactions, but limited mechanistic studies are available regarding their hydrolysis reactions. In this paper, five single-crystal structures related with the reaction of four-step hydrolytic degradation of N,N‧-ethylenedimaleimide have been investigated. On the basis of experimental results, the reaction mechanisms without or with water catalysis are proposed, which could provide some enlightenment into the study of similar hydrolytic degradations.

  18. A key mechanism underlying sensory experience-dependent maturation of neocortical GABAergic circuits in vivo

    OpenAIRE

    Jiao, Yuanyuan; Zhang, Zhi; Zhang, Chunzhao; Wang, Xinjun; Sakata, Kazuko; Lu, Bai; Sun, Qian-Quan

    2011-01-01

    Mechanisms underlying experience-dependent refinement of cortical connections, especially GABAergic inhibitory circuits, are unknown. By using a line of mutant mice that lack activity-dependent BDNF expression (bdnf-KIV), we show that experience regulation of cortical GABAergic network is mediated by activity-driven BDNF expression. Levels of endogenous BDNF protein in the barrel cortex are strongly regulated by sensory inputs from whiskers. There is a severe alteration of excitation and inhi...

  19. Lamins, laminopathies and disease mechanisms: Possible role for proteasomal degradation of key regulatory proteins

    Indian Academy of Sciences (India)

    Veena K Parnaik; Pankaj Chaturvedi; B H Muralikrishna

    2011-08-01

    Lamins are major structural proteins of the nucleus and are essential for nuclear integrity and organization of nuclear functions. Mutations in the human lamin genes lead to highly degenerative genetic diseases that affect a number of different tissues such as muscle, adipose or neuronal tissues, or cause premature ageing syndromes. New findings on the role of lamins in cellular signalling pathways, as well as in ubiquitin-mediated proteasomal degradation, have given important insights into possible mechanisms of pathogenesis.

  20. [Key molecular mechanisms associated with cell malignant transformation in acute myeloid leukemia].

    Science.gov (United States)

    Orlova, N N; Lebedev, T D; Spirin, P V; Prassolov, V S

    2016-01-01

    Cancer, along with cardiovascular disorders, is one of the most important problems of healthcare. Pathologies of the hematopoietic system are the most prevalent in patients under 30 years of age, including acute myeloid leukemia (AML), which is widespread and difficult to treat. The review considers the mechanisms that play a significant role in AML cell malignant transformation and shows the contributions of certain genes to both remission and resistance of AML cells to various treatments.

  1. Quantum mechanics of excitation transport in photosynthetic complexes: a key issues review.

    Science.gov (United States)

    Levi, Federico; Mostarda, Stefano; Rao, Francesco; Mintert, Florian

    2015-07-01

    For a long time microscopic physical descriptions of biological processes have been based on quantum mechanical concepts and tools, and routinely employed by chemical physicists and quantum chemists. However, the last ten years have witnessed new developments on these studies from a different perspective, rooted in the framework of quantum information theory. The process that more, than others, has been subject of intense research is the transfer of excitation energy in photosynthetic light-harvesting complexes, a consequence of the unexpected experimental discovery of oscillating signals in such highly noisy systems. The fundamental interdisciplinary nature of this research makes it extremely fascinating, but can also constitute an obstacle to its advance. Here in this review our objective is to provide an essential summary of the progress made in the theoretical description of excitation energy dynamics in photosynthetic systems from a quantum mechanical perspective, with the goal of unifying the language employed by the different communities. This is initially realized through a stepwise presentation of the fundamental building blocks used to model excitation transfer, including protein dynamics and the theory of open quantum system. Afterwards, we shall review how these models have evolved as a consequence of experimental discoveries; this will lead us to present the numerical techniques that have been introduced to quantitatively describe photo-absorbed energy dynamics. Finally, we shall discuss which mechanisms have been proposed to explain the unusual coherent nature of excitation transport and what insights have been gathered so far on the potential functional role of such quantum features.

  2. Quantum mechanics of excitation transport in photosynthetic complexes: a key issues review

    Science.gov (United States)

    Levi, Federico; Mostarda, Stefano; Rao, Francesco; Mintert, Florian

    2015-07-01

    For a long time microscopic physical descriptions of biological processes have been based on quantum mechanical concepts and tools, and routinely employed by chemical physicists and quantum chemists. However, the last ten years have witnessed new developments on these studies from a different perspective, rooted in the framework of quantum information theory. The process that more, than others, has been subject of intense research is the transfer of excitation energy in photosynthetic light-harvesting complexes, a consequence of the unexpected experimental discovery of oscillating signals in such highly noisy systems. The fundamental interdisciplinary nature of this research makes it extremely fascinating, but can also constitute an obstacle to its advance. Here in this review our objective is to provide an essential summary of the progress made in the theoretical description of excitation energy dynamics in photosynthetic systems from a quantum mechanical perspective, with the goal of unifying the language employed by the different communities. This is initially realized through a stepwise presentation of the fundamental building blocks used to model excitation transfer, including protein dynamics and the theory of open quantum system. Afterwards, we shall review how these models have evolved as a consequence of experimental discoveries; this will lead us to present the numerical techniques that have been introduced to quantitatively describe photo-absorbed energy dynamics. Finally, we shall discuss which mechanisms have been proposed to explain the unusual coherent nature of excitation transport and what insights have been gathered so far on the potential functional role of such quantum features.

  3. Transcriptome Analysis Reveals Key Flavonoid 3'-Hydroxylase and Flavonoid 3',5'-Hydroxylase Genes in Affecting the Ratio of Dihydroxylated to Trihydroxylated Catechins in Camellia sinensis.

    Directory of Open Access Journals (Sweden)

    Kang Wei

    Full Text Available The ratio of dihydroxylated to trihydroxylated catechins (RDTC is an important indicator of tea quality and biochemical marker for the study of genetic diversity. It is reported to be under genetic control but the underlying mechanism is not well understood. Flavonoid 3'-hydroxylase (F3'H and flavonoid 3',5'-hydroxylase (F3'5'H are key enzymes involved in the formation of dihydroxylated and trihydroxylated catechins. The transcriptome and HPLC analysis of tea samples from Longjing43 and Zhonghuang2 under control and shading treatment were performed to assess the F3'H and F3'5'H genes that might affect RDTC. A total of 74.7 million reads of mRNA seq (2×101bp data were generated. After de novo assembly, 109,909 unigenes were obtained, and 39,982 of them were annotated using 7 public databases. Four key F3'H and F3'5'H genes (including CsF3'5'H1, CsF3'H1, CsF3'H2 and CsF3'H3 were identified to be closely correlated with RDTC. Shading treatment had little effect on RDTC, which was attributed to the stable expression of these key F3'H and F3'5'H genes. The correlation of the coexpression of four key genes and RDTC was further confirmed among 13 tea varieties by real time PCR and HPLC analysis. The coexpression of three F3'H genes and a F3'5'H gene may play a key role in affecting RDTC in Camellia sinensis. The current results may establish valuable foundation for further research about the mechanism controlling catechin composition in tea.

  4. Large scale flows in transitional plane Couette flow: a key ingredient of the spot growth mechanism

    CERN Document Server

    Couliou, M

    2015-01-01

    Using Particle Image Velocimetry (PIV) in a new experimental plane Couette flow, we investigate the dynamics of turbulent patches invading formerly laminar flows. We evidence experimentally for the first time in this geometry the existence of large scale flows. These flows appear as soon as laminar and turbulent domains coexist. Spectral analysis is used to study the dynamical evolution of these large scales as well as that of the small scales associated with turbulence. We show that large-scale flows grow before turbulent spots develop and we point out the crucial role they play in the growth mechanism and possibly also in the emergence of organised patterns.

  5. Failure analysis of porcupine quills under axial compression reveals their mechanical response during buckling.

    Science.gov (United States)

    Torres, Fernando G; Troncoso, Omar P; Diaz, John; Arce, Diego

    2014-11-01

    Porcupine quills are natural structures formed by a thin walled conical shell and an inner foam core. Axial compression tests, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and Fourier transform infrared spectroscopy (FT-IR) were all used to compare the characteristics and mechanical properties of porcupine quills with and without core. The failure mechanisms that occur during buckling were analyzed by scanning electron microscopy (SEM), and it was found that delamination buckling is mostly responsible for the decrease in the measured buckling stress of the quills with regard to predicted theoretical values. Our analysis also confirmed that the foam core works as an energy dissipater improving the mechanical response of an empty cylindrical shell, retarding the onset of buckling as well as producing a step wise decrease in force after buckling, instead of an instantaneous decrease in force typical for specimens without core. Cell collapse and cell densification in the inner foam core were identified as the key mechanisms that allow for energy absorption during buckling.

  6. Experimental determination of the key heat transfer mechanisms in pharmaceutical freeze-drying.

    Science.gov (United States)

    Ganguly, Arnab; Nail, Steven L; Alexeenko, Alina

    2013-05-01

    The study is aimed at quantifying the relative contribution of key heat transfer modes in lyophilization. Measurements of vial heat transfer rates in a laboratory-scale freeze-dryer were performed using pure water, which was partially sublimed under various conditions. The separation distance between the shelf and the vial was systematically varied, and sublimation rates were determined gravimetrically. The heat transfer rates were observed to be independent of separation distance between the vial and the shelf and linearly dependent on pressure in the free molecular flow limit, realized at low pressures (120 mTorr), heat transfer rates were independent of pressure and inversely proportional to separation distance. Previous heat transfer studies in conventional freeze-drying cycles have attributed a dominant portion of the total heat transfer to radiation, the rest to conduction, whereas convection has been found to be insignificant. Although the measurements reported here confirm the significance of the radiative and gas conduction components, the convective component has been found to be comparable to the gas conduction contribution at pressures greater than 100 mTorr. The current investigation supports the conclusion that the convective component of the heat transfer cannot be ignored in typical laboratory-scale freeze-drying conditions. Copyright © 2013 Wiley Periodicals, Inc.

  7. Lifestyles and Ageing: Targeting Key Mechanisms to Shift the Balance from Unhealthy to Healthy Ageing.

    Science.gov (United States)

    Grassi, Claudio; Landi, Francesco; Delogu, Giovanni

    2014-01-01

    The increase in life expectancy has dramatically enhanced the prevalence of age-related chronic diseases resulting in growing costs for both society and individuals. Identification of strategies contributing to healthy ageing is thus one of the major challenges of the coming years. Lifestyle has a primary role among non-genetic factors affecting health and lifespan. In particular, nutrition, mental and physical activity impact the molecular and functional mechanisms whose alterations cause the major age-related diseases. A better understanding of mechanisms underlying the beneficial action of correct lifestyles is useful to develop interventions aimed at preventing and/or delaying the onset of chronic degenerative diseases, to identify high-risk populations who could be targeted in intervention trials as well as to identify novel biomarkers of healthy ageing. A multidisciplinary team of basic scientists and clinicians operating at the Catholic University Medical School in Rome is actively working on this topic to determine the ability of healthy lifestyles to promote active ageing and counteract the major age-related diseases affecting brain health, musculoskeletal function and gut microenvironment. This chapter summarizes our strategic approaches, the major results we obtained so far and the main experimental and translational perspectives.

  8. Deformation mechanism of surrounding rocks and key control technology for a roadway driven along goaf in fully mechanized top-coal caving face

    Institute of Scientific and Technical Information of China (English)

    李学华

    2003-01-01

    The variation of the stress in the bolted surrounding rocks structure of the roadway driven along goaf in a fully mechanized top-coal caving face with moderate stable conditions are studied by using numerical calculation. The essential deformation characteristics of the surrounding rocks in this kind of roadway are obtained and the key technology of bolting support used under these conditions is put forward.

  9. Deformation mechanism of surrounding rocks and key control technology for a roadway driven along goaf in fully mechanized top-coal caving face

    Energy Technology Data Exchange (ETDEWEB)

    Li, X. [China University of Mining and Technology, Xuzhou (China). School of Energy Science and Engineering

    2003-06-01

    The variation of the stress in the bolted surrounding rocks structure of the roadway driven along the goaf in a fully mechanized top-coal caving face with moderate stable conditions are studied by using numerical calculation. The essential deformation characteristics of the surrounding rocks in this kind of roadway are obtained and the key technology of bolting support used under these conditions is put forward. 3 refs., 2 figs., 1 tab.

  10. Analysis on mechanism and key factors of surrounding rock instability in deeply inclined roadway affected by argillation and water seepage

    Institute of Scientific and Technical Information of China (English)

    Ma Rui; Li Guichen; Zhang Nong; Liu Cong; Wei Yinghao; Zhang Ming

    2015-01-01

    Based on the characteristics of surrounding rocks for deeply inclined roadway affected by argillation and water seepage, a structure model of layer crack plate was established to analyze the shear sliding insta-bility mechanism. Through solid mechanics analysis of anchored surrounding rock with defect from water seepage, combined with numerical analysis for instability mechanism under water seepage in deeply inclined roadway, key factors were proposed. Results show that with increasing height of layer crack plate, lateral buckling critical load value for high wall of the roadway decreases;there is a multi-stage distribution for tensile stress along the anchor bolt with defect under pulling state condition;groundwater seepage seriously affects the strength of surrounding rock of the roadway, to some extent the plastic zone of the high side rises up to 8 m. Finally some support strategies were proposed for the inclined roadway and successfully applied to Haoyuan coal mine in Tiela mining area, western China.

  11. Original mechanism of failure initiation revealed through modelling of naturally occurring microstructures

    Science.gov (United States)

    Gorbatikh, Larissa; Lomov, Stepan V.; Verpoest, Ignaas

    2010-05-01

    Motivated to reveal original mechanisms of failure resistance, we developed a material model that encompasses most reoccurring microstructural features of natural composites. The interesting result of the work is a notion that material failure is governed by the quality of interactions between hierarchical levels in the material microstructure. With intelligent use of the structure, these interactions can be tuned to create a powerful synergetic effect on the material failure behaviour. For example, while exploring different mechanisms of failure initiation in composites with bimodal size reinforcements (an indirect way to model two levels of hierarchy simultaneously) we found that failure initiation could be shifted from stress concentration sites of the higher level to the lower level. One could say that the material behaviour became insensitive to the presence of reinforcements on the higher level—a phenomenon that is counterintuitive to what is commonly known. The new mechanism of failure initiation could only be activated in composites with a highly controlled structural organization—in the studied case, reinforcements of the lower level needed to establish lamellar pathways between reinforcements of the higher level. These pathways lead to formation of an intriguing network-like microstructure. Intelligent communication between reinforcements in such a network created the necessary synergy to change the failure initiation mechanism in a discontinuous fashion. Another finding was that by establishing such a network, tensile stresses near dangerous stress concentration sites were locally transformed into compressive stresses. Resemblance of the revealed mechanism to phenomena on the nano-scale was also discussed. In the course of this work a new method was developed to investigate interactions between reinforcements and their collective input into effective and local properties of a composite. The reinforcement phase was modelled with the use of rigid

  12. Systems pharmacology exploration of botanic drug pairs reveals the mechanism for treating different diseases

    Science.gov (United States)

    Zhou, Wei; Wang, Jinan; Wu, Ziyin; Huang, Chao; Lu, Aiping; Wang, Yonghua

    2016-11-01

    Multi-herb therapy has been widely used in Traditional Chinese medicine and tailored to meet the specific needs of each individual. However, the potential molecular or systems mechanisms of them to treat various diseases have not been fully elucidated. To address this question, a systems pharmacology approach, integrating pharmacokinetics, pharmacology and systems biology, is used to comprehensively identify the drug-target and drug-disease networks, exemplified by three representative Radix Salviae Miltiorrhizae herb pairs for treating various diseases (coronary heart disease, dysmenorrheal and nephrotic syndrome). First, the compounds evaluation and the multiple targeting technology screen the active ingredients and identify the specific targets for each herb of three pairs. Second, the herb feature mapping reveals the differences in chemistry and pharmacological synergy between pairs. Third, the constructed compound-target-disease network explains the mechanisms of treatment for various diseases from a systematic level. Finally, experimental verification is taken to confirm our strategy. Our work provides an integrated strategy for revealing the mechanism of synergistic herb pairs, and also a rational way for developing novel drug combinations for treatments of complex diseases.

  13. Mechanical properties of a complete microtubule revealed through molecular dynamics simulation.

    Science.gov (United States)

    Wells, David B; Aksimentiev, Aleksei

    2010-07-21

    Microtubules (MTs) are the largest type of cellular filament, essential in processes ranging from mitosis and meiosis to flagellar motility. Many of the processes depend critically on the mechanical properties of the MT, but the elastic moduli, notably the Young's modulus, are not directly revealed in experiment, which instead measures either flexural rigidity or response to radial deformation. Molecular dynamics (MD) is a method that allows the mechanical properties of single biomolecules to be investigated through computation. Typically, MD requires an atomic resolution structure of the molecule, which is unavailable for many systems, including MTs. By combining structural information from cryo-electron microscopy and electron crystallography, we have constructed an all-atom model of a complete MT and used MD to determine its mechanical properties. The simulations revealed nonlinear axial stress-strain behavior featuring a pronounced softening under extension, a possible plastic deformation transition under radial compression, and a distinct asymmetry in response to the two senses of twist. This work demonstrates the possibility of combining different levels of structural information to produce all-atom models suitable for quantitative MD simulations, which extends the range of systems amenable to the MD method and should enable exciting advances in our microscopic knowledge of biology. Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  14. Reciprocal osmotic challenges reveal mechanisms of divergence in phenotypic plasticity in the killifish Fundulus heteroclitus.

    Science.gov (United States)

    Brennan, Reid S; Galvez, Fernando; Whitehead, Andrew

    2015-04-15

    The killifish Fundulus heteroclitus is an estuarine species with broad physiological plasticity, enabling acclimation to diverse stressors. Previous work suggests that freshwater populations expanded their physiology to accommodate low salinity environments; however, it is unknown whether this compromises their tolerance to high salinity. We used a comparative approach to investigate the mechanisms of a derived freshwater phenotype and the fate of an ancestral euryhaline phenotype after invasion of a freshwater environment. We compared physiological and transcriptomic responses to high- and low-salinity stress in fresh and brackish water populations and found an enhanced plasticity to low salinity in the freshwater population coupled with a reduced ability to acclimate to high salinity. Transcriptomic data identified genes with a conserved common response, a conserved salinity-dependent response and responses associated with population divergence. Conserved common acclimation responses revealed stress responses and alterations in cell-cycle regulation as important mechanisms in the general osmotic response. Salinity-specific responses included the regulation of genes involved in ion transport, intracellular calcium, energetic processes and cellular remodeling. Genes diverged between populations were primarily those showing salinity-specific expression and included those regulating polyamine homeostasis and the cell cycle. Additionally, when populations were matched with their native salinity, expression patterns were consistent with the concept of 'transcriptomic resilience', suggesting local adaptation. These findings provide insight into the fate of a plastic phenotype after a shift in environmental salinity and help to reveal mechanisms allowing for euryhalinity.

  15. Resilience and Vulnerability of Arctic and Boreal Ecosystems: Key Mechanisms and Habitats

    Science.gov (United States)

    Juday, G. P.; Trouillier, M.; Morimoto, M.

    2016-12-01

    The northwestern North American boreal forest experienced a rapid regime shift to elevated temperatures in the mid 1970s, with profound consequences for forests. The common tree species aspen (Asp), Alaska birch (Abir), and white (Wspr) and black spruce display both vulnerability and resilience. Tree growth of all species is well modeled by empirical temperature indices, with both positive and negative temperature relationships. Extreme tree stress and mortality have been detected in radial growth and photo monitoring at Bonanza Creek LTER and across interior boreal Alaska. However, trees on formerly marginally cool sites and regions are resilient or vigorous and accelerating in growth. Wspr experiences an exponential decline in photosynthesis between 21 and 28 deg. C, and such temperatures occur more frequently in the interior boreal region. Abir displays extensive evidence of top dieback from hydraulic conductivity failure, and is experiencing extensive defoliation from leaf miners, as did Asp. However, summer precipitation in interior Alaska has been at record levels since 2014, and unprecedented radial growth occurred in Wspr on the warmest and driest sites. The phenology of Wspr height elongation is effectively modeled by an empirically derived model of temperature and precipitation. The modeled date of cessation of growth occurs earlier by about 3 weeks over the past century, which nearly matches the advance of spring growth. A geographically coherent picture is emerging of systematic climate-driven change, overlain with exceptional events (insect outbreaks, droughts, heavy rains) in space, time, or type. One of the most vulnerable boreal forest habitats is old-growth (150+ yrs.) white spruce, which is a key component of biodiversity, providing important structures for birds, lichens, and mammals, and the most favorable opportunity for wood harvest. Resource management law and policy mandates to maintain ecosystems are mismatched with the unfolding reality.

  16. Sucrose is an early modulator of the key hormonal mechanisms controlling bud outgrowth in Rosa hybrida.

    Science.gov (United States)

    Barbier, François; Péron, Thomas; Lecerf, Marion; Perez-Garcia, Maria-Dolores; Barrière, Quentin; Rolčík, Jakub; Boutet-Mercey, Stéphanie; Citerne, Sylvie; Lemoine, Remi; Porcheron, Benoît; Roman, Hanaé; Leduc, Nathalie; Le Gourrierec, José; Bertheloot, Jessica; Sakr, Soulaiman

    2015-05-01

    Sugar has only recently been identified as a key player in triggering bud outgrowth, while hormonal control of bud outgrowth is already well established. To get a better understanding of sugar control, the present study investigated how sugar availability modulates the hormonal network during bud outgrowth in Rosa hybrida. Other plant models, for which mutants are available, were used when necessary. Buds were grown in vitro to manipulate available sugars. The temporal patterns of the hormonal regulatory network were assessed in parallel with bud outgrowth dynamics. Sucrose determined bud entrance into sustained growth in a concentration-dependent manner. Sustained growth was accompanied by sustained auxin production in buds, and sustained auxin export in a DR5::GUS-expressing pea line. Several events occurred ahead of sucrose-stimulated bud outgrowth. Sucrose upregulated early auxin synthesis genes (RhTAR1, RhYUC1) and the auxin efflux carrier gene RhPIN1, and promoted PIN1 abundance at the plasma membrane in a pPIN1::PIN1-GFP-expressing tomato line. Sucrose downregulated both RwMAX2, involved in the strigolactone-transduction pathway, and RhBRC1, a repressor of branching, at an early stage. The presence of sucrose also increased stem cytokinin content, but sucrose-promoted bud outgrowth was not related to that pathway. In these processes, several non-metabolizable sucrose analogues induced sustained bud outgrowth in R. hybrida, Pisum sativum, and Arabidopsis thaliana, suggesting that sucrose was involved in a signalling pathway. In conclusion, we identified potential hormonal candidates for bud outgrowth control by sugar. They are central to future investigations aimed at disentangling the processes that underlie regulation of bud outgrowth by sugar.

  17. Probing molecular mechanisms of the Hsp90 chaperone: biophysical modeling identifies key regulators of functional dynamics.

    Directory of Open Access Journals (Sweden)

    Anshuman Dixit

    Full Text Available Deciphering functional mechanisms of the Hsp90 chaperone machinery is an important objective in cancer biology aiming to facilitate discovery of targeted anti-cancer therapies. Despite significant advances in understanding structure and function of molecular chaperones, organizing molecular principles that control the relationship between conformational diversity and functional mechanisms of the Hsp90 activity lack a sufficient quantitative characterization. We combined molecular dynamics simulations, principal component analysis, the energy landscape model and structure-functional analysis of Hsp90 regulatory interactions to systematically investigate functional dynamics of the molecular chaperone. This approach has identified a network of conserved regions common to the Hsp90 chaperones that could play a universal role in coordinating functional dynamics, principal collective motions and allosteric signaling of Hsp90. We have found that these functional motifs may be utilized by the molecular chaperone machinery to act collectively as central regulators of Hsp90 dynamics and activity, including the inter-domain communications, control of ATP hydrolysis, and protein client binding. These findings have provided support to a long-standing assertion that allosteric regulation and catalysis may have emerged via common evolutionary routes. The interaction networks regulating functional motions of Hsp90 may be determined by the inherent structural architecture of the molecular chaperone. At the same time, the thermodynamics-based "conformational selection" of functional states is likely to be activated based on the nature of the binding partner. This mechanistic model of Hsp90 dynamics and function is consistent with the notion that allosteric networks orchestrating cooperative protein motions can be formed by evolutionary conserved and sparsely connected residue clusters. Hence, allosteric signaling through a small network of distantly connected

  18. Mutual information reveals multiple structural relaxation mechanisms in a model glass former.

    Science.gov (United States)

    Dunleavy, Andrew J; Wiesner, Karoline; Yamamoto, Ryoichi; Royall, C Patrick

    2015-01-22

    Among the key challenges to our understanding of solidification in the glass transition is that it is accompanied by little apparent change in structure. Recently, geometric motifs have been identified in glassy liquids, but a causal link between these motifs and solidification remains elusive. One 'smoking gun' for such a link would be identical scaling of structural and dynamic lengthscales on approaching the glass transition, but this is highly controversial. Here we introduce an information theoretic approach to determine correlations in displacement for particle relaxation encoded in the initial configuration of a glass-forming liquid. We uncover two populations of particles, one inclined to relax quickly, the other slowly. Each population is correlated with local density and geometric motifs. Our analysis further reveals a dynamic lengthscale similar to that associated with structural properties, which may resolve the discrepancy between structural and dynamic lengthscales.

  19. The dynamic nature of RNA as key to understanding riboswitch mechanisms.

    Science.gov (United States)

    Haller, Andrea; Soulière, Marie F; Micura, Ronald

    2011-12-20

    Riboswitches are gene regulation elements within RNA that recognize specific metabolites. They predominantly occur in the untranslated leader regions of bacterial messenger RNA (mRNA). Upon metabolite binding to the aptamer domain, a structural change in the adjoining downstream expression platform signals "on" or "off" for gene expression. Researchers have achieved much progress in characterizing ligand-bound riboswitch states at the molecular level; an impressive number of high-resolution structures of aptamer-ligand complexes is now available. These structures have significantly contributed toward our understanding of how riboswitches interact with their natural ligands and with structurally related analogues. In contrast, relatively little is known about the nature of the unbound (apo) form of riboswitches. Moreover, the details of how changes in the aptamer domain are transduced into conformational changes in the decision-making expression platform remain murky. In this Account, we report on recent efforts aimed at the characterization of free states, ligand recognition, and ligand-induced folding in riboswitches. Riboswitch action is best approached as a cotranscriptional process, which implies sequential folding and release of the aptamer prior to the signaling of the expression platform. Thus, a complex interplay of several factors has to be taken into account, such as speed of transcription, transcriptional pausing, kinetics and thermodynamics of RNA structure formation, and kinetics and thermodynamics of ligand binding. The response mechanism appears to be best described as a process in which ligand recognition critically dictates the folding pathway of the nascent mRNA during its expression; the resulting structures determine the interactions with the transcriptional or translational apparatus. We discuss experimental methods that offer insight into the dynamics of the free riboswitch state. These include probing experiments, such as in-line and

  20. Distinct levels in the nanoscale organization of DNA-histone complex revealed by its mechanical unfolding

    Science.gov (United States)

    Soni, G. V.; Brar, Loveleen; Hameed, Feroz M.; Raychaudhuri, A. K.; Shivashankar, G. V.

    2007-04-01

    Mechanical unfolding of nanoscale DNA-histone complex, using an atomic force microscope, shows a stepwise disassembly of histones from the nucleosome. A quantitative analysis of the rupture jump statistics and the length released per jump reveals insights into the possible histone contacts within the octamer complex. The measured ruptures correlate with the breakage of multiple contacts that stabilize the histone octamer. These results provide a mechanistic basis by which stepwise disassembly of histone proteins may result from an external force exerted by the adenosinetriphosphate (ATP) dependent chromatin remodeling machines to access regulatory sites on DNA.

  1. Utilizing a Key Aptamer Structure-Switching Mechanism for the Ultrahigh Frequency Detection of Cocaine.

    Science.gov (United States)

    Neves, Miguel A D; Blaszykowski, Christophe; Thompson, Michael

    2016-03-15

    Aptasensing of small molecules remains a challenge as detection often requires the use of labels or signal amplification methodologies, resulting in both difficult-to-prepare sensor platforms and multistep, complex assays. Furthermore, many aptasensors rely on the binding mechanism or structural changes associated with target capture by the aptameric probe, resulting in a detection scheme customized to each aptamer. It is in this context that we report herein a sensitive cocaine aptasensor that offers both real-time and label-free measurement capabilities. Detection relies on the electromagnetic piezoelectric acoustic sensor (EMPAS) platform. The sensing interface consists of a S-(11-trichlorosilyl-undecanyl)benzenethiosulfonate (BTS) adlayer-coated quartz disc onto which a structure-switching cocaine aptamer (MN6) is immobilized, completing the preparation of the MN6 cocaine aptasensor (M6CA). The EMPAS system has recently been employed as the foundation of a cocaine aptasensor based on a structurally rigid cocaine aptamer variant (MN4), an aptasensor referred to by analogy as M4CA. M6CA represents a significant increase in terms of analytical performance, compared to not only M4CA but also other cocaine aptamer-based sensors that do not rely on signal amplification, producing an apparent K(d) of 27 ± 6 μM and a 0.3 μM detection limit. Remarkably, the latter is in the range of that achieved by cocaine aptasensors relying on signal amplification. Furthermore, M6CA proved to be capable not only of regaining its cocaine-binding ability via simple buffer flow over the sensing interface (i.e., without the necessity to implement an additional regeneration step, such as in the case of M4CA), but also of detecting cocaine in a multicomponent matrix possessing potentially assay-interfering species. Finally, through observation of the distinct shape of its response profiles to cocaine injection, demonstration was made that the EMPAS system in practice offers the

  2. Elastoviscous Transitions of Articular Cartilage Reveal a Mechanism of Synergy between Lubricin and Hyaluronic Acid.

    Directory of Open Access Journals (Sweden)

    Edward D Bonnevie

    Full Text Available When lubricated by synovial fluid, articular cartilage provides some of the lowest friction coefficients found in nature. While it is known that macromolecular constituents of synovial fluid provide it with its lubricating ability, it is not fully understood how two of the main molecules, lubricin and hyaluronic acid, lubricate and interact with one another. Here, we develop a novel framework for cartilage lubrication based on the elastoviscous transition to show that lubricin and hyaluronic acid lubricate by distinct mechanisms. Such analysis revealed nonspecific interactions between these molecules in which lubricin acts to concentrate hyaluronic acid near the tissue surface and promotes a transition to a low friction regime consistent with the theory of viscous boundary lubrication. Understanding the mechanics of synovial fluid not only provides insight into the progression of diseases such as arthritis, but also may be applicable to the development of new biomimetic lubricants.

  3. Revealing −1 Programmed Ribosomal Frameshifting Mechanisms by Single-Molecule Techniques and Computational Methods

    Directory of Open Access Journals (Sweden)

    Kai-Chun Chang

    2012-01-01

    Full Text Available Programmed ribosomal frameshifting (PRF serves as an intrinsic translational regulation mechanism employed by some viruses to control the ratio between structural and enzymatic proteins. Most viral mRNAs which use PRF adapt an H-type pseudoknot to stimulate −1 PRF. The relationship between the thermodynamic stability and the frameshifting efficiency of pseudoknots has not been fully understood. Recently, single-molecule force spectroscopy has revealed that the frequency of −1 PRF correlates with the unwinding forces required for disrupting pseudoknots, and that some of the unwinding work dissipates irreversibly due to the torsional restraint of pseudoknots. Complementary to single-molecule techniques, computational modeling provides insights into global motions of the ribosome, whose structural transitions during frameshifting have not yet been elucidated in atomic detail. Taken together, recent advances in biophysical tools may help to develop antiviral therapies that target the ubiquitous −1 PRF mechanism among viruses.

  4. Revealing the Mechanism of Low-Energy Electron Yield Enhancement from Sensitizing Nanoparticles

    CERN Document Server

    Verkhovtsev, Alexey V; Solov'yov, Andrey V

    2014-01-01

    We provide a physical explanation for enhancement of the low-energy electron production by sensitizing nanoparticles due to irradiation by fast ions. It is demonstrated that a significant increase in the number of emitted electrons arises from the collective electron excitations in the nanoparticle. We predict a new mechanism of the yield enhancement due to the plasmon excitations and quantitatively estimate its contribution to the electron production. Revealing the nanoscale mechanism of the electron yield enhancement, we provide an efficient tool for evaluating the yield of emitted electron from various sensitizers. It is shown that the number of low-energy electrons generated by the gold and platinum nanoparticles of a given size exceeds that produced by the equivalent volume of water and by other metallic (e.g., gadolinium) nanoparticles by an order of magnitude. This observation emphasizes the sensitization effect of the noble metal nanoparticles and endorses their application in novel technologies of ca...

  5. Elastoviscous Transitions of Articular Cartilage Reveal a Mechanism of Synergy between Lubricin and Hyaluronic Acid.

    Science.gov (United States)

    Bonnevie, Edward D; Galesso, Devis; Secchieri, Cynthia; Cohen, Itai; Bonassar, Lawrence J

    2015-01-01

    When lubricated by synovial fluid, articular cartilage provides some of the lowest friction coefficients found in nature. While it is known that macromolecular constituents of synovial fluid provide it with its lubricating ability, it is not fully understood how two of the main molecules, lubricin and hyaluronic acid, lubricate and interact with one another. Here, we develop a novel framework for cartilage lubrication based on the elastoviscous transition to show that lubricin and hyaluronic acid lubricate by distinct mechanisms. Such analysis revealed nonspecific interactions between these molecules in which lubricin acts to concentrate hyaluronic acid near the tissue surface and promotes a transition to a low friction regime consistent with the theory of viscous boundary lubrication. Understanding the mechanics of synovial fluid not only provides insight into the progression of diseases such as arthritis, but also may be applicable to the development of new biomimetic lubricants.

  6. Edge-melting: nanoscale key-mechanism to explain nanoparticle formation from heated TEM grids

    Science.gov (United States)

    Cesaria, Maura; Taurino, Antonietta; Catalano, Massimo; Caricato, Anna Paola; Martino, Maurizio

    2016-03-01

    In this study, we examine at both experimental and fundamental levels, the experimental evidence of nanoparticle formation in transmission electron microscopy (TEM) metal grids annealed at temperatures lower than the melting point of the corresponding metal bulk material. Our experimental investigation considers the most thermally unstable TEM grids (i.e. Cu-grids) and inspects the possible sources and mechanisms of contamination of thin films, conventionally deposited on carbon-coated Cu-grids. The investigations are supported by morphological-compositional analyses performed in different regions of the TEM sample. Then, a general model is formulated and discussed in order to explain the grid thermal instability, based on the critical role of edge-melting (i.e. melting initiated at edges and corners of the grid bars), the enhanced rate of evaporation from a liquid surface and the polycristallinity of the grid bars. Hence, we totally disregard conventional arguments such as bulk evaporation and metal vapor pressure and, in order to emphasize and clarify the alternative point of view of our model, we also overview the nano-scale melting phenomenology relevant to our discussion and survey the discrepancies reported in the literature.

  7. Frugivores and seed dispersal: mechanisms and consequences for biodiversity of a key ecological interaction.

    Science.gov (United States)

    Jordano, Pedro; Forget, Pierre-Michel; Lambert, Joanna E; Böhning-Gaese, Katrin; Traveset, Anna; Wright, S Joseph

    2011-06-23

    The 5th Symposium on Frugivores and Seed Dispersal, held in Montpellier (France), 13-18 June 2010, brought together more than 220 researchers exemplifying a wide diversity of approaches to the study of frugivory and dispersal of seeds. Following Ted Fleming and Alejandro Estrada's initiative in 1985, this event was a celebration of the 25th anniversary of the first meeting in Veracruz, Mexico. Frugivory and seed dispersal are active research areas that have diversified in multiple directions since 1985 to include evolution (e.g. phylogenetic diversity and dispersal adaptations), physiology (e.g. sensory cues and digestion), landscape ecology (movement patterns), molecular ecology (e.g. gene flow, genetic diversity and structure), community ecology (e.g. mutualistic interaction networks) and conservation biology (effects of hunting, fragmentation, invasion and extinction), among others. This meeting provided an opportunity to assess conceptual and methodological progress, to present ever more sophisticated insights into frugivory in animals and dispersal patterns in plants, and to report the advances made in examining the mechanisms and consequences of seed dispersal for plants and frugivores.

  8. Muscle coordination is key to the power output and mechanical efficiency of limb movements.

    Science.gov (United States)

    Wakeling, J M; Blake, O M; Chan, H K

    2010-02-01

    The purpose of this study was to determine which features of muscle mechanics and muscle coordination affect the power output from a limb during locomotion. Eight subjects were tested while cycling at maximum exertion for 25 min on a stationary dynamometer. Cadence and load were varied to span a range of power outputs and myoelectric activity was measured from 10 muscles in the leg. Cycle-by-cycle variations in muscle coordination, cadence and power output were observed and the EMG intensity across all muscles was used as an estimate of the metabolic cost for each cycle. Data for the cycles at greatest power output were separated into three groups: maximum power, 80% power but lower EMG intensity and 80% power and higher EMG intensity. Torque-angular velocity relations were determined for the ankle and knee joints. During cycling at maximum power output the ankle joint was not extending at the velocity necessary for maximum power output; thus, maximum limb power occurs when some of the individual muscles cannot be generating maximum power output. Increases in EMG intensity occurred with no increase in power output from the limb: these corresponded to decreases in the efficiency and changes in coordination. Increases in power were achieved that were not matched by equivalent increases in EMG intensity, but did occur with changes in coordination. It is proposed that the power output from the limb is limited by the coordination pattern of the muscles rather than the maximum power output from any one muscle itself.

  9. Is phenotypic plasticity a key mechanism for responding to thermal stress in ants?

    Science.gov (United States)

    Oms, Cristela Sánchez; Cerdá, Xim; Boulay, Raphaël

    2017-06-01

    Unlike natural selection, phenotypic plasticity allows organisms to respond quickly to changing environmental conditions. However, plasticity may not always be adaptive. In insects, body size and other morphological measurements have been shown to decrease as temperature increases. This relationship may lead to a physiological conflict in ants, where larger body size and longer legs often confer better thermal resistance. Here, we tested the effect of developmental temperature (20, 24, 28 or 32 °C) on adult thermal resistance in the thermophilic ant species Aphaenogaster senilis. We found that no larval development occurred at 20 °C. However, at higher temperatures, developmental speed increased as expected and smaller adults were produced. In thermal resistance tests, we found that ants reared at 28 and 32 °C had half-lethal temperatures that were 2 °C higher than those of ants reared at 24 °C. Thus, although ants reared at higher temperatures were smaller in size, they were nonetheless more thermoresistant. These results show that A. senilis can exploit phenotypic plasticity to quickly adjust its thermal resistance to local conditions and that this process is independent of morphological adaptations. This mechanism may be particularly relevant given current rapid climate warming.

  10. A key mechanism underlying sensory experience-dependent maturation of neocortical GABAergic circuits in vivo.

    Science.gov (United States)

    Jiao, Yuanyuan; Zhang, Zhi; Zhang, Chunzhao; Wang, Xinjun; Sakata, Kazuko; Lu, Bai; Sun, Qian-Quan

    2011-07-19

    Mechanisms underlying experience-dependent refinement of cortical connections, especially GABAergic inhibitory circuits, are unknown. By using a line of mutant mice that lack activity-dependent BDNF expression (bdnf-KIV), we show that experience regulation of cortical GABAergic network is mediated by activity-driven BDNF expression. Levels of endogenous BDNF protein in the barrel cortex are strongly regulated by sensory inputs from whiskers. There is a severe alteration of excitation and inhibition balance in the barrel cortex of bdnf-KIV mice as a result of reduced inhibitory but not excitatory conductance. Within the inhibitory circuits, the mutant barrel cortex exhibits significantly reduced levels of GABA release only from the parvalbumin-expressing fast-spiking (FS) interneurons, but not other interneuron subtypes. Postnatal deprivation of sensory inputs markedly decreased perisomatic inhibition selectively from FS cells in wild-type but not bdnf-KIV mice. These results suggest that postnatal experience, through activity-driven BDNF expression, controls cortical development by regulating FS cell-mediated perisomatic inhibition in vivo.

  11. Stability of membranous nanostructures: a possible key mechanism in cancer progression

    Directory of Open Access Journals (Sweden)

    Kralj-Iglic V

    2012-07-01

    Full Text Available Veronika Kralj-IglicBiomedical Research Group, Faculty of Health Sciences, University of Ljubljana, Zdravstvena 5, Ljubljana, SloveniaAbstract: Membranous nanostructures, such as nanovesicles and nanotubules, are an important pool of biological membranes. Recent results indicate that they constitute cell-cell communication systems and that cancer development is influenced by these systems. Nanovesicles that are pinched off from cancer cells can move within the circulation and interact with distant cells. It has been suggested and indicated by experimental evidence that nanovesicles can induce metastases from the primary tumor in this way. Therefore, it is of importance to understand better the mechanisms of membrane budding and vesiculation. Here, a theoretical description is presented concerning consistently related lateral membrane composition, orientational ordering of membrane constituents, and a stable shape of nanovesicles and nanotubules. It is shown that the character of stable nanostructures reflects the composition of the membrane and the intrinsic shape of its constituents. An extension of the fluid mosaic model of biological membranes is suggested by taking into account curvature-mediated orientational ordering of the membrane constituents on strongly anisotropically curved regions. Based on experimental data for artificial membranes, a possible antimetastatic effect of plasma constituents via mediation of attractive interaction between membranous structures is suggested. This mediated attractive interaction hypothetically suppresses nanovesiculation by causing adhesion of buds to the mother membrane and preventing them from being pinched off from the membrane.Keywords: nanovesicles, nanotubules, nanotubes, microvesicles, exosomes, metastasis

  12. Key mechanisms of metabolic changes in mountain pine and larch under drought in the Swiss National Park

    Science.gov (United States)

    Churakova, Olga; Bigler, Christof; Bryukhanova, Marina; Siegwolf, Rolf

    2014-05-01

    Forests are of great ecological, economic and social importance worldwide. In many regions they have been recently affected by water deficits during summer droughts due to increasing temperatures and shortage of precipitation (Allen et al. 2010). Climate models predict that drought frequency will continue to increase during the 21st century and beyond (CH 2011). Since the foundation of the Swiss National Park (SNP) in 1914 these forests have not been managed any more, which allows to study natural processes in these forest ecosystems. Since the 1990s, annual and spring temperatures increased in the SNP up to 0.5 ºC and 1.02 ºC, respectively, and average summer temperature increased up to 0.6 ºC. Annual precipitation decreased by 81 mm compared to the mean values (927 mm) from 1917 to 1989. Therefore, detailed studies of drought effects on the physiological functioning of trees over the last decades are needed. Recently, mortality processes of mountain pines were observed in the Swiss National Park (Bigler, Rigling 2013). It is of great interest to investigate and compare the physiological responses of mountain pine and larch to drought and to understand the mechanisms behind the mortality processes. The goal of our study is to investigate the key mechanisms of tree physiological responses to drought in the SNP using state-of-the-art methods of classical dendrochronology, tree physiology, stable isotope, and compound-specific isotope analyses. Long-term responses of mountain pine and larch trees from north- and south-facing sites to drought will be inferred from tree-ring width data. Based on climatic data a drought index will be calculated and reconstructed back in time. New chronologies for stable carbon and oxygen isotope ratios derived from both pine and larch tree-ring cellulose will provide retrospective insight into the long-term whole-plant physiological control of gas exchange derived from estimates of stomatal conductance, photosynthetic rate and

  13. Mitochondrial proteomic analysis reveals the molecular mechanisms underlying reproductive toxicity of zearalenone in MLTC-1 cells.

    Science.gov (United States)

    Li, Yuzhe; Zhang, Boyang; Huang, Kunlun; He, Xiaoyun; Luo, YunBo; Liang, Rui; Luo, Haoshu; Shen, Xiao Li; Xu, Wentao

    2014-10-03

    Zearalenone (ZEA), a Fusarium mycotoxin that contaminates cereal crops worldwide, has been shown to affect the male reproductive system and trigger reactive oxygen species (ROS) generation. However, the mechanisms of its toxicity have not been fully understood. Because mitochondrion is a key organelle involved in producing ROS and generating metabolic intermediates for biosynthesis, an iTRAQ-based mitoproteomics approach was employed to identify the molecular mechanism of zearalenone toxicity using mitochondria of mouse Leydig tumor cells (MLTC-1). A total of 2014 nonredundant proteins were identified, among which 1401 proteins (69.56%) were overlapped. There were 52 differentially expressed proteins in response to ZEA, and they were primarily involved in energy metabolism, molecular transport and endocrine-related functions. Consistent with mitochondrial proteomic analysis, the ATP and intracellular Ca(2+) levels increased after ZEA treatment. The results suggest that lipid metabolism changed significantly after low-dose ZEA exposure, resulting in two alterations. One is the increase in energy production through promoted fatty acid uptake and β-oxidation, along with excessive oxidative stress; the other is an inhibition of steroidogenesis and esterification, possibly resulting in reduced hormone secretion. A hypothetical model of ZEA-induced mitochondrial damage is proposed to provide a framework for the mechanism of ZEA toxicity. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  14. Equations of interdoublet separation during flagella motion reveal mechanisms of wave propagation and instability.

    Science.gov (United States)

    Bayly, Philip V; Wilson, Kate S

    2014-10-07

    The motion of flagella and cilia arises from the coordinated activity of dynein motor protein molecules arrayed along microtubule doublets that span the length of axoneme (the flagellar cytoskeleton). Dynein activity causes relative sliding between the doublets, which generates propulsive bending of the flagellum. The mechanism of dynein coordination remains incompletely understood, although it has been the focus of many studies, both theoretical and experimental. In one leading hypothesis, known as the geometric clutch (GC) model, local dynein activity is thought to be controlled by interdoublet separation. The GC model has been implemented as a numerical simulation in which the behavior of a discrete set of rigid links in viscous fluid, driven by active elements, was approximated using a simplified time-marching scheme. A continuum mechanical model and associated partial differential equations of the GC model have remained lacking. Such equations would provide insight into the underlying biophysics, enable mathematical analysis of the behavior, and facilitate rigorous comparison to other models. In this article, the equations of motion for the flagellum and its doublets are derived from mechanical equilibrium principles and simple constitutive models. These equations are analyzed to reveal mechanisms of wave propagation and instability in the GC model. With parameter values in the range expected for Chlamydomonas flagella, solutions to the fully nonlinear equations closely resemble observed waveforms. These results support the ability of the GC hypothesis to explain dynein coordination in flagella and provide a mathematical foundation for comparison to other leading models.

  15. The mechanics of bacterial cluster formation on plant leaf surfaces as revealed by bioreporter technology.

    Science.gov (United States)

    Tecon, Robin; Leveau, Johan H J

    2012-05-01

    Bacteria that colonize the leaves of terrestrial plants often occur in clusters whose size varies from a few to thousands of cells. For the formation of such bacterial clusters, two non-mutually exclusive but very different mechanisms may be proposed: aggregation of multiple cells or clonal reproduction of a single cell. Here we assessed the contribution of both mechanisms on the leaves of bean plants that were colonized by the bacterium Pantoea agglomerans. In one approach, we used a mixture of green and red fluorescent P. agglomerans cells to populate bean leaves. We observed that this resulted in clusters made up of only one colour as well as two-colour clusters, thus providing evidence for both mechanisms. Another P. agglomerans bioreporter, designed to quantify the reproductive success of bacterial colonizers by proxy to the rate at which green fluorescent protein is diluted from dividing cells, revealed that during the first hours on the leaf surface, many bacteria were dividing, but not staying together and forming clusters, which is suggestive of bacterial relocation. Together, these findings support a dynamic model of leaf surface colonization, where both aggregative and reproductive mechanisms take place. The bioreporter-based approach we employed here should be broadly applicable towards a more quantitative and mechanistic understanding of bacterial colonization of surfaces in general. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

  16. Single-molecule force spectroscopy reveals the individual mechanical unfolding pathways of a surface layer protein.

    Science.gov (United States)

    Horejs, Christine; Ristl, Robin; Tscheliessnig, Rupert; Sleytr, Uwe B; Pum, Dietmar

    2011-08-05

    Surface layers (S-layers) represent an almost universal feature of archaeal cell envelopes and are probably the most abundant bacterial cell proteins. S-layers are monomolecular crystalline structures of single protein or glycoprotein monomers that completely cover the cell surface during all stages of the cell growth cycle, thereby performing their intrinsic function under a constant intra- and intermolecular mechanical stress. In gram-positive bacteria, the individual S-layer proteins are anchored by a specific binding mechanism to polysaccharides (secondary cell wall polymers) that are linked to the underlying peptidoglycan layer. In this work, atomic force microscopy-based single-molecule force spectroscopy and a polyprotein approach are used to study the individual mechanical unfolding pathways of an S-layer protein. We uncover complex unfolding pathways involving the consecutive unfolding of structural intermediates, where a mechanical stability of 87 pN is revealed. Different initial extensibilities allow the hypothesis that S-layer proteins adapt highly stable, mechanically resilient conformations that are not extensible under the presence of a pulling force. Interestingly, a change of the unfolding pathway is observed when individual S-layer proteins interact with secondary cell wall polymers, which is a direct signature of a conformational change induced by the ligand. Moreover, the mechanical stability increases up to 110 pN. This work demonstrates that single-molecule force spectroscopy offers a powerful tool to detect subtle changes in the structure of an individual protein upon binding of a ligand and constitutes the first conformational study of surface layer proteins at the single-molecule level.

  17. Phaseolin expression in tobacco chloroplast reveals an autoregulatory mechanism in heterologous protein translation.

    Science.gov (United States)

    De Marchis, Francesca; Bellucci, Michele; Pompa, Andrea

    2016-02-01

    Plastid DNA engineering is a well-established research area of plant biotechnology, and plastid transgenes often give high expression levels. However, it is still almost impossible to predict the accumulation rate of heterologous protein in transplastomic plants, and there are many cases of unsuccessful transgene expression. Chloroplasts regulate their proteome at the post-transcriptional level, mainly through translation control. One of the mechanisms to modulate the translation has been described in plant chloroplasts for the chloroplast-encoded subunits of multiprotein complexes, and the autoregulation of the translation initiation of these subunits depends on the availability of their assembly partners [control by epistasy of synthesis (CES)]. In Chlamydomonas reinhardtii, autoregulation of endogenous proteins recruited in the assembly of functional complexes has also been reported. In this study, we revealed a self-regulation mechanism triggered by the accumulation of a soluble recombinant protein, phaseolin, in the stroma of chloroplast-transformed tobacco plants. Immunoblotting experiments showed that phaseolin could avoid this self-regulation mechanism when targeted to the thylakoids in transplastomic plants. To inhibit the thylakoid-targeted phaseolin translation as well, this protein was expressed in the presence of a nuclear version of the phaseolin gene with a transit peptide. Pulse-chase and polysome analysis revealed that phaseolin mRNA translation on plastid ribosomes was repressed due to the accumulation in the stroma of the same soluble polypeptide imported from the cytosol. We suggest that translation autoregulation in chloroplast is not limited to heteromeric protein subunits but also involves at least some of the foreign soluble recombinant proteins, leading to the inhibition of plastome-encoded transgene expression in chloroplast.

  18. A catalytic mechanism for cysteine N-terminal nucleophile hydrolases, as revealed by free energy simulations.

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    Alessio Lodola

    Full Text Available The N-terminal nucleophile (Ntn hydrolases are a superfamily of enzymes specialized in the hydrolytic cleavage of amide bonds. Even though several members of this family are emerging as innovative drug targets for cancer, inflammation, and pain, the processes through which they catalyze amide hydrolysis remains poorly understood. In particular, the catalytic reactions of cysteine Ntn-hydrolases have never been investigated from a mechanistic point of view. In the present study, we used free energy simulations in the quantum mechanics/molecular mechanics framework to determine the reaction mechanism of amide hydrolysis catalyzed by the prototypical cysteine Ntn-hydrolase, conjugated bile acid hydrolase (CBAH. The computational analyses, which were confirmed in water and using different CBAH mutants, revealed the existence of a chair-like transition state, which might be one of the specific features of the catalytic cycle of Ntn-hydrolases. Our results offer new insights on Ntn-mediated hydrolysis and suggest possible strategies for the creation of therapeutically useful inhibitors.

  19. Proteomic Analysis Reveals the Leaf Color Regulation Mechanism in Chimera Hosta “Gold Standard” Leaves

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    Juanjuan Yu

    2016-03-01

    Full Text Available Leaf color change of variegated leaves from chimera species is regulated by fine-tuned molecular mechanisms. Hosta “Gold Standard” is a typical chimera Hosta species with golden-green variegated leaves, which is an ideal material to investigate the molecular mechanisms of leaf variegation. In this study, the margin and center regions of young and mature leaves from Hosta “Gold Standard”, as well as the leaves from plants after excess nitrogen fertilization were studied using physiological and comparative proteomic approaches. We identified 31 differentially expressed proteins in various regions and development stages of variegated leaves. Some of them may be related to the leaf color regulation in Hosta “Gold Standard”. For example, cytosolic glutamine synthetase (GS1, heat shock protein 70 (Hsp70, and chloroplastic elongation factor G (cpEF-G were involved in pigment-related nitrogen synthesis as well as protein synthesis and processing. By integrating the proteomics data with physiological results, we revealed the metabolic patterns of nitrogen metabolism, photosynthesis, energy supply, as well as chloroplast protein synthesis, import and processing in various leaf regions at different development stages. Additionally, chloroplast-localized proteoforms involved in nitrogen metabolism, photosynthesis and protein processing implied that post-translational modifications were crucial for leaf color regulation. These results provide new clues toward understanding the mechanisms of leaf color regulation in variegated leaves.

  20. Bactericidal mechanisms revealed for rapid water disinfection by superabsorbent cryogels decorated with silver nanoparticles.

    Science.gov (United States)

    Loo, Siew-Leng; Krantz, William B; Fane, Anthony G; Gao, Yiben; Lim, Teik-Thye; Hu, Xiao

    2015-02-17

    The authors have recently reported the fabrication of superabsorbent cryogels decorated with silver nanoparticles (PSA/AgNP cryogels) that demonstrate rapid water disinfection. This paper provides a systematic elucidation of the bactericidal mechanisms of AgNPs (silver nanoparticles), both generally and in the specific context of cryogels. Direct contact between the PSA/AgNP cryogel interface and the bacterial cells is required to accomplish disinfection. Specifically, the disinfection efficacy is closely correlated to the cell-bound Ag concentration, which constitutes >90% of the Ag released. Cells exposed to PSA/AgNP cryogels show a significant depletion of intracellular adenosine triphosphate (ATP) content and cell-membrane lesions. A positive ROS (reactive oxygen species) scavenging test confirms the involvement of ROS (·O2(-), H2O2, and ·OH) in the bactericidal mechanism. Furthermore, exposed bacterial cells show an enhanced level of thiobarbituric acid reactive substances, indicating the occurrence of cell-membrane peroxidation mediated by ROS. In addition, this study reveals that both Ag(+) and Ag(0) are involved in the bactericidal mechanism of AgNPs via tests conducted using PSA cryogels with bound Ag(+) ions (or PSA/Ag(+) cryogels without reducing Ag(+) to Ag(0)). Significantly, bacterial cells exposed to PSA/Ag(+) cryogels did not show any cell-membrane damage even though the former had a higher cell-bound Ag concentration than that of the PSA/AgNP cryogels, thus indicating the differential action of Ag(+) and Ag(0).

  1. Computational models reveal a passive mechanism for cell migration in the crypt.

    Directory of Open Access Journals (Sweden)

    Sara-Jane Dunn

    Full Text Available Cell migration in the intestinal crypt is essential for the regular renewal of the epithelium, and the continued upward movement of cells is a key characteristic of healthy crypt dynamics. However, the driving force behind this migration is unknown. Possibilities include mitotic pressure, active movement driven by motility cues, or negative pressure arising from cell loss at the crypt collar. It is possible that a combination of factors together coordinate migration. Here, three different computational models are used to provide insight into the mechanisms that underpin cell movement in the crypt, by examining the consequence of eliminating cell division on cell movement. Computational simulations agree with existing experimental results, confirming that migration can continue in the absence of mitosis. Importantly, however, simulations allow us to infer mechanisms that are sufficient to generate cell movement, which is not possible through experimental observation alone. The results produced by the three models agree and suggest that cell loss due to apoptosis and extrusion at the crypt collar relieves cell compression below, allowing cells to expand and move upwards. This finding suggests that future experiments should focus on the role of apoptosis and cell extrusion in controlling cell migration in the crypt.

  2. Transcriptome profiling of a curdlan-producing Agrobacterium reveals conserved regulatory mechanisms of exopolysaccharide biosynthesis

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    Ruffing Anne M

    2012-02-01

    Full Text Available Abstract Background The ability to synthesize exopolysaccharides (EPS is widespread among microorganisms, and microbial EPS play important roles in biofilm formation, pathogen persistence, and applications in the food and medical industries. Although it is well established that EPS synthesis is invariably in response to environmental cues, it remains largely unknown how various environmental signals trigger activation of the biochemical synthesis machinery. Results We report here the transcriptome profiling of Agrobacterium sp. ATCC 31749, a microorganism that produces large amounts of a glucose polymer known as curdlan under nitrogen starvation. Transcriptome analysis revealed a nearly 100-fold upregulation of the curdlan synthesis operon upon transition to nitrogen starvation, thus establishing the prominent role that transcriptional regulation plays in the EPS synthesis. In addition to known mechanisms of EPS regulation such as activation by c-di-GMP, we identify novel mechanisms of regulation in ATCC 31749, including RpoN-independent NtrC regulation and intracellular pH regulation by acidocalcisomes. Furthermore, we show evidence that curdlan synthesis is also regulated by conserved cell stress responses, including polyphosphate accumulation and the stringent response. In fact, the stringent response signal, pppGpp, appears to be indispensible for transcriptional activation of curdlan biosynthesis. Conclusions This study identifies several mechanisms regulating the synthesis of curdlan, an EPS with numerous applications. These mechanisms are potential metabolic engineering targets for improving the industrial production of curdlan from Agrobacterium sp. ATCC 31749. Furthermore, many of the genes identified in this study are highly conserved across microbial genomes, and we propose that the molecular elements identified in this study may serve as universal regulators of microbial EPS synthesis.

  3. Structure of Rhodococcus equi virulence-associated protein B (VapB) reveals an eight-stranded antiparallel β-barrel consisting of two Greek-key motifs

    Energy Technology Data Exchange (ETDEWEB)

    Geerds, Christina [Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld (Germany); Wohlmann, Jens; Haas, Albert [University of Bonn, Ulrich-Haberland Strasse 61a, 53121 Bonn (Germany); Niemann, Hartmut H., E-mail: hartmut.niemann@uni-bielefeld.de [Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld (Germany)

    2014-06-18

    The structure of VapB, a member of the Vap protein family that is involved in virulence of the bacterial pathogen R. equi, was determined by SAD phasing and reveals an eight-stranded antiparallel β-barrel similar to avidin, suggestive of a binding function. Made up of two Greek-key motifs, the topology of VapB is unusual or even unique. Members of the virulence-associated protein (Vap) family from the pathogen Rhodococcus equi regulate virulence in an unknown manner. They do not share recognizable sequence homology with any protein of known structure. VapB and VapA are normally associated with isolates from pigs and horses, respectively. To contribute to a molecular understanding of Vap function, the crystal structure of a protease-resistant VapB fragment was determined at 1.4 Å resolution. The structure was solved by SAD phasing employing the anomalous signal of one endogenous S atom and two bound Co ions with low occupancy. VapB is an eight-stranded antiparallel β-barrel with a single helix. Structural similarity to avidins suggests a potential binding function. Unlike other eight- or ten-stranded β-barrels found in avidins, bacterial outer membrane proteins, fatty-acid-binding proteins and lysozyme inhibitors, Vaps do not have a next-neighbour arrangement but consist of two Greek-key motifs with strand order 41238567, suggesting an unusual or even unique topology.

  4. Trophic cascades triggered by overfishing reveal possible mechanisms of ecosystem regime shifts.

    Science.gov (United States)

    Daskalov, Georgi M; Grishin, Alexander N; Rodionov, Sergei; Mihneva, Vesselina

    2007-06-19

    Large-scale transitions between alternative states in ecosystems are known as regime shifts. Once described as healthy and dominated by various marine predators, the Black Sea ecosystem by the late 20th century had experienced anthropogenic impacts such as heavy fishing, cultural eutrophication, and invasions by alien species. We studied changes related to these "natural experiments" to reveal the mechanisms of regime shifts. Two major shifts were detected, the first related to a depletion of marine predators and the second to an outburst of the alien comb jelly Mnemiopsis leidyi; both shifts were triggered by intense fishing resulting in system-wide trophic cascades. The complex nature of ecosystem responses to human activities calls for more elaborate approaches than currently provided by traditional environmental and fisheries management. This implies challenging existing practices and implementing explanatory models of ecosystem interactions that can better reconcile conservation and ecosystem management ideals.

  5. Mechanism of replication machinery assembly as revealed by the DNA ligase-PCNA-DNA complex architecture.

    Science.gov (United States)

    Mayanagi, Kouta; Kiyonari, Shinichi; Saito, Mihoko; Shirai, Tsuyoshi; Ishino, Yoshizumi; Morikawa, Kosuke

    2009-03-24

    The 3D structure of the ternary complex, consisting of DNA ligase, the proliferating cell nuclear antigen (PCNA) clamp, and DNA, was investigated by single-particle analysis. This report presents the structural view, where the crescent-shaped DNA ligase with 3 distinct domains surrounds the central DNA duplex, encircled by the closed PCNA ring, thus forming a double-layer structure with dual contacts between the 2 proteins. The relative orientations of the DNA ligase domains, which remarkably differ from those of the known crystal structures, suggest that a large domain rearrangement occurs upon ternary complex formation. A second contact was found between the PCNA ring and the middle adenylation domain of the DNA ligase. Notably, the map revealed a substantial DNA tilt from the PCNA ring axis. This structure allows us to propose a switching mechanism for the replication factors operating on the PCNA ring.

  6. Epitope-dependent mechanisms of CD27 neutralization revealed by X-ray crystallography

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    Obmolova, Galina; Teplyakov, Alexey; Malia, Thomas J.; Wunderler, Nicole; Kwok, Deborah; Barone, Linda; Sweet, Raymond; Ort, Tatiana; Scully, Michael; Gilliland, Gary L. (Janssen)

    2017-03-01

    CD27 is a T and B cell co-stimulatory protein of the TNF receptor superfamily dependent on the availability of the TNF-like ligand CD70. Two anti-CD27 neutralizing monoclonal antibodies were obtained from mouse hybridoma and subsequently humanized and optimized for binding the target. The two antibodies are similar in terms of their CD27-binding affinity and ability to block NF-κB signaling, however their clearance rates in monkeys are very different. The pharmacokinetics profiles could be epitope dependent. To identify the epitopes, we determined the crystal structure of the ternary complex between CD27 and the Fab fragments of these non-competing antibodies. The structure reveals the binding modes of the antibodies suggesting that their mechanisms of action are distinctly different and provides a possible explanation of the in vivo data.

  7. Electrophysiological potentials reveal cortical mechanisms for mental imagery, mental simulation, and grounded (embodied cognition

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    Haline E. Schendan

    2012-09-01

    Full Text Available Grounded cognition theory proposes that cognition, including meaning, is grounded in sensorimotor processing. The mechanism for grounding cognition is mental simulation, which is a type of mental imagery that re-enacts modal processing. To reveal top-down, cortical mechanisms for mental simulation of shape, event-related potentials were recorded to face and object pictures preceded by mental imagery of a picture. Mental imagery of the identical face or object (congruous condition facilitated not only categorical perception (VPP/N170 but also later visual knowledge (N3[00] complex and linguistic knowledge (N400 for faces more than objects, and strategic semantic analysis (late positive complex between 200 and 700 ms. The later effects resembled semantic congruity effects with pictures. Mental imagery also facilitated category decisions, as a P3(00 peaked earlier for congruous than incongruous (other category pictures, resembling the case when identical pictures repeat immediately. Thus mental imagery mimics semantic congruity and immediate repetition priming processes with pictures. Perception control results showed the opposite for faces and were in the same direction for objects: Perceptual repetition adapts (and so impairs processing of perceived faces from categorical perception onwards, but primes processing of objects during categorical perception, visual knowledge processes, and strategic semantic analysis. For both imagery and perception, differences between faces and objects support domain-specificity and indicate that cognition is grounded in modal processing. Altogether, this direct neural evidence reveals that top-down processes of mental imagery sustain an imagistic representation that mimics perception well enough to prime subsequent perception and cognition. This also suggests that automatic mental simulation of the visual shape of faces and objects operates between 200 and 400 ms, and strategic mental simulation operates between

  8. Talaromyces marneffei Genomic, Transcriptomic, Proteomic and Metabolomic Studies Reveal Mechanisms for Environmental Adaptations and Virulence

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    Susanna K. P. Lau

    2017-06-01

    Full Text Available Talaromyces marneffei is a thermally dimorphic fungus causing systemic infections in patients positive for HIV or other immunocompromised statuses. Analysis of its ~28.9 Mb draft genome and additional transcriptomic, proteomic and metabolomic studies revealed mechanisms for environmental adaptations and virulence. Meiotic genes and genes for pheromone receptors, enzymes which process pheromones, and proteins involved in pheromone response pathway are present, indicating its possibility as a heterothallic fungus. Among the 14 Mp1p homologs, only Mp1p is a virulence factor binding a variety of host proteins, fatty acids and lipids. There are 23 polyketide synthase genes, one for melanin and two for mitorubrinic acid/mitorubrinol biosynthesis, which are virulence factors. Another polyketide synthase is for biogenesis of the diffusible red pigment, which consists of amino acid conjugates of monascorubin and rubropunctatin. Novel microRNA-like RNAs (milRNAs and processing proteins are present. The dicer protein, dcl-2, is required for biogenesis of two milRNAs, PM-milR-M1 and PM-milR-M2, which are more highly expressed in hyphal cells. Comparative transcriptomics showed that tandem repeat-containing genes were overexpressed in yeast phase, generating protein polymorphism among cells, evading host’s immunity. Comparative proteomics between yeast and hyphal cells revealed that glyceraldehyde-3-phosphate dehydrogenase, up-regulated in hyphal cells, is an adhesion factor for conidial attachment.

  9. Fibrinogen adsorption mechanisms at the gold substrate revealed by QCM-D measurements and RSA modeling.

    Science.gov (United States)

    Kubiak, Katarzyna; Adamczyk, Zbigniew; Cieśla, Michał

    2016-03-01

    Adsorption kinetics of fibrinogen at a gold substrate at various pHs was thoroughly studied using the QCM-D method. The experimental were interpreted in terms of theoretical calculations performed according to the random sequential adsorption model (RSA). In this way, the hydration functions and water factors of fibrinogen monolayers were quantitatively evaluated at various pHs. It was revealed that for the lower range of fibrinogen coverage the hydration function were considerably lower than previously obtained for the silica sensor [33]. The lower hydration of fibrinogen monolayers on the gold sensor was attributed to its higher roughness. However, for higher fibrinogen coverage the hydration functions for both sensors became identical exhibiting an universal behavior. By using the hydration functions, the fibrinogen adsorption/desorption runs derived from QCM-D measurements were converted to the Γd vs. the time relationships. This allowed to precisely determine the maximum coverage that varied between 1.6mgm(-2) at pH 3.5 and 4.5mgm(-2) at pH 7.4 (for ionic strength of 0.15M). These results agree with theoretical eRSA modeling and previous experimental data derived by using ellipsometry, OWLS and TIRF. Various fibrinogen adsorption mechanisms were revealed by exploiting the maximum coverage data. These results allow one to develop a method for preparing fibrinogen monolayers of well-controlled coverage and molecule orientation. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Analysis of the transcriptional differences between indigenous and invasive whiteflies reveals possible mechanisms of whitefly invasion.

    Science.gov (United States)

    Wang, Yong-Liang; Wang, Yu-Jun; Luan, Jun-Bo; Yan, Gen-Hong; Liu, Shu-Sheng; Wang, Xiao-Wei

    2013-01-01

    The whitefly Bemisa tabaci is a species complex of more than 31 cryptic species which include some of the most destructive invasive pests of crops worldwide. Among them, Middle East-Asia Minor 1 (MEAM1) and Mediterranean have invaded many countries and displaced the native whitefly species. The successful invasion of the two species is largely due to their wide range of host plants, high resistance to insecticides and remarkable tolerance to environmental stresses. However, the molecular differences between invasive and indigenous whiteflies remain largely unknown. Here the global transcriptional difference between the two invasive whitefly species (MEAM1, MED) and one indigenous whitefly species (Asia II 3) were analyzed using the Illumina sequencing. Our analysis indicated that 2,422 genes between MEAM1 and MED; 3,073 genes between MEAM1 and Asia II 3; and 3,644 genes between MED and Asia II 3 were differentially expressed. Gene Ontology enrichment analysis revealed that the differently expressed genes between the invasive and indigenous whiteflies were significantly enriched in the term of 'oxidoreductase activity'. Pathway enrichment analysis showed that carbohydrate, amino acid and glycerolipid metabolisms were more active in MEAM1 and MED than in Asia II 3, which may contribute to their differences in biological characteristics. Our analysis also illustrated that the majority of genes involved in 'drug metabolic pathway' were expressed at a higher level in MEAM1 and MED than in Asia II 3. Taken together, these results revealed that the genes related to basic metabolism and detoxification were expressed at an elevated level in the invasive whiteflies, which might be responsible for their higher resistance to insecticides and environmental stresses. The extensive comparison of MEAM1, MED and Asia II 3 gene expression may serve as an invaluable resource for revealing the molecular mechanisms underlying their biological differences and the whitefly invasion.

  11. Structures of ribosome-bound initiation factor 2 reveal the mechanism of subunit association

    Science.gov (United States)

    Sprink, Thiemo; Ramrath, David J. F.; Yamamoto, Hiroshi; Yamamoto, Kaori; Loerke, Justus; Ismer, Jochen; Hildebrand, Peter W.; Scheerer, Patrick; Bürger, Jörg; Mielke, Thorsten; Spahn, Christian M. T.

    2016-01-01

    Throughout the four phases of protein biosynthesis—initiation, elongation, termination, and recycling—the ribosome is controlled and regulated by at least one specified translational guanosine triphosphatase (trGTPase). Although the structural basis for trGTPase interaction with the ribosome has been solved for the last three steps of translation, the high-resolution structure for the key initiation trGTPase, initiation factor 2 (IF2), complexed with the ribosome, remains elusive. We determine the structure of IF2 complexed with a nonhydrolyzable guanosine triphosphate analog and initiator fMet-tRNAiMet in the context of the Escherichia coli ribosome to 3.7-Å resolution using cryo-electron microscopy. The structural analysis reveals previously unseen intrinsic conformational modes of the 70S initiation complex, establishing the mutual interplay of IF2 and initator transfer RNA (tRNA) with the ribsosome and providing the structural foundation for a mechanistic understanding of the final steps of translation initiation. PMID:26973877

  12. Transcriptome profile reveals heat response mechanism at molecular and metabolic levels in rice flag leaf.

    Science.gov (United States)

    Zhang, Xianwen; Rerksiri, Wirat; Liu, Ailing; Zhou, Xiaoyun; Xiong, Hairong; Xiang, Jianhua; Chen, Xinbo; Xiong, Xingyao

    2013-11-10

    Flag leaf is one of the key photosynthesis organs during rice reproductive stage. A time course microarray analysis of rice flag leaf was done after 40°C treatment for 0 min, 20 min, 60 min, 2h, 4h, and 8h. The identified significant heat responsive genes were mainly involved in transcriptional regulation, transport, protein binding, antioxidant, and stress response. KMC analysis discovered the time-dependent gene expression pattern under heat. MapMan analysis demonstrated that, under heat treatment, Hsp genes and genes involved in glycolysis and ubiquitin-proteasome were enhanced, and genes involved in TCA, carotenoid, dihydroflavonol and anthocyanin metabolisms and light-reaction in the photosynthesis were widely repressed. Meanwhile, some rate-limiting enzyme genes in shikimate, lignin, and mevalonic acid metabolisms were up-regulated, revealing the importance of maintaining specific secondary metabolites under heat stress. The present study increased our understanding of heat response in rice flag leaf and provided good candidate genes for crop improvement.

  13. The Thermoanaerobacter glycobiome reveals mechanisms of pentose and hexose co-utilization in bacteria.

    Directory of Open Access Journals (Sweden)

    Lu Lin

    2011-10-01

    Full Text Available Thermoanaerobic bacteria are of interest in cellulosic-biofuel production, due to their simultaneous pentose and hexose utilization (co-utilization and thermophilic nature. In this study, we experimentally reconstructed the structure and dynamics of the first genome-wide carbon utilization network of thermoanaerobes. The network uncovers numerous novel pathways and identifies previously unrecognized but crucial pathway interactions and the associated key junctions. First, glucose, xylose, fructose, and cellobiose catabolism are each featured in distinct functional modules; the transport systems of hexose and pentose are apparently both regulated by transcriptional antiterminators of the BglG family, which is consistent with pentose and hexose co-utilization. Second, glucose and xylose modules cooperate in that the activity of the former promotes the activity of the latter via activating xylose transport and catabolism, while xylose delays cell lysis by sustaining coenzyme and ion metabolism. Third, the vitamin B₁₂ pathway appears to promote ethanologenesis through ethanolamine and 1, 2-propanediol, while the arginine deiminase pathway probably contributes to cell survival in stationary phase. Moreover, by experimentally validating the distinct yet collaborative nature of glucose and xylose catabolism, we demonstrated that these novel network-derived features can be rationally exploited for product-yield enhancement via optimized timing and balanced loading of the carbon supply in a substrate-specific manner. Thus, this thermoanaerobic glycobiome reveals novel genetic features in carbon catabolism that may have immediate industrial implications and provides novel strategies and targets for fermentation and genome engineering.

  14. Changing the spatial pattern of TFL1 expression reveals its key role in the shoot meristem in controlling Arabidopsis flowering architecture.

    Science.gov (United States)

    Baumann, Kim; Venail, Julien; Berbel, Ana; Domenech, Maria Jose; Money, Tracy; Conti, Lucio; Hanzawa, Yoshie; Madueno, Francisco; Bradley, Desmond

    2015-08-01

    Models for the control of above-ground plant architectures show how meristems can be programmed to be either shoots or flowers. Molecular, genetic, transgenic, and mathematical studies have greatly refined these models, suggesting that the phase of the shoot reflects different genes contributing to its repression of flowering, its vegetativeness ('veg'), before activators promote flower development. Key elements of how the repressor of flowering and shoot meristem gene TFL1 acts have now been tested, by changing its spatiotemporal pattern. It is shown that TFL1 can act outside of its normal expression domain in leaf primordia or floral meristems to repress flower identity. These data show how the timing and spatial pattern of TFL1 expression affect overall plant architecture. This reveals that the underlying pattern of TFL1 interactors is complex and that they may be spatially more widespread than TFL1 itself, which is confined to shoots. However, the data show that while TFL1 and floral genes can both act and compete in the same meristem, it appears that the main shoot meristem is more sensitive to TFL1 rather than floral genes. This spatial analysis therefore reveals how a difference in response helps maintain the 'veg' state of the shoot meristem. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  15. Genomic Scars Generated by Polymerase Theta Reveal the Versatile Mechanism of Alternative End-Joining.

    Directory of Open Access Journals (Sweden)

    Robin van Schendel

    2016-10-01

    Full Text Available For more than half a century, genotoxic agents have been used to induce mutations in the genome of model organisms to establish genotype-phenotype relationships. While inaccurate replication across damaged bases can explain the formation of single nucleotide variants, it remained unknown how DNA damage induces more severe genomic alterations. Here, we demonstrate for two of the most widely used mutagens, i.e. ethyl methanesulfonate (EMS and photo-activated trimethylpsoralen (UV/TMP, that deletion mutagenesis is the result of polymerase Theta (POLQ-mediated end joining (TMEJ of double strand breaks (DSBs. This discovery allowed us to survey many thousands of available C. elegans deletion alleles to address the biology of this alternative end-joining repair mechanism. Analysis of ~7,000 deletion breakpoints and their cognate junctions reveals a distinct order of events. We found that nascent strands blocked at sites of DNA damage can engage in one or more cycles of primer extension using a more downstream located break end as a template. Resolution is accomplished when 3' overhangs have matching ends. Our study provides a step-wise and versatile model for the in vivo mechanism of POLQ action, which explains the molecular nature of mutagen-induced deletion alleles.

  16. Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction.

    Science.gov (United States)

    Teramoto, Takamasa; Fujikawa, Yukari; Kawaguchi, Yoshirou; Kurogi, Katsuhisa; Soejima, Masayuki; Adachi, Rumi; Nakanishi, Yuichi; Mishiro-Sato, Emi; Liu, Ming-Cheh; Sakakibara, Yoichi; Suiko, Masahito; Kimura, Makoto; Kakuta, Yoshimitsu

    2013-01-01

    Post-translational protein modification by tyrosine sulfation has an important role in extracellular protein-protein interactions. The protein tyrosine sulfation reaction is catalysed by the Golgi enzyme called the tyrosylprotein sulfotransferase. To date, no crystal structure is available for tyrosylprotein sulfotransferase. Detailed mechanism of protein tyrosine sulfation reaction has thus remained unclear. Here we present the first crystal structure of the human tyrosylprotein sulfotransferase isoform 2 complexed with a substrate peptide (C4P5Y3) derived from complement C4 and 3'-phosphoadenosine-5'-phosphate at 1.9 Å resolution. Structural and complementary mutational analyses revealed the molecular basis for catalysis being an SN2-like in-line displacement mechanism. Tyrosylprotein sulfotransferase isoform 2 appeared to recognize the C4 peptide in a deep cleft by using a short parallel β-sheet type interaction, and the bound C4P5Y3 forms an L-shaped structure. Surprisingly, the mode of substrate peptide recognition observed in the tyrosylprotein sulfotransferase isoform 2 structure resembles that observed for the receptor type tyrosine kinases.

  17. Microstructural mechanisms of analgesia in percutaneous cervical cordotomy revealed by diffusion tensor imaging.

    Science.gov (United States)

    Gebarski, Stephen S; Chiravuri, Srinivas; Foerster, Bradley R; Patil, Parag G

    2017-09-05

    The purpose of this study is to demonstrate the potential of diffusion tensor imaging (DTI) to reveal structural mechanisms underlying spinal ablative procedures, including percutaneous radiofrequency cordotomy (PRFC). PRFC is a surgical procedure that produces analgesia through focal ablation of the lateral spinothalamic tract (STT), thereby interrupting the flow of pain information from the periphery to the brain. To date, studies regarding mechanisms of analgesia after PRFC have been limited to postmortem cadaveric dissection and histology. However, with recent advances in DTI, the opportunity has arisen to study the STT non-invasively in vivo. In this technical note, an individual with successful pain relief following unilateral STT PRFC was examined using DTI, with the contralateral STT serving as an internal control. PRFC substantially reduced rostrocaudal directional DTI signal in the STT from the lesion in the cervical spinal cord through the pons and mesencephalon. Our findings confirm that focal ablation and anterograde degeneration accompany the analgesic effects of PRFC. In vivo imaging of the STT with DTI may contribute to surgical targeting for PRFC procedures, better understanding of the therapeutic and untoward effects of PRFC, and a deeper understanding of spinothalamic contributions to nociception. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells.

    Science.gov (United States)

    Chiarle, Roberto; Zhang, Yu; Frock, Richard L; Lewis, Susanna M; Molinie, Benoit; Ho, Yu-Jui; Myers, Darienne R; Choi, Vivian W; Compagno, Mara; Malkin, Daniel J; Neuberg, Donna; Monti, Stefano; Giallourakis, Cosmas C; Gostissa, Monica; Alt, Frederick W

    2011-09-30

    Whereas chromosomal translocations are common pathogenetic events in cancer, mechanisms that promote them are poorly understood. To elucidate translocation mechanisms in mammalian cells, we developed high-throughput, genome-wide translocation sequencing (HTGTS). We employed HTGTS to identify tens of thousands of independent translocation junctions involving fixed I-SceI meganuclease-generated DNA double-strand breaks (DSBs) within the c-myc oncogene or IgH locus of B lymphocytes induced for activation-induced cytidine deaminase (AID)-dependent IgH class switching. DSBs translocated widely across the genome but were preferentially targeted to transcribed chromosomal regions. Additionally, numerous AID-dependent and AID-independent hot spots were targeted, with the latter comprising mainly cryptic I-SceI targets. Comparison of translocation junctions with genome-wide nuclear run-ons revealed a marked association between transcription start sites and translocation targeting. The majority of translocation junctions were formed via end-joining with short microhomologies. Our findings have implications for diverse fields, including gene therapy and cancer genomics.

  19. Optical tweezers reveal a dynamic mechanical response of cationic peptide-DNA complexes

    Science.gov (United States)

    Lee, Amy; Zheng, Tai; Sucayan, Sarah; Chou, Szu-Ting; Tricoli, Lucas; Hustedt, Jason; Kahn, Jason; Mixson, A. James; Seog, Joonil

    2013-03-01

    Nonviral carriers have been developed to deliver nucleic acids by forming nanoscale complexes; however, there has been limited success in achieving high transfection efficiency. Our hypothesis is that a factor affecting gene delivery efficiency is the mechanical response of the condensed complex. To begin to test this hypothesis, we directly measured the mechanical properties of DNA-carrier complexes using optical tweezers. Histidine-lysine (HK) polymer, Asparagine-lysine (NK) polymer and poly-L-lysine were used to form complexes with a single DNA molecule. As carriers were introduced, a sudden decrease in DNA extension occurrs at a force level which is defined as critical force (Fc). Fc is carrier and concentration dependent. Pulling revealed reduction in DNA extension length for HK-DNA complexes. The characteristics of force profiles vary by agent and can be dynamically manipulated by changes in environmental conditions such as ionic strength of the buffer as well as pH. Heparin can remove cationic reagents which are otherwise irreversibly bound to DNA. The implications for optimizing molecular interactions to enhance transfection efficiency will be discussed.

  20. Mechanical disassembly of single virus particles reveals kinetic intermediates predicted by theory.

    Science.gov (United States)

    Castellanos, Milagros; Pérez, Rebeca; Carrillo, Pablo J P; de Pablo, Pedro J; Mateu, Mauricio G

    2012-06-06

    New experimental approaches are required to detect the elusive transient intermediates predicted by simulations of virus assembly or disassembly. Here, an atomic force microscope (AFM) was used to mechanically induce partial disassembly of single icosahedral T=1 capsids and virions of the minute virus of mice. The kinetic intermediates formed were imaged by AFM. The results revealed that induced disassembly of single minute-virus-of-mice particles is frequently initiated by loss of one of the 20 equivalent capsomers (trimers of capsid protein subunits) leading to a stable, nearly complete particle that does not readily lose further capsomers. With lower frequency, a fairly stable, three-fourths-complete capsid lacking one pentamer of capsomers and a free, stable pentamer were obtained. The intermediates most frequently identified (capsids missing one capsomer, capsids missing one pentamer of capsomers, and free pentamers of capsomers) had been predicted in theoretical studies of reversible capsid assembly based on thermodynamic-kinetic models, molecular dynamics, or oligomerization energies. We conclude that mechanical manipulation and imaging of simple virus particles by AFM can be used to experimentally identify kinetic intermediates predicted by simulations of assembly or disassembly. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  1. Hydrogen-Activation Mechanism of [Fe] Hydrogenase Revealed by Multi-Scale Modeling

    CERN Document Server

    Finkelmann, Arndt Robert; Reiher, Markus

    2014-01-01

    When investigating the mode of hydrogen activation by [Fe] hydrogenases, not only the chemical reactivity at the active site is of importance but also the large-scale conformational change between the so-called open and closed conformations, which leads to a special spatial arrangement of substrate and iron cofactor. To study H2 activation, a complete model of the solvated and cofactor-bound enzyme in complex with the substrate methenyl-H4MPT+ was constructed. Both the closed and open conformations were simulated with classical molecular dynamics on the 100 ns time scale. Quantum-mechanics/molecular-mechanics calculations on snapshots then revealed the features of the active site that enable the facile H2 cleavage. The hydroxyl group of the pyridinol ligand can easily be deprotonated. With the deprotonated hydroxyl group and the structural arrangement in the closed conformation, H2 coordinated to the Fe center is subject to an ionic and orbital push-pull effect and can be rapidly cleaved with a concerted hydr...

  2. Structures of inactive retinoblastoma protein reveal multiple mechanisms for cell cycle control

    Energy Technology Data Exchange (ETDEWEB)

    Burke, Jason R.; Hura, Greg L.; Rubin, Seth M. (UCSC); (LBNL)

    2012-07-18

    Cyclin-dependent kinase (Cdk) phosphorylation of the Retinoblastoma protein (Rb) drives cell proliferation through inhibition of Rb complexes with E2F transcription factors and other regulatory proteins. We present the first structures of phosphorylated Rb that reveal the mechanism of its inactivation. S608 phosphorylation orders a flexible 'pocket' domain loop such that it mimics and directly blocks E2F transactivation domain (E2F{sup TD}) binding. T373 phosphorylation induces a global conformational change that associates the pocket and N-terminal domains (RbN). This first multidomain Rb structure demonstrates a novel role for RbN in allosterically inhibiting the E2F{sup TD}-pocket association and protein binding to the pocket 'LxCxE' site. Together, these structures detail the regulatory mechanism for a canonical growth-repressive complex and provide a novel example of how multisite Cdk phosphorylation induces diverse structural changes to influence cell cycle signaling.

  3. Revealing a double-inversion mechanism for the F⁻+CH₃Cl SN2 reaction.

    Science.gov (United States)

    Szabó, István; Czakó, Gábor

    2015-01-19

    Stereo-specific reaction mechanisms play a fundamental role in chemistry. The back-side attack inversion and front-side attack retention pathways of the bimolecular nucleophilic substitution (SN2) reactions are the textbook examples for stereo-specific chemical processes. Here, we report an accurate global analytic potential energy surface (PES) for the F(-)+CH₃Cl SN2 reaction, which describes both the back-side and front-side attack substitution pathways as well as the proton-abstraction channel. Moreover, reaction dynamics simulations on this surface reveal a novel double-inversion mechanism, in which an abstraction-induced inversion via a FH···CH₂Cl(-) transition state is followed by a second inversion via the usual [F···CH₃···Cl](-) saddle point, thereby opening a lower energy reaction path for retention than the front-side attack. Quasi-classical trajectory computations for the F(-)+CH₃Cl(ν1=0, 1) reactions show that the front-side attack is a fast direct, whereas the double inversion is a slow indirect process.

  4. Genomic Scars Generated by Polymerase Theta Reveal the Versatile Mechanism of Alternative End-Joining

    Science.gov (United States)

    van Schendel, Robin; van Heteren, Jane; Welten, Richard; Tijsterman, Marcel

    2016-01-01

    For more than half a century, genotoxic agents have been used to induce mutations in the genome of model organisms to establish genotype-phenotype relationships. While inaccurate replication across damaged bases can explain the formation of single nucleotide variants, it remained unknown how DNA damage induces more severe genomic alterations. Here, we demonstrate for two of the most widely used mutagens, i.e. ethyl methanesulfonate (EMS) and photo-activated trimethylpsoralen (UV/TMP), that deletion mutagenesis is the result of polymerase Theta (POLQ)-mediated end joining (TMEJ) of double strand breaks (DSBs). This discovery allowed us to survey many thousands of available C. elegans deletion alleles to address the biology of this alternative end-joining repair mechanism. Analysis of ~7,000 deletion breakpoints and their cognate junctions reveals a distinct order of events. We found that nascent strands blocked at sites of DNA damage can engage in one or more cycles of primer extension using a more downstream located break end as a template. Resolution is accomplished when 3’ overhangs have matching ends. Our study provides a step-wise and versatile model for the in vivo mechanism of POLQ action, which explains the molecular nature of mutagen-induced deletion alleles. PMID:27755535

  5. Revealing the reaction mechanisms of Li–O2 batteries using environmental transmission electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Langli; Liu, Bin; Song, Shidong; Xu, Wu; Zhang, Ji-Guang; Wang, Chongmin

    2017-03-27

    The capacity, Coulombic efficiency, rate, and cyclability of a Li-O2 battery critically depend on the electrode reaction mechanism and the structure/morphology of the reaction product as well as their spatial and temporal evolution1-8, which are all further complicated by the choice of different electrolyte. For the case of aprotic cell, the discharge product, Li2O2, is formed through solution and surface mechanisms9,10, but little is known on the formation mechanism of the perplexing morphology of the reaction product11-15. For the case of Li-O2 battery using solid electrolyte, neither electrode reaction mechanism nor the nature of the reaction production is known. Herein, we reveal the full cycle reaction pathway for Li-O2 batteries and its correlation with the nature of the reaction product. Using an aberration-corrected environmental TEM under oxygen environment, we captured, for the first time, the morphology and phase evolution on the carbon nanotube (CNT) cathode of a working solid-state Li-O2 nano-battery16 and directly correlated these features with electrochemical reaction. We found that the oxygen reduction reaction on CNTs initially produces LiO2, which subsequently evolves to Li2O2 and O2 through disproportionation reaction. Surprisingly it is just the releasing of O2 that inflates the particles to a hollow structure with a Li2O outer surface layer and Li2O2 inner-shell, demonstrating that, in general, accommodation of the released O2 coupled with the Li+ ion diffusion and electron transport paths across both spatial and temporal scales critically governs the morphology of the discharging/charging product in Li-O2 system. We anticipate that the direct observation of Li-O2 reaction mechanisms and their correlation with the morphology of the reaction product set foundation for quantitative understanding/modeling of the electrochemical processes in the Li-O2 system, enabling rational design of both solid-state and aprotic Li-O2 batteries.

  6. Global Gene Expression Analysis Reveals Crosstalk between Response Mechanisms to Cold and Drought Stresses in Cassava Seedlings.

    Science.gov (United States)

    Li, Shuxia; Yu, Xiang; Cheng, Zhihao; Yu, Xiaoling; Ruan, Mengbin; Li, Wenbin; Peng, Ming

    2017-01-01

    Abiotic stress negatively impacts cassava (Manihot esculenta) growth and yield. Several molecular mechanisms of plant response to cold and drought have been identified and described in the literature, however, little is known about the crosstalk of the responses of cassava to these two stresses. To elucidate this question, transcriptome analysis of cassava seedlings under cold or PEG-simulated drought stress treatment was performed. Our results showed that 6103 and 7462 transcripts were significantly regulated by cold and drought stress, respectively. Gene Ontology annotation revealed that the abscisic and jasmonic acid signaling pathways shared between the two stresses responses. We further identified 2434 common differentially expressed genes (DEGs), including 1130 up-regulated and 841 down-regulated DEGs by the two stresses. These co-induced or co-suppressed genes are grouped as stress signal perception and transduction, transcription factors (TFs), metabolism as well as transport facilitation according to the function annotation. Furthermore, a large proportion of well characterized protein kinases, TF families and ubiquitin proteasome system related genes, such as RLKs, MAPKs, AP2/ERFBPs, WRKYs, MYBs, E2 enzymes and E3 ligases, including three complexes of interacting proteins were shown as key points of crosstalk between cold and drought stress signaling transduction pathways in a hierarchical manner. Our research provides valuable information and new insights for genetically improving the tolerance of crops to multiple abiotic stresses.

  7. Global Gene Expression Analysis Reveals Crosstalk between Response Mechanisms to Cold and Drought Stresses in Cassava Seedlings

    Directory of Open Access Journals (Sweden)

    Shuxia Li

    2017-07-01

    Full Text Available Abiotic stress negatively impacts cassava (Manihot esculenta growth and yield. Several molecular mechanisms of plant response to cold and drought have been identified and described in the literature, however, little is known about the crosstalk of the responses of cassava to these two stresses. To elucidate this question, transcriptome analysis of cassava seedlings under cold or PEG-simulated drought stress treatment was performed. Our results showed that 6103 and 7462 transcripts were significantly regulated by cold and drought stress, respectively. Gene Ontology annotation revealed that the abscisic and jasmonic acid signaling pathways shared between the two stresses responses. We further identified 2434 common differentially expressed genes (DEGs, including 1130 up-regulated and 841 down-regulated DEGs by the two stresses. These co-induced or co-suppressed genes are grouped as stress signal perception and transduction, transcription factors (TFs, metabolism as well as transport facilitation according to the function annotation. Furthermore, a large proportion of well characterized protein kinases, TF families and ubiquitin proteasome system related genes, such as RLKs, MAPKs, AP2/ERFBPs, WRKYs, MYBs, E2 enzymes and E3 ligases, including three complexes of interacting proteins were shown as key points of crosstalk between cold and drought stress signaling transduction pathways in a hierarchical manner. Our research provides valuable information and new insights for genetically improving the tolerance of crops to multiple abiotic stresses.

  8. Dynamic network of transcription and pathway crosstalk to reveal molecular mechanism of MGd-treated human lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Liyan Shao

    Full Text Available Recent research has revealed various molecular markers in lung cancer. However, the organizational principles underlying their genetic regulatory networks still await investigation. Here we performed Network Component Analysis (NCA and Pathway Crosstalk Analysis (PCA to construct a regulatory network in human lung cancer (A549 cells which were treated with 50 uM motexafin gadolinium (MGd, a metal cation-containing chemotherapeutic drug for 4, 12, and 24 hours. We identified a set of key TFs, known target genes for these TFs, and signaling pathways involved in regulatory networks. Our work showed that putative interactions between these TFs (such as ESR1/Sp1, E2F1/Sp1, c-MYC-ESR, Smad3/c-Myc, and NFKB1/RELA, between TFs and their target genes (such as BMP41/Est1, TSC2/Myc, APE1/Sp1/p53, RARA/HOXA1, and SP1/USF2, and between signaling pathways (such as PPAR signaling pathway and Adipocytokines signaling pathway. These results will provide insights into the regulatory mechanism of MGd-treated human lung cancer cells.

  9. Proteomics Analysis Reveals Post-Translational Mechanisms for Cold-Induced Metabolic Changes in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Tian Li; Alma L.Burlingame; Zhi-Ping Deng; Zhi Yong Wang; Shou-Ling Xu; Juan A.Oses-Prieto; Sunita Putil; Peng Xu; Rui-Ju Wang; Kathy H.Li; David A.Malty; Liz-He An

    2011-01-01

    Cold-induced changes of gene expression and metabolism are critical for plants to survive freezing. Largely by changing gene expression, exposure to a period of non-freezing low temperatures increases plant tolerance to freezing-a phenomenon known as cold acclimation. Cold also induces rapid metabolic changes, which provide instant protection before temperature drops below freezing point. The molecular mechanisms for such rapid metabolic responses to cold remain largely unknown. Here, we use two-dimensional difference gel electrophoresis (2-D DIGE) analysis of sub-cellular fractions of Arabidopsis thaliana proteome coupled with spot identification by tandem mass spectrometry to identify early cold-responsive proteins in Arabidopsis. These proteins include four enzymes involved in starch degradation, three HSP100 proteins, several proteins in the tricarboxylic acid cycle, and sucrose metabolism. Upon cold treatment, the Disproportionating Enzyme 2 (DPE2), a cytosolic transglucosidase metabolizing maltose to glucose, increased rapidly in the centrifugation pellet fraction and decreased in the soluble fraction. Consistent with cold-induced inactivation of DPE2 enzymatic activity, the dpe2 mutant showed increased freezing tolerance without affecting the C-repeat binding transcription factor (CBF) transcriptional pathway. These results support a model that cold-induced inactivation of DPE2 leads to rapid accumulation of maltose, which is a cold-induced compatible solute that protects cells from freezing damage. This study provides evidence for a key role of rapid post-translational regulation of carbohydrate metabolic enzymes in plant protection against sudden temperature drop.

  10. Molecular mechanisms of ethanol-induced pathogenesis revealed by RNA-sequencing.

    Directory of Open Access Journals (Sweden)

    Laura Camarena

    2010-04-01

    Full Text Available Acinetobacter baumannii is a common pathogen whose recent resistance to drugs has emerged as a major health problem. Ethanol has been found to increase the virulence of A. baumannii in Dictyostelium discoideum and Caenorhabditis elegans models of infection. To better understand the causes of this effect, we examined the transcriptional profile of A. baumannii grown in the presence or absence of ethanol using RNA-Seq. Using the Illumina/Solexa platform, a total of 43,453,960 reads (35 nt were obtained, of which 3,596,474 mapped uniquely to the genome. Our analysis revealed that ethanol induces the expression of 49 genes that belong to different functional categories. A strong induction was observed for genes encoding metabolic enzymes, indicating that ethanol is efficiently assimilated. In addition, we detected the induction of genes encoding stress proteins, including upsA, hsp90, groEL and lon as well as permeases, efflux pumps and a secreted phospholipase C. In stationary phase, ethanol strongly induced several genes involved with iron assimilation and a high-affinity phosphate transport system, indicating that A. baumannii makes a better use of the iron and phosphate resources in the medium when ethanol is used as a carbon source. To evaluate the role of phospholipase C (Plc1 in virulence, we generated and analyzed a deletion mutant for plc1. This strain exhibits a modest, but reproducible, reduction in the cytotoxic effect caused by A. baumannii on epithelial cells, suggesting that phospholipase C is important for virulence. Overall, our results indicate the power of applying RNA-Seq to identify key modulators of bacterial pathogenesis. We suggest that the effect of ethanol on the virulence of A. baumannii is multifactorial and includes a general stress response and other specific components such as phospholipase C.

  11. Experimental Study and Computational Simulations of Key Pebble Bed Thermo-mechanics Issues for Design and Safety

    Energy Technology Data Exchange (ETDEWEB)

    Tokuhiro, Akira; Potirniche, Gabriel; Cogliati, Joshua; Ougouag, Abderrafi

    2014-07-08

    An experimental and computational study, consisting of modeling and simulation (M&S), of key thermal-mechanical issues affecting the design and safety of pebble-bed (PB) reactors was conducted. The objective was to broaden understanding and experimentally validate thermal-mechanic phenomena of nuclear grade graphite, specifically, spheres in frictional contact as anticipated in the bed under reactor relevant pressures and temperatures. The contact generates graphite dust particulates that can subsequently be transported into the flowing gaseous coolent. Under postulated depressurization transients and with the potential for leaked fission products to be adsorbed onto graphite 'dust', there is the potential for fission products to escape from the primary volume. This is a design safety concern. Furthermore, earlier safety assessment identified the distinct possibility for the dispersed dust to combust in contact with air if sufficient conditions are met. Both of these phenomena were noted as important to design review and containing uncertainty to warrant study. The team designed and conducted two separate effects tests to study and benchmark the potential dust-generation rate, as well as study the conditions under which a dust explosion may occure in a standardized, instrumented explosion chamber.

  12. In Vivo Time-Resolved Microtomography Reveals the Mechanics of the Blowfly Flight Motor

    Science.gov (United States)

    Mokso, Rajmund; Wicklein, Martina; Müller, Tonya; Doube, Michael; Stampanoni, Marco; Krapp, Holger G.; Taylor, Graham K.

    2014-01-01

    Dipteran flies are amongst the smallest and most agile of flying animals. Their wings are driven indirectly by large power muscles, which cause cyclical deformations of the thorax that are amplified through the intricate wing hinge. Asymmetric flight manoeuvres are controlled by 13 pairs of steering muscles acting directly on the wing articulations. Collectively the steering muscles account for <3% of total flight muscle mass, raising the question of how they can modulate the vastly greater output of the power muscles during manoeuvres. Here we present the results of a synchrotron-based study performing micrometre-resolution, time-resolved microtomography on the 145 Hz wingbeat of blowflies. These data represent the first four-dimensional visualizations of an organism's internal movements on sub-millisecond and micrometre scales. This technique allows us to visualize and measure the three-dimensional movements of five of the largest steering muscles, and to place these in the context of the deforming thoracic mechanism that the muscles actuate. Our visualizations show that the steering muscles operate through a diverse range of nonlinear mechanisms, revealing several unexpected features that could not have been identified using any other technique. The tendons of some steering muscles buckle on every wingbeat to accommodate high amplitude movements of the wing hinge. Other steering muscles absorb kinetic energy from an oscillating control linkage, which rotates at low wingbeat amplitude but translates at high wingbeat amplitude. Kinetic energy is distributed differently in these two modes of oscillation, which may play a role in asymmetric power management during flight control. Structural flexibility is known to be important to the aerodynamic efficiency of insect wings, and to the function of their indirect power muscles. We show that it is integral also to the operation of the steering muscles, and so to the functional flexibility of the insect flight motor

  13. In vivo time-resolved microtomography reveals the mechanics of the blowfly flight motor.

    Directory of Open Access Journals (Sweden)

    Simon M Walker

    2014-03-01

    Full Text Available Dipteran flies are amongst the smallest and most agile of flying animals. Their wings are driven indirectly by large power muscles, which cause cyclical deformations of the thorax that are amplified through the intricate wing hinge. Asymmetric flight manoeuvres are controlled by 13 pairs of steering muscles acting directly on the wing articulations. Collectively the steering muscles account for <3% of total flight muscle mass, raising the question of how they can modulate the vastly greater output of the power muscles during manoeuvres. Here we present the results of a synchrotron-based study performing micrometre-resolution, time-resolved microtomography on the 145 Hz wingbeat of blowflies. These data represent the first four-dimensional visualizations of an organism's internal movements on sub-millisecond and micrometre scales. This technique allows us to visualize and measure the three-dimensional movements of five of the largest steering muscles, and to place these in the context of the deforming thoracic mechanism that the muscles actuate. Our visualizations show that the steering muscles operate through a diverse range of nonlinear mechanisms, revealing several unexpected features that could not have been identified using any other technique. The tendons of some steering muscles buckle on every wingbeat to accommodate high amplitude movements of the wing hinge. Other steering muscles absorb kinetic energy from an oscillating control linkage, which rotates at low wingbeat amplitude but translates at high wingbeat amplitude. Kinetic energy is distributed differently in these two modes of oscillation, which may play a role in asymmetric power management during flight control. Structural flexibility is known to be important to the aerodynamic efficiency of insect wings, and to the function of their indirect power muscles. We show that it is integral also to the operation of the steering muscles, and so to the functional flexibility of the

  14. In vivo time-resolved microtomography reveals the mechanics of the blowfly flight motor.

    Science.gov (United States)

    Walker, Simon M; Schwyn, Daniel A; Mokso, Rajmund; Wicklein, Martina; Müller, Tonya; Doube, Michael; Stampanoni, Marco; Krapp, Holger G; Taylor, Graham K

    2014-03-01

    Dipteran flies are amongst the smallest and most agile of flying animals. Their wings are driven indirectly by large power muscles, which cause cyclical deformations of the thorax that are amplified through the intricate wing hinge. Asymmetric flight manoeuvres are controlled by 13 pairs of steering muscles acting directly on the wing articulations. Collectively the steering muscles account for flight muscle mass, raising the question of how they can modulate the vastly greater output of the power muscles during manoeuvres. Here we present the results of a synchrotron-based study performing micrometre-resolution, time-resolved microtomography on the 145 Hz wingbeat of blowflies. These data represent the first four-dimensional visualizations of an organism's internal movements on sub-millisecond and micrometre scales. This technique allows us to visualize and measure the three-dimensional movements of five of the largest steering muscles, and to place these in the context of the deforming thoracic mechanism that the muscles actuate. Our visualizations show that the steering muscles operate through a diverse range of nonlinear mechanisms, revealing several unexpected features that could not have been identified using any other technique. The tendons of some steering muscles buckle on every wingbeat to accommodate high amplitude movements of the wing hinge. Other steering muscles absorb kinetic energy from an oscillating control linkage, which rotates at low wingbeat amplitude but translates at high wingbeat amplitude. Kinetic energy is distributed differently in these two modes of oscillation, which may play a role in asymmetric power management during flight control. Structural flexibility is known to be important to the aerodynamic efficiency of insect wings, and to the function of their indirect power muscles. We show that it is integral also to the operation of the steering muscles, and so to the functional flexibility of the insect flight motor.

  15. Desiccation tolerance mechanism in resurrection fern-ally Selaginella tamariscina revealed by physiological and proteomic analysis.

    Science.gov (United States)

    Wang, Xiaonan; Chen, Sixue; Zhang, Heng; Shi, Lei; Cao, Fenglin; Guo, Lihai; Xie, Yongming; Wang, Tai; Yan, Xiufeng; Dai, Shaojun

    2010-12-03

    Drought is one of the most severe limitations to plant growth and productivity. Resurrection plants have evolved a unique capability to tolerate desiccation in vegetative tissues. Fern-ally Selaginella tamariscina (Beauv.) is one of the most primitive vascular resurrection plants, which can survive a desiccated state and recover when water becomes available. To better understand the mechanism of desiccation tolerance, we have applied physiological and proteomic analysis. Samples of S. tamariscina were water-deprived for up to seven days followed by 12 h of rewatering. Our results showed that endogenous abscisic acid (ABA) increased to regulate dehydration-responsive genes/proteins and physiological processes. In the course of dehydration, the contents of osmolytes represented by soluble sugars and proline were increased to maintain cell structure integrity. The activities of four antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutathione reductase (GR)) also increased. In contrast, both the rate of photosynthesis and the chlorophyll content decreased, and plasma membrane integrity was lost. We identified 138 desiccation-responsive two-dimensional electrophoresis (2-DE) spots, representing 103 unique proteins. Hierarchical clustering analysis revealed that 83% of the proteins were down-regulated upon dehydration. They were mainly involved in photosynthesis, carbohydrate and energy metabolism, stress and defense, protein metabolism, signaling, membrane/transport, cell structure, and cell division. The dynamic expression changes of the desiccation-responsive proteins provide strong evidence that cell structure modification, photosynthesis reduction, antioxidant system activation, and protein post-transcriptional/translational modifications are essential to the poikilochlorophyllous fern-ally S. tamariscina in response to dehydration. In addition, our comparative analysis of dehydration-responsive proteins in vegetative tissues

  16. The key role of glutamate 172 in the mechanism of type II NADH:quinone oxidoreductase of Staphylococcus aureus.

    Science.gov (United States)

    Sousa, Filipe M; Sena, Filipa V; Batista, Ana P; Athayde, Diogo; Brito, José A; Archer, Margarida; Oliveira, A Sofia F; Soares, Cláudio M; Catarino, Teresa; Pereira, Manuela M

    2017-10-01

    Type II NADH:quinone oxidoreductases (NDH-2s) are membrane bound enzymes that deliver electrons to the respiratory chain by oxidation of NADH and reduction of quinones. In this way, these enzymes also contribute to the regeneration of NAD(+), allowing several metabolic pathways to proceed. As for the other members of the two-Dinucleotide Binding Domains Flavoprotein (tDBDF) superfamily, the enzymatic mechanism of NDH-2s is still little explored and elusive. In this work we addressed the role of the conserved glutamate 172 (E172) residue in the enzymatic mechanism of NDH-2 from Staphylococcus aureus. We aimed to test our earlier hypothesis that E172 plays a key role in proton transfer to allow the protonation of the quinone. For this we performed a complete biochemical characterization of the enzyme's variants E172A, E172Q and E172S. Our steady state kinetic measurements show a clear decrease in the overall reaction rate, and our substrate interaction studies indicate the binding of the two substrates is also affected by these mutations. Interestingly our fast kinetic results show quinone reduction is more affected than NADH oxidation. We have also determined the X-ray crystal structure of the E172S mutant (2.55Ǻ) and compared it with the structure of the wild type (2.32Ǻ). Together these results support our hypothesis for E172 being of central importance in the catalytic mechanism of NDH-2, which may be extended to other members of the tDBDF superfamily. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Mechanisms of suspended animation are revealed by transcript profiling of diapause in the flesh fly

    National Research Council Canada - National Science Library

    Gregory J. Ragland; David L. Denlinger; Daniel A. Hahn

    2010-01-01

    .... We used transcriptional profiling to identify key groups of genes and pathways differentially regulated during pupal diapause, dynamically regulated across diapause development, and differentially...

  18. The gating mechanism of the human aquaporin 5 revealed by molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Lorant Janosi

    Full Text Available Aquaporins are protein channels located across the cell membrane with the role of conducting water or other small sugar alcohol molecules (aquaglyceroporins. The high-resolution X-ray structure of the human aquaporin 5 (HsAQP5 shows that HsAQP5, as all the other known aquaporins, exhibits tetrameric structure. By means of molecular dynamics simulations we analyzed the role of spontaneous fluctuations on the structural behavior of the human AQP5. We found that different conformations within the tetramer lead to a distribution of monomeric channel structures, which can be characterized as open or closed. The switch between the two states of a channel is a tap-like mechanism at the cytoplasmic end which regulates the water passage through the pore. The channel is closed by a translation of the His67 residue inside the pore. Moreover, water permeation rate calculations revealed that the selectivity filter, located at the other end of the channel, regulates the flow rate of water molecules when the channel is open, by locally modifying the orientation of His173. Furthermore, the calculated permeation rates of a fully open channel are in good agreement with the reported experimental value.

  19. Sac1--Vps74 structure reveals a mechanism to terminate phosphoinositide signaling in the Golgi apparatus

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Yiying; Deng, Yongqiang; Horenkamp, Florian; Reinisch, Karin M.; Burd, Christopher G. [Yale-MED

    2014-08-25

    Sac1 is a phosphoinositide phosphatase of the endoplasmic reticulum and Golgi apparatus that controls organelle membrane composition principally via regulation of phosphatidylinositol 4-phosphate signaling. We present a characterization of the structure of the N-terminal portion of yeast Sac1, containing the conserved Sac1 homology domain, in complex with Vps74, a phosphatidylinositol 4-kinase effector and the orthologue of human GOLPH3. The interface involves the N-terminal subdomain of the Sac1 homology domain, within which mutations in the related Sac3/Fig4 phosphatase have been linked to Charcot–Marie–Tooth disorder CMT4J and amyotrophic lateral sclerosis. Disruption of the Sac1–Vps74 interface results in a broader distribution of phosphatidylinositol 4-phosphate within the Golgi apparatus and failure to maintain residence of a medial Golgi mannosyltransferase. The analysis prompts a revision of the membrane-docking mechanism for GOLPH3 family proteins and reveals how an effector of phosphoinositide signaling serves a dual function in signal termination.

  20. Choreography of Transcriptomes and Lipidomes of Nannochloropsis Reveals the Mechanisms of Oil Synthesis in Microalgae.

    Science.gov (United States)

    Li, Jing; Han, Danxiang; Wang, Dongmei; Ning, Kang; Jia, Jing; Wei, Li; Jing, Xiaoyan; Huang, Shi; Chen, Jie; Li, Yantao; Hu, Qiang; Xu, Jian

    2014-04-01

    To reveal the molecular mechanisms of oleaginousness in microalgae, transcriptomic and lipidomic dynamics of the oleaginous microalga Nannochloropsis oceanica IMET1 under nitrogen-replete (N+) and N-depleted (N-) conditions were simultaneously tracked. At the transcript level, enhanced triacylglycerol (TAG) synthesis under N- conditions primarily involved upregulation of seven putative diacylglycerol acyltransferase (DGAT) genes and downregulation of six other DGAT genes, with a simultaneous elevation of the other Kennedy pathway genes. Under N- conditions, despite downregulation of most de novo fatty acid synthesis genes, the pathways that shunt carbon precursors from protein and carbohydrate metabolic pathways into glycerolipid synthesis were stimulated at the transcript level. In particular, the genes involved in supplying carbon precursors and energy for de novo fatty acid synthesis, including those encoding components of the pyruvate dehydrogenase complex (PDHC), glycolysis, and PDHC bypass, and suites of specific transporters, were substantially upregulated under N- conditions, resulting in increased overall TAG production. Moreover, genes involved in the citric acid cycle and β-oxidation in mitochondria were greatly enhanced to utilize the carbon skeletons derived from membrane lipids and proteins to produce additional TAG or its precursors. This temporal and spatial regulation model of oil accumulation in microalgae provides a basis for improving our understanding of TAG synthesis in microalgae and will also enable more rational genetic engineering of TAG production.

  1. Sac1-Vps74 structure reveals a mechanism to terminate phosphoinositide signaling in the Golgi apparatus.

    Science.gov (United States)

    Cai, Yiying; Deng, Yongqiang; Horenkamp, Florian; Reinisch, Karin M; Burd, Christopher G

    2014-08-18

    Sac1 is a phosphoinositide phosphatase of the endoplasmic reticulum and Golgi apparatus that controls organelle membrane composition principally via regulation of phosphatidylinositol 4-phosphate signaling. We present a characterization of the structure of the N-terminal portion of yeast Sac1, containing the conserved Sac1 homology domain, in complex with Vps74, a phosphatidylinositol 4-kinase effector and the orthologue of human GOLPH3. The interface involves the N-terminal subdomain of the Sac1 homology domain, within which mutations in the related Sac3/Fig4 phosphatase have been linked to Charcot-Marie-Tooth disorder CMT4J and amyotrophic lateral sclerosis. Disruption of the Sac1-Vps74 interface results in a broader distribution of phosphatidylinositol 4-phosphate within the Golgi apparatus and failure to maintain residence of a medial Golgi mannosyltransferase. The analysis prompts a revision of the membrane-docking mechanism for GOLPH3 family proteins and reveals how an effector of phosphoinositide signaling serves a dual function in signal termination.

  2. Large-scale analysis by SAGE reveals new mechanisms of v-erbA oncogene action

    Directory of Open Access Journals (Sweden)

    Faure Claudine

    2007-10-01

    Full Text Available Abstract Background: The v-erbA oncogene, carried by the Avian Erythroblastosis Virus, derives from the c-erbAα proto-oncogene that encodes the nuclear receptor for triiodothyronine (T3R. v-ErbA transforms erythroid progenitors in vitro by blocking their differentiation, supposedly by interference with T3R and RAR (Retinoic Acid Receptor. However, v-ErbA target genes involved in its transforming activity still remain to be identified. Results: By using Serial Analysis of Gene Expression (SAGE, we identified 110 genes deregulated by v-ErbA and potentially implicated in the transformation process. Bioinformatic analysis of promoter sequence and transcriptional assays point out a potential role of c-Myb in the v-ErbA effect. Furthermore, grouping of newly identified target genes by function revealed both expected (chromatin/transcription and unexpected (protein metabolism functions potentially deregulated by v-ErbA. We then focused our study on 15 of the new v-ErbA target genes and demonstrated by real time PCR that in majority their expression was activated neither by T3, nor RA, nor during differentiation. This was unexpected based upon the previously known role of v-ErbA. Conclusion: This paper suggests the involvement of a wealth of new unanticipated mechanisms of v-ErbA action.

  3. Patterned biofilm formation reveals a mechanism for structural heterogeneity in bacterial biofilms.

    Science.gov (United States)

    Gu, Huan; Hou, Shuyu; Yongyat, Chanokpon; De Tore, Suzanne; Ren, Dacheng

    2013-09-03

    Bacterial biofilms are ubiquitous and are the major cause of chronic infections in humans and persistent biofouling in industry. Despite the significance of bacterial biofilms, the mechanism of biofilm formation and associated drug tolerance is still not fully understood. A major challenge in biofilm research is the intrinsic heterogeneity in the biofilm structure, which leads to temporal and spatial variation in cell density and gene expression. To understand and control such structural heterogeneity, surfaces with patterned functional alkanthiols were used in this study to obtain Escherichia coli cell clusters with systematically varied cluster size and distance between clusters. The results from quantitative imaging analysis revealed an interesting phenomenon in which multicellular connections can be formed between cell clusters depending on the size of interacting clusters and the distance between them. In addition, significant differences in patterned biofilm formation were observed between wild-type E. coli RP437 and some of its isogenic mutants, indicating that certain cellular and genetic factors are involved in interactions among cell clusters. In particular, autoinducer-2-mediated quorum sensing was found to be important. Collectively, these results provide missing information that links cell-to-cell signaling and interaction among cell clusters to the structural organization of bacterial biofilms.

  4. Revealing Atomic-Level Mechanisms of Protein Allostery with Molecular Dynamics Simulations.

    Directory of Open Access Journals (Sweden)

    Samuel Hertig

    2016-06-01

    Full Text Available Molecular dynamics (MD simulations have become a powerful and popular method for the study of protein allostery, the widespread phenomenon in which a stimulus at one site on a protein influences the properties of another site on the protein. By capturing the motions of a protein's constituent atoms, simulations can enable the discovery of allosteric binding sites and the determination of the mechanistic basis for allostery. These results can provide a foundation for applications including rational drug design and protein engineering. Here, we provide an introduction to the investigation of protein allostery using molecular dynamics simulation. We emphasize the importance of designing simulations that include appropriate perturbations to the molecular system, such as the addition or removal of ligands or the application of mechanical force. We also demonstrate how the bidirectional nature of allostery-the fact that the two sites involved influence one another in a symmetrical manner-can facilitate such investigations. Through a series of case studies, we illustrate how these concepts have been used to reveal the structural basis for allostery in several proteins and protein complexes of biological and pharmaceutical interest.

  5. Revealing Atomic-Level Mechanisms of Protein Allostery with Molecular Dynamics Simulations.

    Science.gov (United States)

    Hertig, Samuel; Latorraca, Naomi R; Dror, Ron O

    2016-06-01

    Molecular dynamics (MD) simulations have become a powerful and popular method for the study of protein allostery, the widespread phenomenon in which a stimulus at one site on a protein influences the properties of another site on the protein. By capturing the motions of a protein's constituent atoms, simulations can enable the discovery of allosteric binding sites and the determination of the mechanistic basis for allostery. These results can provide a foundation for applications including rational drug design and protein engineering. Here, we provide an introduction to the investigation of protein allostery using molecular dynamics simulation. We emphasize the importance of designing simulations that include appropriate perturbations to the molecular system, such as the addition or removal of ligands or the application of mechanical force. We also demonstrate how the bidirectional nature of allostery-the fact that the two sites involved influence one another in a symmetrical manner-can facilitate such investigations. Through a series of case studies, we illustrate how these concepts have been used to reveal the structural basis for allostery in several proteins and protein complexes of biological and pharmaceutical interest.

  6. Structures of the NLRP14 pyrin domain reveal a conformational switch mechanism regulating its molecular interactions

    Energy Technology Data Exchange (ETDEWEB)

    Eibl, Clarissa; Hessenberger, Manuel; Wenger, Julia; Brandstetter, Hans, E-mail: hans.brandstetter@sbg.ac.at [University of Salzburg, Billrothstrasse 11, 5020 Salzburg (Austria)

    2014-07-01

    Pyrin domains (PYDs) recruit downstream effector molecules in NLR signalling. A specific charge-relay system suggests a the formation of a signalling complex involving a PYD dimer. The cytosolic tripartite NLR receptors serve as important signalling platforms in innate immunity. While the C-terminal domains act as sensor and activation modules, the N-terminal death-like domain, e.g. the CARD or pyrin domain, is thought to recruit downstream effector molecules by homotypic interactions. Such homotypic complexes have been determined for all members of the death-domain superfamily except for pyrin domains. Here, crystal structures of human NLRP14 pyrin-domain variants are reported. The wild-type protein as well as the clinical D86V mutant reveal an unexpected rearrangement of the C-terminal helix α6, resulting in an extended α5/6 stem-helix. This reordering mediates a novel symmetric pyrin-domain dimerization mode. The conformational switching is controlled by a charge-relay system with a drastic impact on protein stability. How the identified charge relay allows classification of NLRP receptors with respect to distinct recruitment mechanisms is discussed.

  7. Population genetics of two key mosquito vectors of Rift Valley Fever virus reveals new insights into the changing disease outbreak patterns in Kenya.

    Directory of Open Access Journals (Sweden)

    David P Tchouassi

    2014-12-01

    Full Text Available Rift Valley fever (RVF outbreaks in Kenya have increased in frequency and range to include northeastern Kenya where viruses are increasingly being isolated from known (Aedes mcintoshi and newly-associated (Ae. ochraceus vectors. The factors contributing to these changing outbreak patterns are unclear and the population genetic structure of key vectors and/or specific virus-vector associations, in particular, are under-studied. By conducting mitochondrial and nuclear DNA analyses on >220 Kenyan specimens of Ae. mcintoshi and Ae. ochraceus, we uncovered high levels of vector complexity which may partly explain the disease outbreak pattern. Results indicate that Ae. mcintoshi consists of a species complex with one of the member species being unique to the newly-established RVF outbreak-prone northeastern region of Kenya, whereas Ae. ochraceus is a homogeneous population that appears to be undergoing expansion. Characterization of specimens from a RVF-prone site in Senegal, where Ae. ochraceus is a primary vector, revealed direct genetic links between the two Ae. ochraceus populations from both countries. Our data strongly suggest that unlike Ae. mcintoshi, Ae. ochraceus appears to be a relatively recent, single 'introduction' into Kenya. These results, together with increasing isolations from this vector, indicate that Ae. ochraceus will likely be of greater epidemiological importance in future RVF outbreaks in Kenya. Furthermore, the overall vector complexity calls into question the feasibility of mosquito population control approaches reliant on genetic modification.

  8. RNA interference targeting CYP76AH1 in hairy roots of Salvia miltiorrhiza reveals its key role in the biosynthetic pathway of tanshinones.

    Science.gov (United States)

    Ma, Ying; Ma, Xiao-Hui; Meng, Fan-Yun; Zhan, Zhi-Lai; Guo, Juan; Huang, Lu-Qi

    2016-08-19

    Plant cytochrome P450s (CYPs) are well known as the largest family of enzymes that contribute to both primary metabolism and the chemical diversity of plant secondary metabolites. It is important to elucidate the in vivo role of CYPs in secondary metabolism, in order to apply them in the production of valuable metabolites in medicinal plants via metabolic engineering. CYP76AH1 has been suggested to catalyze the conversion of the carbon skeleton miltiradiene into the intermediate ferruginol, which is involved in the biosynthesis of tanshinones, the chief bioactive ingredients of Salvia miltiorrhiza. However, its role in planta remains to be elucidated. In this work, we constructed a CYP76AH1 RNAi system for hairy roots. Metabolic analysis of RNAi-AH1 hairy root lines showed a significantly increased accumulation of miltiradiene compared to the control lines. At the same time, the concentration of ferruginol decreased revealing the in vivo catalytic activity of CYP76AH1. The content of tanshinones decreased significantly after silencing of CYP76AH1, which verified its key role in the biosynthesis of tanshinones, and indicated that it could be used as a target for metabolic engineering.

  9. DNA stable-isotope probing of oil sands tailings pond enrichment cultures reveals different key players for toluene degradation under methanogenic and sulfidogenic conditions.

    Science.gov (United States)

    Laban, Nidal Abu; Dao, Anh; Foght, Julia

    2015-05-01

    Oil sands tailings ponds are anaerobic repositories of fluid wastes produced by extraction of bitumen from oil sands ores. Diverse indigenous microbiota biodegrade hydrocarbons (including toluene) in situ, producing methane, carbon dioxide and/or hydrogen sulfide, depending on electron acceptor availability. Stable-isotope probing of cultures enriched from tailings associated specific taxa and functional genes to (13)C6- and (12)C7-toluene degradation under methanogenic and sulfate-reducing conditions. Total DNA was subjected to isopycnic ultracentrifugation followed by gradient fraction analysis using terminal restriction fragment length polymorphism (T-RFLP) and construction of 16S rRNA, benzylsuccinate synthase (bssA) and dissimilatory sulfite reductase (dsrB) gene clone libraries. T-RFLP analysis plus sequencing and in silico digestion of cloned taxonomic and functional genes revealed that Clostridiales, particularly Desulfosporosinus (136 bp T-RF) contained bssA genes and were key toluene degraders during methanogenesis dominated by Methanosaeta. Deltaproteobacterial Desulfobulbaceae (157 bp T-RF) became dominant under sulfidogenic conditions, likely because the Desulfosporosinus T-RF 136 apparently lacks dsrB and therefore, unlike its close relatives, is presumed incapable of dissimilatory sulfate reduction. We infer incomplete oxidation of toluene by Desulfosporosinus in syntrophic association with Methanosaeta under methanogenic conditions, and complete toluene oxidation by Desulfobulbaceae during sulfate reduction. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. Comparative transcriptome combined with morpho-physiological analyses revealed key factors for differential cadmium accumulation in two contrasting sweet sorghum genotypes.

    Science.gov (United States)

    Feng, Juanjuan; Jia, Weitao; Lv, Sulian; Bao, Hexigeduleng; Miao, Fangfang; Zhang, Xuan; Wang, Jinhui; Li, Jihong; Li, Dongsheng; Zhu, Cheng; Li, Shizhong; Li, Yinxin

    2017-07-13

    Cadmium (Cd) is a widespread soil contaminant threatening human health. As an ideal energy plant, sweet sorghum (Sorghum bicolor (L.) Moench) has great potential in phytoremediation of Cd-polluted soils, although the molecular mechanisms are largely unknown. In this study, key factors responsible for differential Cd accumulation between two contrasting sweet sorghum genotypes (high-Cd accumulation one H18, and low-Cd accumulation one L69) were investigated. H18 exhibited a much higher ability of Cd uptake and translocation than L69. Furthermore, Cd uptake through symplasmic pathway and Cd concentrations in xylem sap were both higher in H18 than those in L69. Root anatomy observation found the endodermal apoplasmic barriers were much stronger in L69, which may restrict the Cd loading into xylem. The molecular mechanisms underlying these morpho-physiological traits were further dissected by comparative transcriptome analysis. Many genes involved in cell wall modification and heavy metal transport were found to be Cd-responsive DEGs and/or DEGs between these two genotypes. KEGG pathway analysis found phenylpropanoid biosynthesis pathway was over-represented, indicating this pathway may play important roles in differential Cd accumulation between two genotypes. Based on these results, a schematic representation of main processes involved in differential Cd uptake and translocation in H18 and L69 is proposed, which suggests that higher Cd accumulation in H18 depends on a multilevel coordination of efficient Cd uptake and transport, including efficient root uptake and xylem loading, less root cell wall binding, and weaker endodermal apoplasmic barriers. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  11. Proteomic analyses reveal the key roles of BrlA and AbaA in biogenesis of gliotoxin in Aspergillus fumigatus

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Kwang-Soo, E-mail: shinks@dju.kr [Division of Life Science, Daejeon University, Daejeon, 300-716 (Korea, Republic of); Kim, Young Hwan [Biomedical Omics Team, Korea Basic Science Institute (KBSI), Ohcang, 368-883 (Korea, Republic of); Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, 305-764 (Korea, Republic of); Department of Bio-Analytical Science, University of Science and Technology, Daejeon, 305-333 (Korea, Republic of); Yu, Jae-Hyuk, E-mail: jyu1@wisc.edu [Departments of Bacteriology and Genetics, The University of Wisconsin–Madison, Madison, WI, 53706 (United States)

    2015-07-31

    The opportunistic human pathogenic fungus Aspergillus fumigatus primarily reproduces by forming a large number of asexual spores (conidia). Sequential activation of the central regulators BrlA, AbaA and WetA is necessary for the fungus to undergo asexual development. In this study, to address the presumed roles of these key developmental regulators during proliferation of the fungus, we analyzed and compared the proteomes of vegetative cells of wild type (WT) and individual mutant strains. Approximately 1300 protein spots were detectable from 2-D electrophoresis gels. Among these, 13 proteins exhibiting significantly altered accumulation levels were further identified by ESI-MS/MS. Markedly, we found that the GliM and GliT proteins associated with gliotoxin (GT) biosynthesis and self-protection of the fungus from GT were significantly down-regulated in the ΔabaA and ΔbrlA mutants. Moreover, mRNA levels of other GT biosynthetic genes including gliM, gliP, gliT, and gliZ were significantly reduced in both mutant strains, and no and low levels of GT were detectable in the ΔbrlA and ΔabaA mutant strains, respectively. As GliT is required for the protection of the fungus from GT, growth of the ΔbrlA mutant with reduced levels of GliT was severely impaired by exogenous GT. Our studies demonstrate that AbaA and BrlA positively regulate expression of the GT biosynthetic gene cluster in actively growing vegetative cells, and likely bridge morphological and chemical development during the life-cycle of A. fumigatus. - Highlights: • Proteome analyses of WT and mutants reveal 13 differentially expressed proteins. • The GliT and GliM proteins are significantly down-regulated by ΔabaA and ΔbrlA. • Expression of other gliotoxin biosynthetic genes is lowered by ΔabaA and ΔbrlA. • Growth of ΔbrlA strain lacking GliT is completely inhibited by exogenous gliotoxin. • BrlA and AbaA play key roles in biogenesis of gliotoxin in Aspergillus fumigatus.

  12. Epitope mapping for monoclonal antibody reveals the activation mechanism for αVβ3 integrin.

    Directory of Open Access Journals (Sweden)

    Tetsuji Kamata

    Full Text Available Epitopes for a panel of anti-αVβ3 monoclonal antibodies (mAbs were investigated to explore the activation mechanism of αVβ3 integrin. Experiments utilizing αV/αIIb domain-swapping chimeras revealed that among the nine mAbs tested, five recognized the ligand-binding β-propeller domain and four recognized the thigh domain, which is the upper leg of the αV chain. Interestingly, the four mAbs included function-blocking as well as non-functional mAbs, although they bound at a distance from the ligand-binding site. The epitopes for these four mAbs were further determined using human-to-mouse αV chimeras. Among the four, P3G8 recognized an amino acid residue, Ser-528, located on the side of the thigh domain, while AMF-7, M9, and P2W7 all recognized a common epitope, Ser-462, that was located close to the α-genu, where integrin makes a sharp bend in the crystal structure. Fibrinogen binding studies for cells expressing wild-type αVβ3 confirmed that AMF-7, M9, and P2W7 were inhibitory, while P3G8 was non-functional. However, these mAbs were all unable to block binding when αVβ3 was constrained in its extended conformation. These results suggest that AMF-7, M9, and P2W7 block ligand binding allosterically by stabilizing the angle of the bend in the bent conformation. Thus, a switchblade-like movement of the integrin leg is indispensable for the affinity regulation of αVβ3 integrin.

  13. Informal payments and the quality of health care: Mechanisms revealed by Tanzanian health workers.

    Science.gov (United States)

    Mæstad, Ottar; Mwisongo, Aziza

    2011-02-01

    Informal payments for health services are common in many transitional and developing countries. The aim of this paper is to investigate the nature of informal payments in the health sector of Tanzania and to identify mechanisms through which informal payments may affect the quality of health care. Our focus is on the effect of informal payments on health worker behaviours, in particular the interpersonal dynamics among health workers at their workplaces. We organised eight focus groups with 58 health workers representing different cadres and levels of care in one rural and one urban district in Tanzania. We found that health workers at all levels receive informal payments in a number of different contexts. Health workers sometimes share the payments received, but only partially, and more rarely within the cadre than across cadres. Our findings indicate that health workers are involved in 'rent-seeking' activities, such as creating artificial shortages and deliberately lowering the quality of service, in order to extract extra payments from patients or to bargain for a higher share of the payments received by their colleagues. The discussions revealed that many health workers think that the distribution of informal payments is grossly unfair. The findings suggest that informal payments can impact negatively on the quality of health care through rent-seeking behaviours and through frustrations created by the unfair allocation of payments. Interestingly, the presence of corruption may also induce non-corrupt workers to reduce the quality of care. Positive impacts can occur because informal payments may induce health workers to increase their efforts, and maybe more so if there is competition among health workers about receiving the payments. Moreover, informal payments add to health workers' incomes and might thus contribute to retention of health workers within the health sector.

  14. Proton Gradients as a Key Physical Factor in the Evolution of the Forced Transport Mechanism Across the Lipid Membrane.

    Science.gov (United States)

    Strbak, Oliver; Kanuchova, Zuzana; Krafcik, Andrej

    2016-11-01

    A critical phase in the transition from prebiotic chemistry to biological evolution was apparently an asymmetric ion flow across the lipid membrane. Due to imbalance in the ion flow, the early lipid vesicles could selectively take the necessary molecules from the environment, and release the side-products from the vesicle. Natural proton gradients played a definitively crucial role in this process, since they remain the basis of energy transfer in the present-day cells. On the basis of this supposition, and the premise of the early vesicle membrane's impermeability to protons, we have shown that the emergence of the proton gradient in the lipid vesicle could be a key physical factor in the evolution of the forced transport mechanism (pore formation and active transport) across the lipid bilayer. This driven flow of protons across the membrane is the result of the electrochemical proton gradient and osmotic pressures on the integrity of the lipid vesicle. At a critical number of new lipid molecules incorporated into the vesicle, the energies associated with the creation of the proton gradient exceed the bending stiffness of the lipid membrane, and overlap the free energy of the lipid bilayer pore formation.

  15. Environmental Conditions Influence Induction of Key ABC-Transporter Genes Affecting Glyphosate Resistance Mechanism in Conyza canadensis

    Directory of Open Access Journals (Sweden)

    Eleni Tani

    2016-04-01

    Full Text Available Conyza canadensis has been reported to be the most frequent weed species that evolved resistance to glyphosate in various parts of the world. The objective of the present study was to investigate the effect of environmental conditions (temperature and light on the expression levels of the EPSPS gene and two major ABC-transporter genes (M10 and M11 on glyphosate susceptible (GS and glyphosate resistant (GR horseweed populations, collected from several regions across Greece. Real-time PCR was conducted to determine the expression level of the aforementioned genes when glyphosate was applied at normal (1×; 533 g·a.e.·ha−1 and high rates (4×, 8×, measured at an early one day after treatment (DAT and a later stage (four DAT of expression. Plants were exposed to light or dark conditions, at three temperature regimes (8, 25, 35 °C. GR plants were made sensitive when exposed to 8 °C with light; those sensitized plants behaved biochemically (shikimate accumulation and molecularly (expression of EPSPS and ABC-genes like the GS plants. Results from the current study show the direct link between the environmental conditions and the induction level of the above key genes that likely affect the efficiency of the proposed mechanism of glyphosate resistance.

  16. Environmental Conditions Influence Induction of Key ABC-Transporter Genes Affecting Glyphosate Resistance Mechanism in Conyza canadensis.

    Science.gov (United States)

    Tani, Eleni; Chachalis, Demosthenis; Travlos, Ilias S; Bilalis, Dimitrios

    2016-04-20

    Conyza canadensis has been reported to be the most frequent weed species that evolved resistance to glyphosate in various parts of the world. The objective of the present study was to investigate the effect of environmental conditions (temperature and light) on the expression levels of the EPSPS gene and two major ABC-transporter genes (M10 and M11) on glyphosate susceptible (GS) and glyphosate resistant (GR) horseweed populations, collected from several regions across Greece. Real-time PCR was conducted to determine the expression level of the aforementioned genes when glyphosate was applied at normal (1×; 533 g·a.e.·ha(-1)) and high rates (4×, 8×), measured at an early one day after treatment (DAT) and a later stage (four DAT) of expression. Plants were exposed to light or dark conditions, at three temperature regimes (8, 25, 35 °C). GR plants were made sensitive when exposed to 8 °C with light; those sensitized plants behaved biochemically (shikimate accumulation) and molecularly (expression of EPSPS and ABC-genes) like the GS plants. Results from the current study show the direct link between the environmental conditions and the induction level of the above key genes that likely affect the efficiency of the proposed mechanism of glyphosate resistance.

  17. Proton Gradients as a Key Physical Factor in the Evolution of the Forced Transport Mechanism Across the Lipid Membrane

    Science.gov (United States)

    Strbak, Oliver; Kanuchova, Zuzana; Krafcik, Andrej

    2016-11-01

    A critical phase in the transition from prebiotic chemistry to biological evolution was apparently an asymmetric ion flow across the lipid membrane. Due to imbalance in the ion flow, the early lipid vesicles could selectively take the necessary molecules from the environment, and release the side-products from the vesicle. Natural proton gradients played a definitively crucial role in this process, since they remain the basis of energy transfer in the present-day cells. On the basis of this supposition, and the premise of the early vesicle membrane's impermeability to protons, we have shown that the emergence of the proton gradient in the lipid vesicle could be a key physical factor in the evolution of the forced transport mechanism (pore formation and active transport) across the lipid bilayer. This driven flow of protons across the membrane is the result of the electrochemical proton gradient and osmotic pressures on the integrity of the lipid vesicle. At a critical number of new lipid molecules incorporated into the vesicle, the energies associated with the creation of the proton gradient exceed the bending stiffness of the lipid membrane, and overlap the free energy of the lipid bilayer pore formation.

  18. Anaerobic biodegradability and methanogenic toxicity of key constituents in copper chemical mechanical planarization effluents of the semiconductor industry.

    Science.gov (United States)

    Hollingsworth, Jeremy; Sierra-Alvarez, Reyes; Zhou, Michael; Ogden, Kimberly L; Field, Jim A

    2005-06-01

    Copper chemical mechanical planarization (CMP) effluents can account for 30-40% of the water discharge in semiconductor manufacturing. CMP effluents contain high concentrations of soluble copper and a complex mixture of organic constituents. The aim of this study is to perform a preliminary assessment of the treatability of CMP effluents in anaerobic sulfidogenic bioreactors inoculated with anaerobic granular sludge by testing individual compounds expected in the CMP effluents. Of all the compounds tested (copper (II), benzotriazoles, polyethylene glycol (M(n) 300), polyethylene glycol (M(n) 860) monooleate, perfluoro-1-octane sulfonate, citric acid, oxalic acid and isopropanol) only copper was found to be inhibitory to methanogenic activity at the concentrations tested. Most of the organic compounds tested were biodegradable with the exception of perfluoro-1-octane sulfonate and benzotriazoles under sulfate reducing conditions and with the exception of the same compounds as well as Triton X-100 under methanogenic conditions. The susceptibility of key components in CMP effluents to anaerobic biodegradation combined with their low microbial inhibition suggest that CMP effluents should be amenable to biological treatment in sulfate reducing bioreactors.

  19. Edwardsiella comparative phylogenomics reveal the new intra/inter-species taxonomic relationships, virulence evolution and niche adaptation mechanisms.

    Directory of Open Access Journals (Sweden)

    Minjun Yang

    Full Text Available Edwardsiella bacteria are leading fish pathogens causing huge losses to aquaculture industries worldwide. E. tarda is a broad-host range pathogen that infects more than 20 species of fish and other animals including humans while E. ictaluri is host-adapted to channel catfish causing enteric septicemia of catfish (ESC. Thus, these two species consist of a useful comparative system for studying the intricacies of pathogen evolution. Here we present for the first time the phylogenomic comparisons of 8 genomes of E. tarda and E. ictaluri isolates. Genome-based phylogenetic analysis revealed that E. tarda could be separate into two kinds of genotypes (genotype I, EdwGI and genotype II, EdwGII based on the sequence similarity. E. tarda strains of EdwGI were clustered together with the E. ictaluri lineage and showed low sequence conservation to E. tarda strains of EdwGII. Multilocus sequence analysis (MLSA of 48 distinct Edwardsiella strains also supports the new taxonomic relationship of the lineages. We identified the type III and VI secretion systems (T3SS and T6SS as well as iron scavenging related genes that fulfilled the criteria of a key evolutionary factor likely facilitating the virulence evolution and adaptation to a broad range of hosts in EdwGI E. tarda. The surface structure-related genes may underlie the adaptive evolution of E. ictaluri in the host specification processes. Virulence and competition assays of the null mutants of the representative genes experimentally confirmed their contributive roles in the evolution/niche adaptive processes. We also reconstructed the hypothetical evolutionary pathway to highlight the virulence evolution and niche adaptation mechanisms of Edwardsiella. This study may facilitate the development of diagnostics, vaccines, and therapeutics for this under-studied pathogen.

  20. Transcriptomic Analysis Reveals Mechanisms of Sterile and Fertile Flower Differentiation and Development in Viburnum macrocephalum f. keteleeri

    Science.gov (United States)

    Lu, Zhaogeng; Xu, Jing; Li, Weixing; Zhang, Li; Cui, Jiawen; He, Qingsong; Wang, Li; Jin, Biao

    2017-01-01

    Sterile and fertile flowers are an important evolutionary developmental (evo-devo) phenotype in angiosperm flowers, playing important roles in pollinator attraction and sexual reproductive success. However, the gene regulatory mechanisms underlying fertile and sterile flower differentiation and development remain largely unknown. Viburnum macrocephalum f. keteleeri, which possesses fertile and sterile flowers in a single inflorescence, is a useful candidate species for investigating the regulatory networks in differentiation and development. We developed a de novo-assembled flower reference transcriptome. Using RNA sequencing (RNA-seq), we compared the expression patterns of fertile and sterile flowers isolated from the same inflorescence over its rapid developmental stages. The flower reference transcriptome consisted of 105,683 non-redundant transcripts, of which 5,675 transcripts showed significant differential expression between fertile and sterile flowers. Combined with morphological and cytological changes between fertile and sterile flowers, we identified expression changes of many genes potentially involved in reproductive processes, phytohormone signaling, and cell proliferation and expansion using RNA-seq and qRT-PCR. In particular, many transcription factors (TFs), including MADS-box family members and ABCDE-class genes, were identified, and expression changes in TFs involved in multiple functions were analyzed and highlighted to determine their roles in regulating fertile and sterile flower differentiation and development. Our large-scale transcriptional analysis of fertile and sterile flowers revealed the dynamics of transcriptional networks and potentially key components in regulating differentiation and development of fertile and sterile flowers in Viburnum macrocephalum f. keteleeri. Our data provide a useful resource for Viburnum transcriptional research and offer insights into gene regulation of differentiation of diverse evo-devo processes in

  1. Atomic force microscopy stiffness tomography on living Arabidopsis thaliana cells reveals the mechanical properties of surface and deep cell-wall layers during growth.

    Science.gov (United States)

    Radotić, Ksenija; Roduit, Charles; Simonović, Jasna; Hornitschek, Patricia; Fankhauser, Christian; Mutavdžić, Dragosav; Steinbach, Gabor; Dietler, Giovanni; Kasas, Sandor

    2012-08-08

    Cell-wall mechanical properties play a key role in the growth and the protection of plants. However, little is known about genuine wall mechanical properties and their growth-related dynamics at subcellular resolution and in living cells. Here, we used atomic force microscopy (AFM) stiffness tomography to explore stiffness distribution in the cell wall of suspension-cultured Arabidopsis thaliana as a model of primary, growing cell wall. For the first time that we know of, this new imaging technique was performed on living single cells of a higher plant, permitting monitoring of the stiffness distribution in cell-wall layers as a function of the depth and its evolution during the different growth phases. The mechanical measurements were correlated with changes in the composition of the cell wall, which were revealed by Fourier-transform infrared (FTIR) spectroscopy. In the beginning and end of cell growth, the average stiffness of the cell wall was low and the wall was mechanically homogenous, whereas in the exponential growth phase, the average wall stiffness increased, with increasing heterogeneity. In this phase, the difference between the superficial and deep wall stiffness was highest. FTIR spectra revealed a relative increase in the polysaccharide/lignin content.

  2. RNA-Seq reveals dynamic changes of gene expression in key stages of intestine regeneration in the sea cucumber Apostichopus japonicus. [corrected].

    Directory of Open Access Journals (Sweden)

    Lina Sun

    Full Text Available BACKGROUND: Sea cucumbers (Holothuroidea; Echinodermata have the capacity to regenerate lost tissues and organs. Although the histological and cytological aspects of intestine regeneration have been extensively studied, little is known of the genetic mechanisms involved. There has, however, been a renewed effort to develop a database of Expressed Sequence Tags (ESTs in Apostichopus japonicus, an economically-important species that occurs in China. This is important for studies on genetic breeding, molecular markers and special physiological phenomena. We have also constructed a library of ESTs obtained from the regenerative body wall and intestine of A. japonicus. The database has increased to ~30000 ESTs. RESULTS: We used RNA-Seq to determine gene expression profiles associated with intestinal regeneration in A. japonicus at 3, 7, 14 and 21 days post evisceration (dpe. This was compared to profiles obtained from a normally-functioning intestine. Approximately 5 million (M reads were sequenced in every library. Over 2400 up-regulated genes (>10% and over 1000 down-regulated genes (~5% were observed at 3 and 7dpe (log2Ratio ≥ 1, FDR ≤ 0.001. Specific "Go terms" revealed that the DEGs (Differentially Expressed Genes performed an important function at every regeneration stage. Besides some expected pathways (for example, Ribosome and Spliceosome pathway term, the "Notch signaling pathway," the "ECM-receptor interaction" and the "Cytokine-cytokine receptor interaction" were significantly enriched. We also investigated the expression profiles of developmental genes, ECM-associated genes and Cytoskeletal genes. Twenty of the most important differentially expressed genes (DEGs were verified by Real-time PCR, which resulted in a trend concordance of almost 100% between the two techniques. CONCLUSION: Our studies demonstrated dynamic changes in global gene expression during intestine regeneration and presented a series of candidate genes and enriched

  3. Communication: Slow supramolecular mode in amine and thiol derivatives of 2-ethyl-1-hexanol revealed by combined dielectric and shear-mechanical studies.

    Science.gov (United States)

    Adrjanowicz, K; Jakobsen, B; Hecksher, T; Kaminski, K; Dulski, M; Paluch, M; Niss, K

    2015-11-14

    In this paper, we present results of dielectric and shear-mechanical studies for amine (2-ethyl-1-hexylamine) and thiol (2-ethyl-1-hexanethiol) derivatives of the monohydroxy alcohol, 2-ethyl-1-hexanol. The amine and thiol can form hydrogen bonds weaker in strength than those of the alcohol. The combination of dielectric and shear-mechanical data enables us to reveal the presence of a relaxation mode slower than the α-relaxation. This mode is analogous to the Debye mode seen in monohydroxy alcohols and demonstrates that supramolecular structures are present for systems with lower hydrogen bonding strength. We report some key features accompanying the decrease in the strength of the hydrogen bonding interactions on the relaxation dynamics close to the glass-transition. This includes changes (i) in the amplitude of the Debye and α-relaxations and (ii) the separation between primary and secondary modes.

  4. Two Antagonistic MALT1 Auto-Cleavage Mechanisms Reveal a Role for TRAF6 to Unleash MALT1 Activation.

    Science.gov (United States)

    Ginster, Stefanie; Bardet, Maureen; Unterreiner, Adeline; Malinverni, Claire; Renner, Florian; Lam, Stephen; Freuler, Felix; Gerrits, Bertran; Voshol, Johannes; Calzascia, Thomas; Régnier, Catherine H; Renatus, Martin; Nikolay, Rainer; Israël, Laura; Bornancin, Frédéric

    2017-01-01

    The paracaspase MALT1 has arginine-directed proteolytic activity triggered by engagement of immune receptors. Recruitment of MALT1 into activation complexes is required for MALT1 proteolytic function. Here, co-expression of MALT1 in HEK293 cells, either with activated CARD11 and BCL10 or with TRAF6, was used to explore the mechanism of MALT1 activation at the molecular level. This work identified a prominent self-cleavage site of MALT1 isoform A (MALT1A) at R781 (R770 in MALT1B) and revealed that TRAF6 can activate MALT1 independently of the CBM. Intramolecular cleavage at R781/R770 removes a C-terminal TRAF6-binding site in both MALT1 isoforms, leaving MALT1B devoid of the two key interaction sites with TRAF6. A previously identified auto-proteolysis site of MALT1 at R149 leads to deletion of the death-domain, thereby abolishing interaction with BCL10. By using MALT1 isoforms and cleaved fragments thereof, as well as TRAF6 WT and mutant forms, this work shows that TRAF6 induces N-terminal auto-proteolytic cleavage of MALT1 at R149 and accelerates MALT1 protein turnover. The MALT1 fragment generated by N-terminal self-cleavage at R149 was labile and displayed enhanced signaling properties that required an intact K644 residue, previously shown to be a site for mono-ubiquitination of MALT1. Conversely, C-terminal self-cleavage at R781/R770 hampered the ability for self-cleavage at R149 and stabilized MALT1 by hindering interaction with TRAF6. C-terminal self-cleavage had limited impact on MALT1A but severely reduced MALT1B proteolytic and signaling functions. It also abrogated NF-κB activation by N-terminally cleaved MALT1A. Altogether, this study provides further insights into mechanisms that regulate the scaffolding and activation cycle of MALT1. It also emphasizes the reduced functional capacity of MALT1B as compared to MALT1A.

  5. Two Antagonistic MALT1 Auto-Cleavage Mechanisms Reveal a Role for TRAF6 to Unleash MALT1 Activation

    Science.gov (United States)

    Renner, Florian; Lam, Stephen; Freuler, Felix; Gerrits, Bertran; Voshol, Johannes; Calzascia, Thomas; Régnier, Catherine H.; Renatus, Martin; Nikolay, Rainer; Israël, Laura; Bornancin, Frédéric

    2017-01-01

    The paracaspase MALT1 has arginine-directed proteolytic activity triggered by engagement of immune receptors. Recruitment of MALT1 into activation complexes is required for MALT1 proteolytic function. Here, co-expression of MALT1 in HEK293 cells, either with activated CARD11 and BCL10 or with TRAF6, was used to explore the mechanism of MALT1 activation at the molecular level. This work identified a prominent self-cleavage site of MALT1 isoform A (MALT1A) at R781 (R770 in MALT1B) and revealed that TRAF6 can activate MALT1 independently of the CBM. Intramolecular cleavage at R781/R770 removes a C-terminal TRAF6-binding site in both MALT1 isoforms, leaving MALT1B devoid of the two key interaction sites with TRAF6. A previously identified auto-proteolysis site of MALT1 at R149 leads to deletion of the death-domain, thereby abolishing interaction with BCL10. By using MALT1 isoforms and cleaved fragments thereof, as well as TRAF6 WT and mutant forms, this work shows that TRAF6 induces N-terminal auto-proteolytic cleavage of MALT1 at R149 and accelerates MALT1 protein turnover. The MALT1 fragment generated by N-terminal self-cleavage at R149 was labile and displayed enhanced signaling properties that required an intact K644 residue, previously shown to be a site for mono-ubiquitination of MALT1. Conversely, C-terminal self-cleavage at R781/R770 hampered the ability for self-cleavage at R149 and stabilized MALT1 by hindering interaction with TRAF6. C-terminal self-cleavage had limited impact on MALT1A but severely reduced MALT1B proteolytic and signaling functions. It also abrogated NF-κB activation by N-terminally cleaved MALT1A. Altogether, this study provides further insights into mechanisms that regulate the scaffolding and activation cycle of MALT1. It also emphasizes the reduced functional capacity of MALT1B as compared to MALT1A. PMID:28052131

  6. PDE11A regulates social behaviors and is a key mechanism by which social experience sculpts the brain.

    Science.gov (United States)

    Hegde, Shweta; Ji, Hao; Oliver, David; Patel, Neema S; Poupore, Nicolas; Shtutman, Michael; Kelly, Michy P

    2016-10-29

    Despite the fact that appropriate social behaviors are vital to thriving in one's environment, little is understood of the molecular mechanisms controlling social behaviors or how social experience sculpts these signaling pathways. Here, we determine if Phosphodiesterase 11A (PDE11A), an enzyme that is enriched in the ventral hippocampal formation (VHIPP) and that breaks down cAMP and cGMP, regulates social behaviors. PDE11 wild-type (WT), heterozygous (HT), and knockout (KO) mice were tested in various social approach assays and gene expression differences were measured by RNA sequencing. The effect of social isolation on PDE11A4 compartmentalization and subsequent social interactions and social memory was also assessed. Deletion of PDE11A triggered age- and sex-dependent deficits in social approach in specific social contexts but not others. Mice appear to detect altered social behaviors of PDE11A KO mice, because C57BL/6J mice prefer to spend time with a sex-matched PDE11A WT vs. its KO littermate; whereas, a PDE11A KO prefers to spend time with a novel PDE11A KO vs. its WT littermate. Not only is PDE11A required for intact social interactions, we found that 1month of social isolation vs. group housing decreased PDE11A4 protein expression specifically within the membrane fraction of VHIPP. This isolation-induced decrease in PDE11A4 expression appears functional because social isolation impairs subsequent social approach behavior and social memory in a PDE11A genotype-dependent manner. Pathway analyses following RNA sequencing suggests PDE11A is a key regulator of the oxytocin pathway and membrane signaling, consistent with its pivotal role in regulating social behavior.

  7. Structures of down syndrome kinases, DYRKs, reveal mechanisms of kinase activation and substrate recognition

    DEFF Research Database (Denmark)

    Soundararajan, M.; Roos, A.K.; Savitsky, P.

    2013-01-01

    Dual-specificity tyrosine-(Y)-phosphorylation-regulated kinases (DYRKs) play key roles in brain development, regulation of splicing, and apoptosis, and are potential drug targets for neurodegenerative diseases and cancer. We present crystal structures of one representative member of each DYRK sub...

  8. Decoupling kinematics and mechanics reveals coding properties of trigeminal ganglion neurons in the rat vibrissal system.

    Science.gov (United States)

    Bush, Nicholas E; Schroeder, Christopher L; Hobbs, Jennifer A; Yang, Anne Et; Huet, Lucie A; Solla, Sara A; Hartmann, Mitra Jz

    2016-06-27

    Tactile information available to the rat vibrissal system begins as external forces that cause whisker deformations, which in turn excite mechanoreceptors in the follicle. Despite the fundamental mechanical origin of tactile information, primary sensory neurons in the trigeminal ganglion (Vg) have often been described as encoding the kinematics (geometry) of object contact. Here we aimed to determine the extent to which Vg neurons encode the kinematics vs. mechanics of contact. We used models of whisker bending to quantify mechanical signals (forces and moments) at the whisker base while simultaneously monitoring whisker kinematics and recording single Vg units in both anesthetized rats and awake, body restrained rats. We employed a novel manual stimulation technique to deflect whiskers in a way that decouples kinematics from mechanics, and used Generalized Linear Models (GLMs) to show that Vg neurons more directly encode mechanical signals when the whisker is deflected in this decoupled stimulus space.

  9. Mutation of putative N-linked glycosylation sites on the human nucleotide receptor P2X7 reveals a key residue important for receptor function.

    Science.gov (United States)

    Lenertz, Lisa Y; Wang, Ziyi; Guadarrama, Arturo; Hill, Lindsay M; Gavala, Monica L; Bertics, Paul J

    2010-06-08

    The nucleotide receptor P2X(7) is an immunomodulatory cation channel and a potential therapeutic target. P2X(7) is expressed in immune cells such as monocytes and macrophages and is activated by extracellular ATP following tissue injury or infection. Ligand binding to P2X(7) can stimulate ERK1/2, the transcription factor CREB, enzymes linked to the production of reactive oxygen species and interleukin-1 isoforms, and the formation of a nonspecific pore. However, little is known about the biochemistry of P2X(7), including whether the receptor is N-linked glycosylated and if this modification affects receptor function. Here we provide evidence that P2X(7) is sensitive to the glycosidases EndoH and PNGase F and that the human receptor appears glycosylated at N187, N202, N213, N241, and N284. Mutation of N187 results in weakened P2X(7) agonist-induced phosphorylation of ERK1/2, CREB, and p90 ribosomal S6 kinase, as well as a decreased level of pore formation. In further support of a role for glycosylation in receptor function, treatment of RAW 264.7 macrophages with the N-linked glycosylation synthesis inhibitor tunicamycin attenuates P2X(7) agonist-induced, but not phorbol ester-induced, ERK1/2 phosphorylation. Interestingly, residue N187 belongs to an N-linked glycosylation consensus sequence found in six of the seven P2X family members, suggesting this site is fundamentally important to P2X receptor function. To address the mechanism whereby N187 mutation attenuates receptor activity, we developed a live cell proteinase K digestion assay that demonstrated altered cell surface expression of P2X(7) N187A. This is the first report to map human P2X(7) glycosylation sites and reveal residue N187 is critical for receptor trafficking and function.

  10. Novel mechanisms for superoxide-scavenging activity of human manganese superoxide dismutase determined by the K68 key acetylation site.

    Science.gov (United States)

    Lu, Jiaqi; Cheng, Kuoyuan; Zhang, Bo; Xu, Huan; Cao, Yuanzhao; Guo, Fei; Feng, Xudong; Xia, Qing

    2015-08-01

    Superoxide is the primary reactive oxygen species generated in the mitochondria. Manganese superoxide dismutase (SOD2) is the major enzymatic superoxide scavenger present in the mitochondrial matrix and one of the most crucial reactive oxygen species-scavenging enzymes in the cell. SOD2 is activated by sirtuin 3 (SIRT3) through NAD(+)-dependent deacetylation. However, the exact acetylation sites of SOD2 are ambiguous and the mechanisms underlying the deacetylation-mediated SOD2 activation largely remain unknown. We are the first to characterize SOD2 mutants of the acetylation sites by investigating the relative enzymatic activity, structures, and electrostatic potential of SOD2 in this study. These SOD2 mutations affected the superoxide-scavenging activity in vitro and in HEK293T cells. The lysine 68 (K68) site is the most important acetylation site contributing to SOD2 activation and plays a role in cell survival after paraquat treatment. The molecular basis underlying the regulation of SOD2 activity by K68 was investigated in detail. Molecular dynamics simulations revealed that K68 mutations induced a conformational shift of residues located in the active center of SOD2 and altered the charge distribution on the SOD2 surface. Thus, the entry of the superoxide anion into the coordinated core of SOD2 was inhibited. Our results provide a novel mechanistic insight, whereby SOD2 acetylation affects the structure and charge distribution of SOD2, its tetramerization, and p53-SOD2 interactions of SOD2 in the mitochondria, which may play a role in nuclear-mitochondrial communication during aging.

  11. A role of TRPA1 in mechanical hyperalgesia is revealed by pharmacological inhibition

    Directory of Open Access Journals (Sweden)

    Huynh Truc

    2007-12-01

    Full Text Available Abstract Mechanical hyperalgesia is a clinically-relevant form of pain sensitization that develops through largely unknown mechanisms. TRPA1, a Transient Receptor Potential ion channel, is a sensor of pungent chemicals that may play a role in acute noxious mechanosensation and cold thermosensation. We have developed a specific small molecule TRPA1 inhibitor (AP18 that can reduce cinnameldehyde-induced nociception in vivo. Interestingly, AP18 is capable of reversing CFA-induced mechanical hyperalgesia in mice. Although TRPA1-deficient mice develop normal CFA-induced hyperalgeisa, AP18 is ineffective in the knockout mice, consistent with an on-target mechanism. Therefore, TRPA1 plays a role in sensitization of nociception, and that compensation in TRPA1-deficient mice masks this requirement.

  12. Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire.

    Science.gov (United States)

    The P. ultimum DAOM BR144 (=CBS 805.95 = ATCC200006) genome (42.8 Mb) encodes 15,290 genes, and has extensive sequence similarity and synteny with related Phytophthora spp., including the potato late blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86 % o...

  13. Multitechnique approach to reveal the mechanism of copper(II)-catalyzed arylation reactions

    NARCIS (Netherlands)

    Tromp, M.; van Strijdonck, G.P.F.; van Berkel, S.S.; van den Hoogenband, A.; Feiters, M.C.; de Bruin, B.; Fiddy, S.G.; van der Eerden, A.M.J.; van Bokhoven, J.A.; van Leeuwen, P.W.N.M.; Koningsberger, D.C.

    2010-01-01

    Multiple in situ and time-resolved spectroscopic techniques (EDXAFS, UV−vis, EPR, and NMR), with a focus on simultaneously acquired EDXAFS and time-resolved UV−vis, are described to reveal detailed structural and electronic information on reaction intermediates of an important Cu(II)-catalyzed N-ary

  14. Mechanical properties of ultra thin metallic films revealed by synchrotron techniques

    Energy Technology Data Exchange (ETDEWEB)

    Gruber, Patric Alfons

    2007-07-20

    A prerequisite for the study of the scaling behavior of mechanical properties of ultra thin films is a suitable testing technique. Therefore synchrotron-based in situ testing techniques were developed and optimized in order to characterize the stress evolution in ultra thin metallic films on compliant polymer substrates during isothermal tensile tests. Experimental procedures for polycrystalline as well as single crystalline films were established. These techniques were used to systematically investigate the influence of microstructure, film thickness (20 to 1000 nm) and temperature (-150 to 200 C) on the mechanical properties. Passivated and unpassivated Au and Cu films as well as single crystalline Au films on polyimide substrates were tested. Special care was also dedicated to the microstructural characterization of the samples which was very important for the correct interpretation of the results of the mechanical tests. Down to a film thickness of about 100 to 200 nm the yield strength increased for all film systems (passivated and unpassivated) and microstructures (polycrystalline and singlecrystalline). The influence of different interfaces was smaller than expected. This could be explained by a dislocation source model based on the nucleation of perfect dislocations. For polycrystalline films the film thickness as well as the grain size distribution had to be considered. For smaller film thicknesses the increase in flow stress was weaker and the deformation behavior changed because the nucleation of perfect dislocations became unfavorable. Instead, the film materials used alternative mechanisms to relieve the high stresses. For regular and homogeneous deformation the total strain was accommodated by the nucleation and motion of partial dislocations. If the deformation was localized due to initial cracks in a brittle interlayer or local delamination, dislocation plasticity was not effective enough to relieve the stress concentration and the films showed

  15. An Authentication and Key Management Mechanism for Resource Constrained Devices in IEEE 802.11-based IoT Access Networks

    Directory of Open Access Journals (Sweden)

    Ki-Wook Kim

    2017-09-01

    Full Text Available Many Internet of Things (IoT services utilize an IoT access network to connect small devices with remote servers. They can share an access network with standard communication technology, such as IEEE 802.11ah. However, an authentication and key management (AKM mechanism for resource constrained IoT devices using IEEE 802.11ah has not been proposed as yet. We therefore propose a new AKM mechanism for an IoT access network, which is based on IEEE 802.11 key management with the IEEE 802.1X authentication mechanism. The proposed AKM mechanism does not require any pre-configured security information between the access network domain and the IoT service domain. It considers the resource constraints of IoT devices, allowing IoT devices to delegate the burden of AKM processes to a powerful agent. The agent has sufficient power to support various authentication methods for the access point, and it performs cryptographic functions for the IoT devices. Performance analysis shows that the proposed mechanism greatly reduces computation costs, network costs, and memory usage of the resource-constrained IoT device as compared to the existing IEEE 802.11 Key Management with the IEEE 802.1X authentication mechanism.

  16. An Authentication and Key Management Mechanism for Resource Constrained Devices in IEEE 802.11-based IoT Access Networks.

    Science.gov (United States)

    Kim, Ki-Wook; Han, Youn-Hee; Min, Sung-Gi

    2017-09-21

    Many Internet of Things (IoT) services utilize an IoT access network to connect small devices with remote servers. They can share an access network with standard communication technology, such as IEEE 802.11ah. However, an authentication and key management (AKM) mechanism for resource constrained IoT devices using IEEE 802.11ah has not been proposed as yet. We therefore propose a new AKM mechanism for an IoT access network, which is based on IEEE 802.11 key management with the IEEE 802.1X authentication mechanism. The proposed AKM mechanism does not require any pre-configured security information between the access network domain and the IoT service domain. It considers the resource constraints of IoT devices, allowing IoT devices to delegate the burden of AKM processes to a powerful agent. The agent has sufficient power to support various authentication methods for the access point, and it performs cryptographic functions for the IoT devices. Performance analysis shows that the proposed mechanism greatly reduces computation costs, network costs, and memory usage of the resource-constrained IoT device as compared to the existing IEEE 802.11 Key Management with the IEEE 802.1X authentication mechanism.

  17. Fungicidal mechanisms of cathelicidins LL-37 and CATH-2 revealed by live-cell imaging

    NARCIS (Netherlands)

    Ordonez Alvarez, Soledad; Amarullah, Ilham H; Wubbolts, Richard W; Veldhuizen, Edwin J A; Haagsman, Henk P

    2014-01-01

    Antifungal mechanisms of action of two cathelicidins, chicken CATH-2 and human LL-37, were studied and compared with the mode of action of the salivary peptide histatin 5 (Hst5). Candida albicans was used as a model organism for fungal pathogens. Analysis by live-cell imaging showed that the peptide

  18. Operation mechanism of a molecular machine revealed using time-resolved vibrational spectroscopy

    NARCIS (Netherlands)

    M.R. Panman; P. Bodis; D.J. Shaw; B.H. Bakker; A.C. Newton; E.R. Kay; A.M. Brouwer; W.J. Buma; D.A. Leigh; S. Woutersen

    2010-01-01

    Rotaxanes comprise macrocycles that can shuttle between docking stations along an axle. We explored the nanosecond shuttling mechanism by reversing the relative binding affinities of two stations through ultraviolet-induced transient reduction. We monitored the ensuing changes in the CO-stretching b

  19. Correlating chemical sensitivity and basal gene expression reveals mechanism of action | Office of Cancer Genomics

    Science.gov (United States)

    Changes in cellular gene expression in response to small-molecule or genetic perturbations have yielded signatures that can connect unknown mechanisms of action (MoA) to ones previously established. We hypothesized that differential basal gene expression could be correlated with patterns of small-molecule sensitivity across many cell lines to illuminate the actions of compounds whose MoA are unknown.

  20. The mechanics of bacterial cluster formation on plant leaf surfaces as revealed by bioreporter technology

    NARCIS (Netherlands)

    Tecon, R.; Leveau, J.H.J.

    2012-01-01

    Bacteria that colonize the leaves of terrestrial plants often occur in clusters whose size varies from a few to thousands of cells. For the formation of such bacterial clusters, two non-mutually exclusive but very different mechanisms may be proposed: aggregation of multiple cells or clonal

  1. Mechanical Unfolding of an Autotransporter Passenger Protein Reveals the Secretion Starting Point and Processive Transport Intermediates

    NARCIS (Netherlands)

    Baclayon, Marian; van Ulsen, Peter; Mouhib, Halima; Shabestari, Maryam Hashemi; Verzijden, Timo; Abeln, Sanne; Roos, Wouter H; Wuite, Gijs J L

    2016-01-01

    The backbone of secreted autotransporter passenger proteins generally attains a stable β-helical structure. The secretion of passengers across the outer membrane was proposed to be driven by sequential folding of this structure at the cell surface. This mechanism would require a relatively-stable in

  2. Ex vivo stretch reveals altered mechanical properties of isolated dystrophin-deficient hearts.

    Science.gov (United States)

    Barnabei, Matthew S; Metzger, Joseph M

    2012-01-01

    Duchenne muscular dystrophy (DMD) is a progressive and fatal disease of muscle wasting caused by loss of the cytoskeletal protein dystrophin. In the heart, DMD results in progressive cardiomyopathy and dilation of the left ventricle through mechanisms that are not fully understood. Previous reports have shown that loss of dystrophin causes sarcolemmal instability and reduced mechanical compliance of isolated cardiac myocytes. To expand upon these findings, here we have subjected the left ventricles of dystrophin-deficient mdx hearts to mechanical stretch. Unexpectedly, isolated mdx hearts showed increased left ventricular (LV) compliance compared to controls during stretch as LV volume was increased above normal end diastolic volume. During LV chamber distention, sarcomere lengths increased similarly in mdx and WT hearts despite greater excursions in volume of mdx hearts. This suggests that the mechanical properties of the intact heart cannot be modeled as a simple extrapolation of findings in single cardiac myocytes. To explain these findings, a model is proposed in which disruption of the dystrophin-glycoprotein complex perturbs cell-extracellular matrix contacts and promotes the apparent slippage of myocytes past each other during LV distension. In comparison, similar increases in LV compliance were obtained in isolated hearts from β-sarcoglycan-null and laminin-α(2) mutant mice, but not in dysferlin-null mice, suggesting that increased whole-organ compliance in mdx mice is a specific effect of disrupted cell-extracellular matrix contacts and not a general consequence of cardiomyopathy via membrane defect processes. Collectively, these findings suggest a novel and cell-death independent mechanism for the progressive pathological LV dilation that occurs in DMD.

  3. Ex vivo stretch reveals altered mechanical properties of isolated dystrophin-deficient hearts.

    Directory of Open Access Journals (Sweden)

    Matthew S Barnabei

    Full Text Available Duchenne muscular dystrophy (DMD is a progressive and fatal disease of muscle wasting caused by loss of the cytoskeletal protein dystrophin. In the heart, DMD results in progressive cardiomyopathy and dilation of the left ventricle through mechanisms that are not fully understood. Previous reports have shown that loss of dystrophin causes sarcolemmal instability and reduced mechanical compliance of isolated cardiac myocytes. To expand upon these findings, here we have subjected the left ventricles of dystrophin-deficient mdx hearts to mechanical stretch. Unexpectedly, isolated mdx hearts showed increased left ventricular (LV compliance compared to controls during stretch as LV volume was increased above normal end diastolic volume. During LV chamber distention, sarcomere lengths increased similarly in mdx and WT hearts despite greater excursions in volume of mdx hearts. This suggests that the mechanical properties of the intact heart cannot be modeled as a simple extrapolation of findings in single cardiac myocytes. To explain these findings, a model is proposed in which disruption of the dystrophin-glycoprotein complex perturbs cell-extracellular matrix contacts and promotes the apparent slippage of myocytes past each other during LV distension. In comparison, similar increases in LV compliance were obtained in isolated hearts from β-sarcoglycan-null and laminin-α(2 mutant mice, but not in dysferlin-null mice, suggesting that increased whole-organ compliance in mdx mice is a specific effect of disrupted cell-extracellular matrix contacts and not a general consequence of cardiomyopathy via membrane defect processes. Collectively, these findings suggest a novel and cell-death independent mechanism for the progressive pathological LV dilation that occurs in DMD.

  4. The origins of enhanced activity in factor VIIa analogs and the interplay between key allosteric sites revealed by hydrogen exchange mass spectrometry

    DEFF Research Database (Denmark)

    Rand, Kasper D; Andersen, Mette D; Olsen, Ole H;

    2008-01-01

    to investigate the conformational effects of site-directed mutagenesis at key positions in FVIIa and the origins of enhanced intrinsic activity of FVIIa analogs. The differences in hydrogen exchange of two highly active variants, FVIIa(DVQ) and FVIIa(VEAY), imply that enhanced catalytic efficiency was attained...

  5. A stepwise retro-imino-ene as a key step in the mechanism of allene formation via the Crabbé acetylene homologation.

    Science.gov (United States)

    González, Marta; Rodríguez, Roi Álvarez; Cid, Maria Magdalena; López, Carlos Silva

    2012-05-15

    The mechanism of the acetylene homologation procedure accidentally discovered and further developed by Crabbé and coworkers is unknown. Kinetic isotope effect (KIE) experiments, however, suggest that an intramolecular hydrogen shift is the key step of the transformation. In this work, we present a computational study of this mechanism. We found that the reaction proceeds via an unexpected stepwise retro-imino-ene rearrangement. This mechanism justifies the role of Cu(I) as a reaction catalyst and is also compatible with the KIE experiments reported.

  6. Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium

    Energy Technology Data Exchange (ETDEWEB)

    Wernimont, Amy K; Artz, Jennifer D.; Jr, Patrick Finerty; Lin, Yu-Hui; Amani, Mehrnaz; Allali-Hassani, Abdellah; Senisterra, Guillermo; Vedadi, Masoud; Tempel, Wolfram; Mackenzie, Farrell; Chau, Irene; Lourido, Sebastian; Sibley, L. David; Hui, Raymond (Toronto); (WU-MED)

    2010-09-21

    Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.

  7. A comparative mechanical analysis of plant and animal cells reveals convergence across kingdoms.

    Science.gov (United States)

    Durand-Smet, Pauline; Chastrette, Nicolas; Guiroy, Axel; Richert, Alain; Berne-Dedieu, Annick; Szecsi, Judit; Boudaoud, Arezki; Frachisse, Jean-Marie; Bendahmane, Mohammed; Bendhamane, Mohammed; Hamant, Oliver; Asnacios, Atef

    2014-11-18

    Plant and animals have evolved different strategies for their development. Whether this is linked to major differences in their cell mechanics remains unclear, mainly because measurements on plant and animal cells relied on independent experiments and setups, thus hindering any direct comparison. In this study we used the same micro-rheometer to compare animal and plant single cell rheology. We found that wall-less plant cells exhibit the same weak power law rheology as animal cells, with comparable values of elastic and loss moduli. Remarkably, microtubules primarily contributed to the rheological behavior of wall-less plant cells whereas rheology of animal cells was mainly dependent on the actin network. Thus, plant and animal cells evolved different molecular strategies to reach a comparable cytoplasmic mechanical core, suggesting that evolutionary convergence could include the internal biophysical properties of cells.

  8. Biomembrane interactions reveal the mechanism of action of surface-immobilized host defense IDR-1010 peptide.

    Science.gov (United States)

    Gao, Guangzheng; Cheng, John T J; Kindrachuk, Jason; Hancock, Robert E W; Straus, Suzana K; Kizhakkedathu, Jayachandran N

    2012-02-24

    Dissecting the mechanism of action of surface-tethered antimicrobial and immunomodulatory peptides is critical to the design of optimized anti-infection coatings on biomedical devices. To address this, we compared the biomembrane interactions of host defense peptide IDR-1010cys (1) in free form, (2) as a soluble polymer conjugate, and (3) with one end tethered to a solid support with model bacterial and mammalian lipid membranes. Our results show that IDR-1010cys in all three distinct forms interacted with bacterial and mammalian lipid vesicles, but the extent of the interactions as monitored by the induction of secondary structure varied. The enhanced interaction of surface-tethered peptides is well correlated with their very good antimicrobial activities. Our results demonstrate that there may be a difference in the mechanism of action of surface-tethered versus free IDR-1010cys.

  9. fMRI Study Revealing Neural Mechanisms of the Functions of SOA in Spatial Orienting

    Institute of Scientific and Technical Information of China (English)

    Yin Tian; Qian Zhang; De-Zhong Yao

    2009-01-01

    It is well documented that orienting attention plays an important role in visual search. However, it remains unclear how the executive brain regions will act when two different stimulus onset asynchrony (SOA) are used in visual search. In this work, event-related fMRI was used to investigate neural mechanisms on the functions of SOA in endogenous and exogenous orienting. The results showed that in the endogenous orienting, long SOA versus short SOA resulted in widespread cortical activation mainly including right medial frontal gyrus and bilateral middle frontal gyri. Conversely, in exogenous orienting, long SOA compared to short SOA resulted in only activations in bilateral middle frontal gyri. These findings indicated that these two spatial orienting involved different brain areas and neural mechanisms.

  10. Crystal structure of an invertebrate cytolysin pore reveals unique properties and mechanism of assembly

    Science.gov (United States)

    Podobnik, Marjetka; Savory, Peter; Rojko, Nejc; Kisovec, Matic; Wood, Neil; Hambley, Richard; Pugh, Jonathan; Wallace, E. Jayne; McNeill, Luke; Bruce, Mark; Liko, Idlir; Allison, Timothy M.; Mehmood, Shahid; Yilmaz, Neval; Kobayashi, Toshihide; Gilbert, Robert J. C.; Robinson, Carol V.; Jayasinghe, Lakmal; Anderluh, Gregor

    2016-05-01

    The invertebrate cytolysin lysenin is a member of the aerolysin family of pore-forming toxins that includes many representatives from pathogenic bacteria. Here we report the crystal structure of the lysenin pore and provide insights into its assembly mechanism. The lysenin pore is assembled from nine monomers via dramatic reorganization of almost half of the monomeric subunit structure leading to a β-barrel pore ~10 nm long and 1.6-2.5 nm wide. The lysenin pore is devoid of additional luminal compartments as commonly found in other toxin pores. Mutagenic analysis and atomic force microscopy imaging, together with these structural insights, suggest a mechanism for pore assembly for lysenin. These insights are relevant to the understanding of pore formation by other aerolysin-like pore-forming toxins, which often represent crucial virulence factors in bacteria.

  11. Diversity of Chemical Mechanisms in Thioredoxin Catalysis Revealed by Single-Molecule Force Spectroscopy

    Science.gov (United States)

    Perez-Jimenez, Raul; Li, Jingyuan; Kosuri, Pallav; Sanchez-Romero, Inmaculada; Wiita, Arun P.; Rodriguez-Larrea, David; Chueca, Ana; Holmgren, Arne; Miranda-Vizuete, Antonio; Becker, Katja; Cho, Seung-Hyun; Beckwith, Jon; Gelhaye, Eric; Jacquot, Jean P.; Gaucher, Eric; Sanchez-Ruiz, Jose M.; Berne, Bruce J.; Fernandez, Julio M.

    2009-01-01

    Thioredoxins are oxido-reductase enzymes present in all organisms, catalyzing the reduction of disulfide bonds in proteins. By applying a calibrated force to a substrate disulfide, the chemical mechanisms of Trx catalysis can be examined in detail at the single molecule level. Here we use single molecule force-clamp spectroscopy to explore the chemical evolution of Trx catalysis by probing the chemistry of eight different thioredoxin enzymes. While all Trxs show a characteristic Michaelis-Menten mechanism detected when the disulfide bond is stretched at low forces, two different chemical behaviors distinguish bacterial from eukaryotic-origin Trxs at high forces. Eukaryotic-origin Trxs reduce disulfide bonds through a single-electron transfer reaction (SET) whereas bacterial-origin Trxs exhibit both nucleophilic substitution (SN2) and SET reactions. A computational analysis of Trx structures identifies the evolution of the binding groove as an important factor controlling the chemistry of Trx catalysis. PMID:19597482

  12. The cellular and molecular mechanisms of tissue repair and regeneration as revealed by studies in Xenopus

    Science.gov (United States)

    Li, Jingjing; Zhang, Siwei

    2016-01-01

    Abstract Survival of any living organism critically depends on its ability to repair and regenerate damaged tissues and/or organs during its lifetime following injury, disease, or aging. Various animal models from invertebrates to vertebrates have been used to investigate the molecular and cellular mechanisms of wound healing and tissue regeneration. It is hoped that such studies will form the framework for identifying novel clinical treatments that will improve the healing and regenerative capacity of humans. Amongst these models, Xenopus stands out as a particularly versatile and powerful system. This review summarizes recent findings using this model, which have provided fundamental knowledge of the mechanisms responsible for efficient and perfect tissue repair and regeneration.

  13. Diversity of Chemical Mechanisms in Thioredoxin Catalysis Revealed by Single-Molecule Force Spectroscopy

    OpenAIRE

    2009-01-01

    Thioredoxins (Trxs) are oxidoreductase enzymes, present in all organisms, that catalyze the reduction of disulfide bonds in proteins. By applying a calibrated force to a substrate disulfide, the chemical mechanisms of Trx catalysis can be examined in detail at the single-molecule level. Here we use single-molecule force-clamp spectroscopy to explore the chemical evolution of Trx catalysis by probing the chemistry of eight different Trx enzymes. All Trxs show a characteristic Michaelis-Menten ...

  14. Single-molecule imaging reveals a common mechanism shared by G-quadruplex–resolving helicases

    Science.gov (United States)

    Tippana, Ramreddy; Hwang, Helen; Opresko, Patricia L.; Bohr, Vilhelm A.; Myong, Sua

    2016-01-01

    G-quadruplex (GQ) is a four stranded DNA secondary structure that arises from a guanine rich sequence. Stable formation of GQ in genomic DNA can be counteracted by the resolving activity of specialized helicases including RNA helicase AU (associated with AU rich elements) (RHAU) (G4 resolvase 1), Bloom helicase (BLM), and Werner helicase (WRN). However, their substrate specificity and the mechanism involved in GQ unfolding remain uncertain. Here, we report that RHAU, BLM, and WRN exhibit distinct GQ conformation specificity, but use a common mechanism of repetitive unfolding that leads to disrupting GQ structure multiple times in succession. Such unfolding activity of RHAU leads to efficient annealing exclusively within the same DNA molecule. The same resolving activity is sufficient to dislodge a stably bound GQ ligand, including BRACO-19, NMM, and Phen-DC3. Our study demonstrates a plausible biological scheme where different helicases are delegated to resolve specific GQ structures by using a common repetitive unfolding mechanism that provides a robust resolving power. PMID:27407146

  15. Revealing the physicochemical mechanism for ultrasonic separation of alcohol-water mixtures

    Science.gov (United States)

    Kirpalani, D. M.; Toll, F.

    2002-08-01

    The selective separation of ethanol from ethanol-water mixtures by ultrasonic atomization has been reported recently by Sato, Matsuura, and Fujii [J. Chem. Phys. 114, 2382 (2001)]. In that work, experimental data were reported that confirmed the generation of an ethanol-rich droplet mist and attempted to explain the selective separation in terms of parametric decay instability of the capillary wave formed during sonication. In the present work, an alternate mechanism based on the conjunction theory has been postulated for the process of ultrasonic atomization. This mechanism involves the formation of cavitating bubbles in the liquid during sonication and their eventual collapse at the liquid surface into a cloud of microbubbles that moves upwards in a capillary fountain jet. The selective separation of alcohols has been explained as a corollary effect of the physical mechanism resulting in a surface excess of alcohol molecules formed at the surface of the microbubbles. The alcohol molecules vaporize into the microbubbles and release an alcohol-rich mist on their collapse in regions of high accumulation of acoustic energy.

  16. Mechanisms of cell cycle control revealed by a systematic and quantitative overexpression screen in S. cerevisiae.

    Directory of Open Access Journals (Sweden)

    Wei Niu

    2008-07-01

    Full Text Available Regulation of cell cycle progression is fundamental to cell health and reproduction, and failures in this process are associated with many human diseases. Much of our knowledge of cell cycle regulators derives from loss-of-function studies. To reveal new cell cycle regulatory genes that are difficult to identify in loss-of-function studies, we performed a near-genome-wide flow cytometry assay of yeast gene overexpression-induced cell cycle delay phenotypes. We identified 108 genes whose overexpression significantly delayed the progression of the yeast cell cycle at a specific stage. Many of the genes are newly implicated in cell cycle progression, for example SKO1, RFA1, and YPR015C. The overexpression of RFA1 or YPR015C delayed the cell cycle at G2/M phases by disrupting spindle attachment to chromosomes and activating the DNA damage checkpoint, respectively. In contrast, overexpression of the transcription factor SKO1 arrests cells at G1 phase by activating the pheromone response pathway, revealing new cross-talk between osmotic sensing and mating. More generally, 92%-94% of the genes exhibit distinct phenotypes when overexpressed as compared to their corresponding deletion mutants, supporting the notion that many genes may gain functions upon overexpression. This work thus implicates new genes in cell cycle progression, complements previous screens, and lays the foundation for future experiments to define more precisely roles for these genes in cell cycle progression.

  17. From Blame to Punishment: Disrupting Prefrontal Cortex Activity Reveals Norm Enforcement Mechanisms.

    Science.gov (United States)

    Buckholtz, Joshua W; Martin, Justin W; Treadway, Michael T; Jan, Katherine; Zald, David H; Jones, Owen; Marois, René

    2015-09-23

    The social welfare provided by cooperation depends on the enforcement of social norms. Determining blameworthiness and assigning a deserved punishment are two cognitive cornerstones of norm enforcement. Although prior work has implicated the dorsolateral prefrontal cortex (DLPFC) in norm-based judgments, the relative contribution of this region to blameworthiness and punishment decisions remains poorly understood. Here, we used repetitive transcranial magnetic stimulation (rTMS) and fMRI to determine the specific role of DLPFC function in norm-enforcement behavior. DLPFC rTMS reduced punishment for wrongful acts without affecting blameworthiness ratings, and fMRI revealed punishment-selective DLPFC recruitment, suggesting that these two facets of norm-based decision making are neurobiologically dissociable. Finally, we show that DLPFC rTMS affects punishment decision making by altering the integration of information about culpability and harm. Together, these findings reveal a selective, causal role for DLPFC in norm enforcement: representational integration of the distinct information streams used to make punishment decisions.

  18. Revealing Nanoscale Passivation and Corrosion Mechanisms of Reactive Battery Materials in Gas Environments.

    Science.gov (United States)

    Li, Yuzhang; Li, Yanbin; Sun, Yongming; Butz, Benjamin; Yan, Kai; Koh, Ai Leen; Zhao, Jie; Pei, Allen; Cui, Yi

    2017-08-09

    Lithium (Li) metal is a high-capacity anode material (3860 mAh g(-1)) that can enable high-energy batteries for electric vehicles and grid-storage applications. However, Li metal is highly reactive and repeatedly consumed when exposed to liquid electrolyte (during battery operation) or the ambient environment (throughout battery manufacturing). Studying these corrosion reactions on the nanoscale is especially difficult due to the high chemical reactivity of both Li metal and its surface corrosion films. Here, we directly generate pure Li metal inside an environmental transmission electron microscope (TEM), revealing the nanoscale passivation and corrosion process of Li metal in oxygen (O2), nitrogen (N2), and water vapor (H2O). We find that while dry O2 and N2 (99.9999 vol %) form uniform passivation layers on Li, trace water vapor (∼1 mol %) disrupts this passivation and forms a porous film on Li metal that allows gas to penetrate and continuously react with Li. To exploit the self-passivating behavior of Li in dry conditions, we introduce a simple dry-N2 pretreatment of Li metal to form a protective layer of Li nitride prior to battery assembly. The fast ionic conductivity and stable interface of Li nitride results in improved battery performance with dendrite-free cycling and low voltage hysteresis. Our work reveals the detailed process of Li metal passivation/corrosion and demonstrates how this mechanistic insight can guide engineering solutions for Li metal batteries.

  19. Quantum Key Distribution

    Science.gov (United States)

    Seshu, Ch.

    Quantum Key Distribution (QKD) uses Quantum Mechanics to guarantee secure communication. It enables two parties to produce a shared random bit string known only to them, which can be used as a key to encrypt and decrypt messages.

  20. Differential co-expression and regulation analyses reveal different mechanisms underlying major depressive disorder and subsyndromal symptomatic depression.

    Science.gov (United States)

    Xu, Fan; Yang, Jing; Chen, Jin; Wu, Qingyuan; Gong, Wei; Zhang, Jianguo; Shao, Weihua; Mu, Jun; Yang, Deyu; Yang, Yongtao; Li, Zhiwei; Xie, Peng

    2015-04-03

    Recent depression research has revealed a growing awareness of how to best classify depression into depressive subtypes. Appropriately subtyping depression can lead to identification of subtypes that are more responsive to current pharmacological treatment and aid in separating out depressed patients in which current antidepressants are not particularly effective. Differential co-expression analysis (DCEA) and differential regulation analysis (DRA) were applied to compare the transcriptomic profiles of peripheral blood lymphocytes from patients with two depressive subtypes: major depressive disorder (MDD) and subsyndromal symptomatic depression (SSD). Six differentially regulated genes (DRGs) (FOSL1, SRF, JUN, TFAP4, SOX9, and HLF) and 16 transcription factor-to-target differentially co-expressed gene links or pairs (TF2target DCLs) appear to be the key differential factors in MDD; in contrast, one DRG (PATZ1) and eight TF2target DCLs appear to be the key differential factors in SSD. There was no overlap between the MDD target genes and SSD target genes. Venlafaxine (Efexor™, Effexor™) appears to have a significant effect on the gene expression profile of MDD patients but no significant effect on the gene expression profile of SSD patients. DCEA and DRA revealed no apparent similarities between the differential regulatory processes underlying MDD and SSD. This bioinformatic analysis may provide novel insights that can support future antidepressant R&D efforts.

  1. GC/MS-based metabolomic studies reveal key roles of glycine in regulating silk synthesis in silkworm, Bombyx mori.

    Science.gov (United States)

    Chen, Quanmei; Liu, Xinyu; Zhao, Ping; Sun, Yanhui; Zhao, Xinjie; Xiong, Ying; Xu, Guowang; Xia, Qingyou

    2015-02-01

    Metabolic profiling of silkworm, especially the factors that affect silk synthesis at the metabolic level, is little known. Herein, metabolomic method based on gas chromatography-mass spectrometry was applied to identify key metabolic changes in silk synthesis deficient silkworms. Forty-six differential metabolites were identified in Nd group with the defect of silk synthesis. Significant changes in the levels of glycine and uric acid (up-regulation), carbohydrates and free fatty acids (down-regulation) were observed. The further metabolomics of silk synthesis deficient silkworms by decreasing silk proteins synthesis using knocking out fibroin heavy chain gene or extirpating silk glands operation showed that the changes of the metabolites were almost consistent with those of the Nd group. Furthermore, the increased silk yields by supplying more glycine or its related metabolite confirmed that glycine is a key metabolite to regulate silk synthesis. These findings provide important insights into the regulation between metabolic profiling and silk synthesis.

  2. Neural mechanisms of phonemic restoration for speech comprehension revealed by magnetoencephalography.

    Science.gov (United States)

    Sunami, Kishiko; Ishii, Akira; Takano, Sakurako; Yamamoto, Hidefumi; Sakashita, Tetsushi; Tanaka, Masaaki; Watanabe, Yasuyoshi; Yamane, Hideo

    2013-11-01

    In daily communication, we can usually still hear the spoken words as if they had not been masked and can comprehend the speech when spoken words are masked by background noise. This phenomenon is known as phonemic restoration. Since little is known about the neural mechanisms underlying phonemic restoration for speech comprehension, we aimed to identify the neural mechanisms using magnetoencephalography (MEG). Twelve healthy male volunteers with normal hearing participated in the study. Participants were requested to carefully listen to and understand recorded spoken Japanese stories, which were either played forward (forward condition) or in reverse (reverse condition), with their eyes closed. Several syllables of spoken words were replaced by 300-ms white-noise stimuli with an inter-stimulus interval of 1.6-20.3s. We compared MEG responses to white-noise stimuli during the forward condition with those during the reverse condition using time-frequency analyses. Increased 3-5 Hz band power in the forward condition compared with the reverse condition was continuously observed in the left inferior frontal gyrus [Brodmann's areas (BAs) 45, 46, and 47] and decreased 18-22 Hz band powers caused by white-noise stimuli were seen in the left transverse temporal gyrus (BA 42) and superior temporal gyrus (BA 22). These results suggest that the left inferior frontal gyrus and left transverse and superior temporal gyri are involved in phonemic restoration for speech comprehension. Our findings may help clarify the neural mechanisms of phonemic restoration as well as develop innovative treatment methods for individuals suffering from impaired speech comprehension, particularly in noisy environments.

  3. Growth and nitrogen uptake characteristics reveal outbreak mechanism of the opportunistic macroalga Gracilaria tenuistipitata.

    Directory of Open Access Journals (Sweden)

    Chao Wang

    Full Text Available Macroalgae has bloomed in the brackish lake of Shenzhen Bay, China continuously from 2010 to 2014. Gracilaria tenuistipitata was identified as the causative macroalgal species. The aim of this study was to explore the outbreak mechanism of G. tenuistipitata, by studying the effects of salinity and nitrogen sources on growth, and the different nitrogen sources uptake characteristic. Our experimental design was based on environmental conditions observed in the bloom areas, and these main factors were simulated in the laboratory. Results showed that salinity 12 to 20 ‰ was suitable for G. tenuistipitata growth. When the nitrogen sources' (NH4+, NO3- concentrations reached 40 µM or above, the growth rate of G. tenuistipitata was significantly higher. Algal biomass was higher (approximately 1.4 times when cultured with NH4+ than that with NO3- addition. Coincidentally, macroalgal bloom formed during times of moderate salinity (∼12 ‰ and high nitrogen conditions. The NH4+ and NO3- uptake characteristic was studied to understand the potential mechanism of G. tenuistipitata bloom. NH4+ uptake was best described by a linear, rate-unsaturated response, with the slope decreasing with time intervals. In contrast, NO3- uptake followed a rate-saturating mechanism best described by the Michaelis-Menten model, with kinetic parameters Vmax = 37.2 µM g-1 DM h-1 and Ks = 61.5 µM. Further, based on the isotope 15N tracer method, we found that 15N from NH4+ accumulated faster and reached an atom% twice than that of 15N from NO3-, suggesting when both NH4+ and NO3- were available, NH4+ was assimilated more rapidly. The results of the present study indicate that in the estuarine environment, the combination of moderate salinity with high ammonium may stimulate bloom formation.

  4. Growth and nitrogen uptake characteristics reveal outbreak mechanism of the opportunistic macroalga Gracilaria tenuistipitata.

    Science.gov (United States)

    Wang, Chao; Lei, Anping; Zhou, Kai; Hu, Zhengyu; Hao, Wenlong; Yang, Junda

    2014-01-01

    Macroalgae has bloomed in the brackish lake of Shenzhen Bay, China continuously from 2010 to 2014. Gracilaria tenuistipitata was identified as the causative macroalgal species. The aim of this study was to explore the outbreak mechanism of G. tenuistipitata, by studying the effects of salinity and nitrogen sources on growth, and the different nitrogen sources uptake characteristic. Our experimental design was based on environmental conditions observed in the bloom areas, and these main factors were simulated in the laboratory. Results showed that salinity 12 to 20 ‰ was suitable for G. tenuistipitata growth. When the nitrogen sources' (NH4+, NO3-) concentrations reached 40 µM or above, the growth rate of G. tenuistipitata was significantly higher. Algal biomass was higher (approximately 1.4 times) when cultured with NH4+ than that with NO3- addition. Coincidentally, macroalgal bloom formed during times of moderate salinity (∼12 ‰) and high nitrogen conditions. The NH4+ and NO3- uptake characteristic was studied to understand the potential mechanism of G. tenuistipitata bloom. NH4+ uptake was best described by a linear, rate-unsaturated response, with the slope decreasing with time intervals. In contrast, NO3- uptake followed a rate-saturating mechanism best described by the Michaelis-Menten model, with kinetic parameters Vmax = 37.2 µM g-1 DM h-1 and Ks = 61.5 µM. Further, based on the isotope 15N tracer method, we found that 15N from NH4+ accumulated faster and reached an atom% twice than that of 15N from NO3-, suggesting when both NH4+ and NO3- were available, NH4+ was assimilated more rapidly. The results of the present study indicate that in the estuarine environment, the combination of moderate salinity with high ammonium may stimulate bloom formation.

  5. Localizing gene regulation reveals a staggered wood decay mechanism for the brown rot fungus Postia placenta.

    Science.gov (United States)

    Zhang, Jiwei; Presley, Gerald N; Hammel, Kenneth E; Ryu, Jae-San; Menke, Jon R; Figueroa, Melania; Hu, Dehong; Orr, Galya; Schilling, Jonathan S

    2016-09-27

    Wood-degrading brown rot fungi are essential recyclers of plant biomass in forest ecosystems. Their efficient cellulolytic systems, which have potential biotechnological applications, apparently depend on a combination of two mechanisms: lignocellulose oxidation (LOX) by reactive oxygen species (ROS) and polysaccharide hydrolysis by a limited set of glycoside hydrolases (GHs). Given that ROS are strongly oxidizing and nonselective, these two steps are likely segregated. A common hypothesis has been that brown rot fungi use a concentration gradient of chelated metal ions to confine ROS generation inside wood cell walls before enzymes can infiltrate. We examined an alternative: that LOX components involved in ROS production are differentially expressed by brown rot fungi ahead of GH components. We used spatial mapping to resolve a temporal sequence in Postia placenta, sectioning thin wood wafers colonized directionally. Among sections, we measured gene expression by whole-transcriptome shotgun sequencing (RNA-seq) and assayed relevant enzyme activities. We found a marked pattern of LOX up-regulation in a narrow (5-mm, 48-h) zone at the hyphal front, which included many genes likely involved in ROS generation. Up-regulation of GH5 endoglucanases and many other GHs clearly occurred later, behind the hyphal front, with the notable exceptions of two likely expansins and a GH28 pectinase. Our results support a staggered mechanism for brown rot that is controlled by differential expression rather than microenvironmental gradients. This mechanism likely results in an oxidative pretreatment of lignocellulose, possibly facilitated by expansin- and pectinase-assisted cell wall swelling, before cellulases and hemicellulases are deployed for polysaccharide depolymerization.

  6. Network Analysis Reveals the Recognition Mechanism for Mannose-binding Lectins

    Science.gov (United States)

    Zhao, Yunjie; Jian, Yiren; Zeng, Chen; Computational Biophysics Lab Team

    The specific carbohydrate binding of mannose-binding lectin (MBL) protein in plants makes it a very useful molecular tool for cancer cell detection and other applications. The biological states of most MBL proteins are dimeric. Using dynamics network analysis on molecular dynamics (MD) simulations on the model protein of MBL, we elucidate the short- and long-range driving forces behind the dimer formation. The results are further supported by sequence coevolution analysis. We propose a general framework for deciphering the recognition mechanism underlying protein-protein interactions that may have potential applications in signaling pathways.

  7. State-dependent mechanisms of LTP expression revealed by optical quantal analysis.

    Science.gov (United States)

    Ward, Bonnie; McGuinness, Lindsay; Akerman, Colin J; Fine, Alan; Bliss, Tim V P; Emptage, Nigel J

    2006-11-22

    The expression mechanism of long-term potentiation (LTP) remains controversial. Here we combine electrophysiology and Ca(2+) imaging to examine the role of silent synapses in LTP expression. Induction of LTP fails to change p(r) at these synapses but instead mediates an unmasking process that is sensitive to the inhibition of postsynaptic membrane fusion. Once unmasked, however, further potentiation of formerly silent synapses leads to an increase in p(r). The state of the synapse thus determines how LTP is expressed.

  8. Structures of pattern recognition receptors reveal molecular mechanisms of autoinhibition, ligand recognition and oligomerization.

    Science.gov (United States)

    Chuenchor, Watchalee; Jin, Tengchuan; Ravilious, Geoffrey; Xiao, T Sam

    2014-02-01

    Pattern recognition receptors (PRRs) are essential sentinels for pathogens or tissue damage and integral components of the innate immune system. Recent structural studies have provided unprecedented insights into the molecular mechanisms of ligand recognition and signal transduction by several PRR families at distinct subcellular compartments. Here we highlight some of the recent discoveries and summarize the common themes that are emerging from these exciting studies. Better mechanistic understanding of the structure and function of the PRRs will improve future prospects of therapeutic targeting of these important innate immune receptors.

  9. Global Transcriptomic Analysis Reveals the Mechanism of Phelipanche aegyptiaca Seed Germination

    Directory of Open Access Journals (Sweden)

    Zhaoqun Yao

    2016-07-01

    Full Text Available Phelipanche aegyptiaca is one of the most destructive root parasitic plants of Orobanchaceae. This plant has significant impacts on crop yields worldwide. Conditioned and host root stimulants, in particular, strigolactones, are needed for unique seed germination. However, no extensive study on this phenomenon has been conducted because of insufficient genomic information. Deep RNA sequencing, including de novo assembly and functional annotation was performed on P. aegyptiaca germinating seeds. The assembled transcriptome was used to analyze transcriptional dynamics during seed germination. Key gene categories involved were identified. A total of 274,964 transcripts were determined, and 53,921 unigenes were annotated according to the NR, GO, COG, KOG, and KEGG databases. Overall, 5324 differentially expressed genes among dormant, conditioned, and GR24-treated seeds were identified. GO and KEGG enrichment analyses demonstrated numerous DEGs related to DNA, RNA, and protein repair and biosynthesis, as well as carbohydrate and energy metabolism. Moreover, ABA and ethylene were found to play important roles in this process. GR24 application resulted in dramatic changes in ABA and ethylene-associated genes. Fluridone, a carotenoid biosynthesis inhibitor, alone could induce P. aegyptiaca seed germination. In addition, conditioning was probably not the indispensable stage for P. aegyptiaca, because the transcript level variation of MAX2 and KAI2 genes (relate to strigolactone signaling was not up-regulated by conditioning treatment.

  10. Analysis of Hsp90 cochaperone interactions reveals a novel mechanism for TPR protein recognition.

    Science.gov (United States)

    Chadli, Ahmed; Bruinsma, Elizabeth S; Stensgard, Bridget; Toft, David

    2008-03-01

    The chaperone Hsp90 is required for the appropriate regulation of numerous key signaling molecules, including the progesterone receptor (PR). Many important cochaperones bind Hsp90 through their tetratricopeptide repeat (TPR) domains. Two such proteins, GCUNC45 and FKBP52, assist PR chaperoning and are thought to interact sequentially with PR-Hsp90 complexes. TPR proteins bind to the C-terminal MEEVD sequence of Hsp90, but GCUNC45 has been shown also to bind to a novel site near the N-terminus. We now show that FKBP52 is also able to bind to this site, and that these two cochaperones act competitively, through Hsp90, to modulate PR activity. The N-terminal site involves noncontiguous amino acids within or near the ATP binding pocket of Hsp90. TPR interactions at this site are thus strongly regulated by nucleotide binding and Hsp90 conformation. We propose an expanded model for client chaperoning in which the coordinated use of TPR recognition sites at both N- and C-terminal ends of Hsp90 enhances its ability to coordinate interactions with multiple TPR partners.

  11. Molecular architecture of synaptic actin cytoskeleton in hippocampal neurons reveals a mechanism of dendritic spine morphogenesis.

    Science.gov (United States)

    Korobova, Farida; Svitkina, Tatyana

    2010-01-01

    Excitatory synapses in the brain play key roles in learning and memory. The formation and functions of postsynaptic mushroom-shaped structures, dendritic spines, and possibly of presynaptic terminals, rely on actin cytoskeleton remodeling. However, the cytoskeletal architecture of synapses remains unknown hindering the understanding of synapse morphogenesis. Using platinum replica electron microscopy, we characterized the cytoskeletal organization and molecular composition of dendritic spines, their precursors, dendritic filopodia, and presynaptic boutons. A branched actin filament network containing Arp2/3 complex and capping protein was a dominant feature of spine heads and presynaptic boutons. Surprisingly, the spine necks and bases, as well as dendritic filopodia, also contained a network, rather than a bundle, of branched and linear actin filaments that was immunopositive for Arp2/3 complex, capping protein, and myosin II, but not fascin. Thus, a tight actin filament bundle is not necessary for structural support of elongated filopodia-like protrusions. Dynamically, dendritic filopodia emerged from densities in the dendritic shaft, which by electron microscopy contained branched actin network associated with dendritic microtubules. We propose that dendritic spine morphogenesis begins from an actin patch elongating into a dendritic filopodium, which tip subsequently expands via Arp2/3 complex-dependent nucleation and which length is modulated by myosin II-dependent contractility.

  12. Transcriptomic analysis reveals the metabolic mechanism of L-ascorbic acid in Ziziphus jujuba Mill.

    Directory of Open Access Journals (Sweden)

    Chunmei eZhang

    2016-02-01

    Full Text Available Chinese jujube (Ziziphus jujuba Mill. is the most economically important member of the Rhamnaceae family and contains a high concentration of ascorbic acid (AsA. To explore the metabolic mechanism of AsA accumulation, we investigated the abundance of AsA in the fruit development stages, the leaf and flower of Z. jujuba cv Junzao, and the mature fruit of one type of wild jujube (Z. jujuba var. spinosa Hu, Yanchuan sour jujube. And the expression patterns of genes involved in AsA biosynthesis, degradation and recycling were analyzed. The result showed that AsA biosynthesis during early fruit development (the enlargement stage is the main reason for jujube high accumulation. The L-galactose pathway plays a predominant role in the biosynthesis of AsA during jujube fruit development, and the genes GMP1, GME1, GGP, and GaLDH involved in the determination of AsA concentration during fruit development and in different genotypes; the myo-inositol pathway along with the genes GME2 and GMP2 in the L-galactose pathway play a compensatory role in maintaining AsA accumulation during the ripening stage. These findings enhance our understanding of the molecular mechanism in regulating AsA accumulation for jujube.

  13. Cell metabolomics reveals the neurotoxicity mechanism of cadmium in PC12 cells.

    Science.gov (United States)

    Zong, Li; Xing, Junpeng; Liu, Shu; Liu, Zhiqiang; Song, Fengrui

    2017-08-17

    The heavy metals such as cadmium (Cd) can induce neurotoxicity. Extensive studies about the effects of Cd on human health have been reported, however, a systematic investigation on the molecular mechanisms of the effects of Cd on central nervous system is still needed. In this paper, the neuronal PC-12 cells were treated with a series of concentrations of CdCl2 for 48h. Then the cytotoxicity was evaluated by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. The IC15 value (15% inhibiting concentration) was selected for further mechanism studies. After PC-12 cells incubated with CdCl2 at a dose of IC15 for 48h, the intracellular and extracellular metabolites were profiled using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS)-based cell metabolomics approach. As found, the effects of the heavy metal Cd produced on the PC-12 cell viability were dose-dependent. The metabolic changes were involved in the glycolysis and gluconeogenesis, biopterin metabolism, tryptophan metabolism, tyrosine metabolism, glycerophospholipid metabolism, and fatty acids beta-oxidation. These could cause the perturbation of cell membrane, redox balance, energy supply, cellular detoxification, further affecting the cellular proliferation and apoptosis and other cellular activities. Copyright © 2017. Published by Elsevier Inc.

  14. Substrate complexes of human dipeptidyl peptidase III reveal the mechanism of enzyme inhibition

    Science.gov (United States)

    Kumar, Prashant; Reithofer, Viktoria; Reisinger, Manuel; Wallner, Silvia; Pavkov-Keller, Tea; Macheroux, Peter; Gruber, Karl

    2016-01-01

    Human dipeptidyl-peptidase III (hDPP III) is a zinc-dependent hydrolase cleaving dipeptides off the N-termini of various bioactive peptides. Thus, the enzyme is likely involved in a number of physiological processes such as nociception and is also implicated in several forms of cancer. We present high-resolution crystal structures of hDPP III in complex with opioid peptides (Met-and Leu-enkephalin, endomorphin-2) as well as with angiotensin-II and the peptide inhibitor IVYPW. These structures confirm the previously reported large conformational change of the enzyme upon ligand binding and show that the structure of the closed conformation is independent of the nature of the bound peptide. The overall peptide-binding mode is also conserved ensuring the correct positioning of the scissile peptide bond with respect to the catalytic zinc ion. The structure of the angiotensin-II complex shows, how longer peptides are accommodated in the binding cleft of hDPP III. Differences in the binding modes allow a distinction between real substrates and inhibitory peptides or “slow” substrates. The latter displace a zinc bound water molecule necessitating the energetically much less favoured anhydride mechanism as opposed to the favoured promoted-water mechanism. The structural data also form the necessary framework for the design of specific hDPP III inhibitors. PMID:27025154

  15. Proteomic analysis reveals resistance mechanism against biofuel hexane in Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Liu, Jie; Chen, Lei; Wang, Jiangxin; Qiao, Jianjun; Zhang, Weiwen

    2012-09-07

    Recent studies have demonstrated that photosynthetic cyanobacteria could be an excellent cell factory to produce renewable biofuels and chemicals due to their capability to utilize solar energy and CO2 as the sole energy and carbon sources. Biosynthesis of carbon-neutral biofuel alkanes with good chemical and physical properties has been proposed. However, to make the process economically feasible, one major hurdle to improve the low cell tolerance to alkanes needed to be overcome. Towards the goal to develop robust and high-alkane-tolerant hosts, in this study, the responses of model cyanobacterial Synechocystis PCC 6803 to hexane, a representative of alkane, were investigated using a quantitative proteomics approach with iTRAQ - LC-MS/MS technologies. In total, 1,492 unique proteins were identified, representing about 42% of all predicted protein in the Synechocystis genome. Among all proteins identified, a total of 164 and 77 proteins were found up- and down-regulated, respectively. Functional annotation and KEGG pathway enrichment analyses showed that common stress responses were induced by hexane in Synechocystis. Notably, a large number of transporters and membrane-bound proteins, proteins against oxidative stress and proteins related to sulfur relay system and photosynthesis were induced, suggesting that they are possibly the major protection mechanisms against hexane toxicity. The study provided the first comprehensive view of the complicated molecular mechanism employed by cyanobacterial model species, Synechocystis to defend against hexane stress. The study also provided a list of potential targets to engineer Synechocystis against hexane stress.

  16. Reconstitution of a eukaryotic replisome reveals suppression mechanisms that define leading/lagging strand operation

    Science.gov (United States)

    Georgescu, Roxana E; Schauer, Grant D; Yao, Nina Y; Langston, Lance D; Yurieva, Olga; Zhang, Dan; Finkelstein, Jeff; O'Donnell, Mike E

    2015-01-01

    We have reconstituted a eukaryotic leading/lagging strand replisome comprising 31 distinct polypeptides. This study identifies a process unprecedented in bacterial replisomes. While bacteria and phage simply recruit polymerases to the fork, we find that suppression mechanisms are used to position the distinct eukaryotic polymerases on their respective strands. Hence, Pol ε is active with CMG on the leading strand, but it is unable to function on the lagging strand, even when Pol δ is not present. Conversely, Pol δ-PCNA is the only enzyme capable of extending Okazaki fragments in the presence of Pols ε and α. We have shown earlier that Pol δ-PCNA is suppressed on the leading strand with CMG (Georgescu et al., 2014). We propose that CMG, the 11-subunit helicase, is responsible for one or both of these suppression mechanisms that spatially control polymerase occupancy at the fork. DOI: http://dx.doi.org/10.7554/eLife.04988.001 PMID:25871847

  17. Transcriptional Network Analysis Reveals Drought Resistance Mechanisms of AP2/ERF Transgenic Rice

    Directory of Open Access Journals (Sweden)

    Hongryul Ahn

    2017-06-01

    Full Text Available This study was designed to investigate at the molecular level how a transgenic version of rice “Nipponbare” obtained a drought-resistant phenotype. Using multi-omics sequencing data, we compared wild-type rice (WT and a transgenic version (erf71 that had obtained a drought-resistant phenotype by overexpressing OsERF71, a member of the AP2/ERF transcription factor (TF family. A comprehensive bioinformatics analysis pipeline, including TF networks and a cascade tree, was developed for the analysis of multi-omics data. The results of the analysis showed that the presence of OsERF71 at the source of the network controlled global gene expression levels in a specific manner to make erf71 survive longer than WT. Our analysis of the time-series transcriptome data suggests that erf71 diverted more energy to survival-critical mechanisms related to translation, oxidative response, and DNA replication, while further suppressing energy-consuming mechanisms, such as photosynthesis. To support this hypothesis further, we measured the net photosynthesis level under physiological conditions, which confirmed the further suppression of photosynthesis in erf71. In summary, our work presents a comprehensive snapshot of transcriptional modification in transgenic rice and shows how this induced the plants to acquire a drought-resistant phenotype.

  18. Leaf Proteome Analysis Reveals Prospective Drought and Heat Stress Response Mechanisms in Soybean

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    Aayudh Das

    2016-01-01

    Full Text Available Drought and heat are among the major abiotic stresses that affect soybean crops worldwide. During the current investigation, the effect of drought, heat, and drought plus heat stresses was compared in the leaves of two soybean varieties, Surge and Davison, combining 2D-DIGE proteomic data with physiology and biochemical analyses. We demonstrated how 25 differentially expressed photosynthesis-related proteins affect RuBisCO regulation, electron transport, Calvin cycle, and carbon fixation during drought and heat stress. We also observed higher abundance of heat stress-induced EF-Tu protein in Surge. It is possible that EF-Tu might have activated heat tolerance mechanisms in the soybean. Higher level expressions of heat shock-related protein seem to be regulating the heat tolerance mechanisms. This study identifies the differential expression of various abiotic stress-responsive proteins that regulate various molecular processes and signaling cascades. One inevitable outcome from the biochemical and proteomics assays of this study is that increase of ROS levels during drought stress does not show significant changes at the phenotypic level in Davison and this seems to be due to a higher amount of carbonic anhydrase accumulation in the cell which aids the cell to become more resistant to cytotoxic concentrations of H2O2.

  19. [Heat-responsive mechanisms in plants revealed by proteomic analysis: A review].

    Science.gov (United States)

    Liu, Jun-ming; Zhao, Qi; Yin, Ze-peng; Xu, Chen-xi; Wang, Quan-hua; Dai, Shao-jun

    2015-08-01

    Heat stress is a major abiotic stress that limits plant growth and productivity. In recent years, proteomic investigations provide more information for understanding the sophisticated heat-responsive molecular mechanism in plants at systematic biological level. The heat-responsive proteomic patterns in several plants, i. e., model plants (Arabidopsis thaliana), staple food crops (soybean, rice and wheat), heat-tolerant plants (Agrostis stolonifera, Portulaca oleracea, and Carissa spinarum), grapevine, Populus euphratica, Medicago sativa, and Pinellia ternate, were reported. A total of 838 heat-responsive proteins have been identified in these studies. Among them, 534 proteins were induced and the expression of 304 proteins was reduced in plants under heat stress. In this paper, the diverse protein patterns in plants under various heat stress conditions (30-45 °C for 0-10 d) were analyzed integratively. This provided new evidences and clues for further interpreting the signaling and metabolic pathways, e.g., signaling, stress and defense, carbohydrate and energy metabolism, photosynthesis, transcription, protein synthesis and fate, membrane and transport, in heat-responsive networks, and laid a foundation for a holistic understanding of the molecular regulatory mechanism in plants in response to heat stress.

  20. Transcriptome Profiling Reveals the Antitumor Mechanism of Polysaccharide from Marine Algae Gracilariopsis lemaneiformis.

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    Yani Kang

    Full Text Available Seaweed is one of the important biomass producers and possesses active metabolites with potential therapeutic effects against tumors. The red alga Gracilariopsis lemaneiformis (Gp. lemaneiformis possesses antitumor activity, and the polysaccharide of Gp. lemaneiformis (PGL has been demonstrated to be an ingredient with marked anticancer activity. However, the anticancer mechanism of PGL remains to be elucidated. In this study, we analyzed the inhibitory effect of PGL on the cell growth of 3 human cancer cell lines and found that PGL inhibited cell proliferation, reduced cell viability, and altered cell morphology in a time- and concentration-dependent manner. Our transcriptome analysis indicates that PGL can regulate the expression of 758 genes, which are involved in apoptosis, the cell cycle, nuclear division, and cell death. Furthermore, we demonstrated that PGL induced apoptosis and cell cycle arrest and modulated the expression of related genes in the A549 cell line. Our work provides a framework to understand the effects of PGL on cancer cells, and can serve as a resource for delineating the antitumor mechanisms of Gp. lemaneiformis.

  1. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    Energy Technology Data Exchange (ETDEWEB)

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); Roszak, Aleksander W. [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel, E-mail: daniel.walker@glasgow.ac.uk [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom)

    2015-06-30

    The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

  2. Competition between inverse piezoelectric effect and deformation potential mechanism in undoped GaAs revealed by ultrafast acoustics

    Directory of Open Access Journals (Sweden)

    Pezeril T.

    2013-03-01

    Full Text Available By using the picosecond ultrasonics technique, piezoelectric effect in GaAs undoped sample at both faces (A[111] and B[-1-1-1] is experimentally studied. We demonstrate that piezoelectric generation of sound can dominate in GaAs material over the deformation potential mechanism even in the absence of static externally applied or built-in electric field in the semiconductor material. In that case, the Dember field, caused by the separation of photo-generated electrons and holes in the process of supersonic diffusion, is sufficient for the dominance of the piezoelectric mechanism during the optoacoustic excitation. The experimental results on the sample at both faces reveal that in one case (A face, the two mechanisms, piezoelectric effect and deformation potential, can compensate each other leading to a large decrease of the measured Brillouin oscillation magnitude.

  3. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment.

    Science.gov (United States)

    Fyfe, Cameron D; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W; Cogdell, Richard J; Wall, Daniel M; Burchmore, Richard J S; Byron, Olwyn; Walker, Daniel

    2015-07-01

    Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

  4. Deformation Mechanism of the Northern Tibetan Plateau as Revealed by Magnetotelluric Data

    Science.gov (United States)

    Zhang, Letian; Wei, Wenbo; Jin, Sheng; Ye, Gaofeng; Xie, Chengliang

    2017-04-01

    As a unique geologic unit on the northern margin of the Tibetan Plateau, the Qaidam Basin plays a significant role in constraining the vertical uplift and horizontal expansion of the northern and northeastern Tibetan Plateau. However, due to its complex evolution history and difficult logistic condition, deformation mechanism of the lithosphere beneath the Qaidam Basin is still highly debated. To better understand the lithospheric electrical structure and deformation mechanism of the Qaidam Basin, A 250 km long, NE-SW directed Magnetotelluric (MT) profile was finished in the northern portion of the Basin, which is roughly perpendicular to the thrust fault systems on the western and eastern margins of the Basin, as well as anticlinorium systems within the Basin. The profile consists of 20 broad-band MT stations and 5 long-period MT stations. Original time series data is processed with regular robust routines. Dimensionality and regional strike direction are determined for the dataset through data analysis. Based on the analysis results, 2D inversions were performed to produce a preferred model of the lithospheric electrical structure beneath the northern Qaidam Basin. Uncertainty analysis of the 2D inversion model was also conducted based on a data resampling approach. The outcome 2D electrical model was further used to estimate the distribution of temperature and melt fraction in the upper mantle based on laboratory-determined relationships between the electrical conductivity and temperature of nominally anhydrous minerals and basaltic melt by using the mixing law of Hashin-Shtrikman's bounds. All these results suggest that: (1) the crust-mantle boundary is imaged as a conductive layer beneath the western Qaidam Basin, with its temperature estimated to be 1200-1300 °C and melt fraction 5-8%, indicating decoupling deformation of the crust and upper mantle. (2) A large-scale east-dipping conductor is imaged beneath the eastern Qaidam Basin. This conductor extends

  5. Deep sequencing the transcriptome reveals seasonal adaptive mechanisms in a hibernating mammal.

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    Marshall Hampton

    Full Text Available Mammalian hibernation is a complex phenotype involving metabolic rate reduction, bradycardia, profound hypothermia, and a reliance on stored fat that allows the animal to survive for months without food in a state of suspended animation. To determine the genes responsible for this phenotype in the thirteen-lined ground squirrel (Ictidomys tridecemlineatus we used the Roche 454 platform to sequence mRNA isolated at six points throughout the year from three key tissues: heart, skeletal muscle, and white adipose tissue (WAT. Deep sequencing generated approximately 3.7 million cDNA reads from 18 samples (6 time points ×3 tissues with a mean read length of 335 bases. Of these, 3,125,337 reads were assembled into 140,703 contigs. Approximately 90% of all sequences were matched to proteins in the human UniProt database. The total number of distinct human proteins matched by ground squirrel transcripts was 13,637 for heart, 12,496 for skeletal muscle, and 14,351 for WAT. Extensive mitochondrial RNA sequences enabled a novel approach of using the transcriptome to construct the complete mitochondrial genome for I. tridecemlineatus. Seasonal and activity-specific changes in mRNA levels that met our stringent false discovery rate cutoff (1.0 × 10(-11 were used to identify patterns of gene expression involving various aspects of the hibernation phenotype. Among these patterns are differentially expressed genes encoding heart proteins AT1A1, NAC1 and RYR2 controlling ion transport required for contraction and relaxation at low body temperatures. Abundant RNAs in skeletal muscle coding ubiquitin pathway proteins ASB2, UBC and DDB1 peak in October, suggesting an increase in muscle proteolysis. Finally, genes in WAT that encode proteins involved in lipogenesis (ACOD, FABP4 are highly expressed in August, but gradually decline in expression during the seasonal transition to lipolysis.

  6. Particle size studies to reveal crystallization mechanisms of the metal organic framework HKUST-1 during sonochemical synthesis.

    Science.gov (United States)

    Armstrong, Mitchell R; Senthilnathan, Sethuraman; Balzer, Christopher J; Shan, Bohan; Chen, Liang; Mu, Bin

    2017-01-01

    Systematic studies of key operating parameters for the sonochemical synthesis of the metal organic framework (MOF) HKUST-1(also called CuBTC) were performed including reaction time, reactor volume, sonication amplitude, sonication tip size, solvent composition, and reactant concentrations analyzed through SEM particle size analysis. Trends in the particle size and size distributions show reproducible control of average particle sizes between 1 and 4μm. These results along with complementary studies in sonofragmentation and temperature control were conducted to compare these results to kinetic crystal growth models found in literature to develop a plausible hypothetical mechanism for ultrasound-assisted growth of metal-organic-frameworks composed of a competitive mechanism including constructive solid-on-solid (SOS) crystal growth and a deconstructive sonofragmentation. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Large scale integration of drug-target information reveals poly-pharmacological drug action mechanisms in tumor cell line growth inhibition assays

    Science.gov (United States)

    Knight, Richard A.; Gostev, Mikhail; Ilisavskii, Sergei; Willis, Anne E.; Melino, Gerry; Antonov, Alexey V.

    2014-01-01

    Understanding therapeutic mechanisms of drug anticancer cytotoxicity represents a key challenge in preclinical testing. Here we have performed a meta-analysis of publicly available tumor cell line growth inhibition assays (~ 70 assays from 6 independent experimental groups covering ~ 500 000 molecules) with the primary goal of understanding molecular therapeutic mechanisms of cancer cytotoxicity. To implement this we have collected currently available information on protein targets for molecules that were tested in the assays. We used a statistical methodology to identify protein targets overrepresented among molecules exhibiting cancer cytotoxicity with the particular focus of identifying overrepresented patterns consisting of several proteins (i.e. proteins “A” and “B” and “C”). Our analysis demonstrates that targeting individual proteins can result in a significant increase (up to 50-fold) of the observed odds for a molecule to be an efficient inhibitor of tumour cell line growth. However, further insight into potential molecular mechanisms reveals a multi-target mode of action: targeting a pattern of several proteins drastically increases the observed odds (up to 500-fold) for a molecule to be tumour cytotoxic. In contrast, molecules targeting only one protein but not targeting an additional set of proteins tend to be nontoxic. Our findings support a poly-pharmacology drug discovery paradigm, demonstrating that anticancer cytotoxicity is a product, in most cases, of multi-target mode of drug action PMID:24553133

  8. Functional chromatography reveals three natural products that target the same protein with distinct mechanisms of action

    Science.gov (United States)

    Kang, MinJin; Wu, Tongde; Wijeratne, E. M. Kithsiri; Lau, Eric C.; Mason, Damian J.; Mesa, Celestina; Tillotson, Joseph; Zhang, Donna D.; Gunatilaka, A. A. Leslie; La Clair, James J.

    2014-01-01

    Access to lead compounds with defined molecular targets continues to be a barrier to the translation of natural product resources. As a solution, we have developed a system that uses discreet, recombinant proteins as the vehicles for natural product isolation. Here, we describe the use of this functional chromatographic method to identify natural products that bind to the AAA+ chaperone, p97, a promising cancer target. Application of this method to a panel of fungal and plant extracts identified rheoemodin, 1-hydroxydehydroherbarin and phomapyrrolidone A as distinct p97 modulators. Excitingly, each of these molecules displayed a unique mechanism of p97 modulation. This discovery provides strong support for the application of functional chromatography to the discovery of protein modulators that would likely escape traditional high-throughput or phenotypic screening platforms. PMID:25125376

  9. Comparative transcriptomic analysis reveals phenol tolerance mechanism of evolved Chlorella strain.

    Science.gov (United States)

    Zhou, Lin; Cheng, Dujia; Wang, Liang; Gao, Juan; Zhao, Quanyu; Wei, Wei; Sun, Yuhan

    2017-03-01

    The growth of microalgae is inhibited by high concentration phenol due to reactive oxygen species. An evolved strain tolerated to 500mg/L phenol, Chlorella sp. L5, was obtained in previous study. In this study, comparative transcriptomic analysis was performed for Chlorella sp. L5 and its original strain (Chlorella sp. L3). The tolerance mechanism of Chlorella sp. L5 for high concentration phenol was explored on genome scale. It was identified that the up-regulations of the related genes according to antioxidant enzymes (SOD, APX, CAT and GR) and carotenoids (astaxanthin, lutein and lycopene) biosynthesis had critical roles to tolerate high concentration phenol. In addition, most of genes of PS I, PS II, photosynthetic electron transport chain and starch biosynthesis were also up-regulated. It was consistent to the experimental results of total carbohydrate contents of Chlorella sp. L3 and Chlorella sp. L5 under 0mg/L and 500mg/L phenol.

  10. Using Cellular Proteins to Reveal Mechanisms of HIV Infection | Center for Cancer Research

    Science.gov (United States)

    A vital step in HIV infection is the insertion of viral DNA into the genome of the host cell. In order for the insertion to occur, viral nucleic acid must be transported through the membrane that separates the main cellular compartment (the cytoplasm) from the nucleus, where the host DNA is located. Scientists are actively studying the mechanism used to transport viral DNA into the nucleus in the hopes of targeting this step with future anti-HIV treatments. Up to this point, researchers have identified some of the viral components that play a role in nuclear transport, but they have not determined how viral interactions with other molecules in the cell contribute to the process.

  11. Tribology. Mechanisms of antiwear tribofilm growth revealed in situ by single-asperity sliding contacts.

    Science.gov (United States)

    Gosvami, N N; Bares, J A; Mangolini, F; Konicek, A R; Yablon, D G; Carpick, R W

    2015-04-03

    Zinc dialkyldithiophosphates (ZDDPs) form antiwear tribofilms at sliding interfaces and are widely used as additives in automotive lubricants. The mechanisms governing the tribofilm growth are not well understood, which limits the development of replacements that offer better performance and are less likely to degrade automobile catalytic converters over time. Using atomic force microscopy in ZDDP-containing lubricant base stock at elevated temperatures, we monitored the growth and properties of the tribofilms in situ in well-defined single-asperity sliding nanocontacts. Surface-based nucleation, growth, and thickness saturation of patchy tribofilms were observed. The growth rate increased exponentially with either applied compressive stress or temperature, consistent with a thermally activated, stress-assisted reaction rate model. Although some models rely on the presence of iron to catalyze tribofilm growth, the films grew regardless of the presence of iron on either the tip or substrate, highlighting the critical role of stress and thermal activation.

  12. Evolution Reveals A Glutathione-dependent Mechanism Of 3-hydroxypropionic Acid Detoxification

    DEFF Research Database (Denmark)

    Kildegaard, Kanchana Rueksomtawin; Hallström, Björn M.; Blicher, Thomas H.;

    Biologically produced 3-hydroxypropionic acid (3HP) is a potential source for sustainable acrylates and can also find direct use as monomer in the production of biodegradable polymers. For industrial-scale production, high titer, rate and yield are essential; thus there is a need for robust cell...... factories tolerant to high concentration of 3HP, preferably at low pH. Through adaptive laboratory evolution we selected S. cerevisiae strains with improved tolerance to 3HP at pH 3.5. Genome sequencing of three independent clones identified single-nucleotide changes in the SFA1 gene encoding S...... as a glutathione-dependent route for detoxification of 3-hydroxypropionic aldehyde (reuterin). The identified molecular response to 3HP and reuterin may well be a general mechanism for handling resistance to organic acids and aldehydes by living cells...

  13. Revealing the reaction mechanisms of Li-O2 batteries using environmental transmission electron microscopy

    Science.gov (United States)

    Luo, Langli; Liu, Bin; Song, Shidong; Xu, Wu; Zhang, Ji-Guang; Wang, Chongmin

    2017-07-01

    The performances of a Li-O2 battery depend on a complex interplay between the reaction mechanism at the cathode, the chemical structure and the morphology of the reaction products, and their spatial and temporal evolution; all parameters that, in turn, are dependent on the choice of the electrolyte. In an aprotic cell, for example, the discharge product, Li2O2, forms through a combination of solution and surface chemistries that results in the formation of a baffling toroidal morphology. In a solid electrolyte, neither the reaction mechanism at the cathode nor the nature of the reaction product is known. Here we report the full-cycle reaction pathway for Li-O2 batteries and show how this correlates with the morphology of the reaction products. Using aberration-corrected environmental transmission electron microscopy (TEM) under an oxygen environment, we image the product morphology evolution on a carbon nanotube (CNT) cathode of a working solid-state Li-O2 nanobattery and correlate these features with the electrochemical reaction at the electrode. We find that the oxygen-reduction reaction (ORR) on CNTs initially produces LiO2, which subsequently disproportionates into Li2O2 and O2. The release of O2 creates a hollow nanostructure with Li2O outer-shell and Li2O2 inner-shell surfaces. Our findings show that, in general, the way the released O2 is accommodated is linked to lithium-ion diffusion and electron-transport paths across both spatial and temporal scales; in turn, this interplay governs the morphology of the discharging/charging products in Li-O2 cells.

  14. Mechanism of silver nanoparticles action on insect pigmentation reveals intervention of copper homeostasis.

    Directory of Open Access Journals (Sweden)

    Najealicka Armstrong

    Full Text Available Silver nanoparticles (AgNPs, like almost all nanoparticles, are potentially toxic beyond a certain concentration because the survival of the organism is compromised due to scores of pathophysiological abnormalities past that concentration. However, the mechanism of AgNP toxicity remains undetermined. Instead of applying a toxic dose, we attempted to monitor the effects of AgNPs at a nonlethal concentration on wild type Drosophila melanogaster by exposing them throughout their development. All adult flies raised in AgNP doped food showed that up to 50 mg/L concentration AgNP has no negative influence on median survival; however, these flies appeared uniformly lighter in body color due to the loss of melanin pigments in their cuticle. Additionally, fertility and vertical movement ability were compromised due to AgNP feeding. Determination of the amount of free ionic silver (Ag(+ led us to claim that the observed biological effects have resulted from the AgNPs and not from Ag(+. Biochemical analysis suggests that the activity of copper dependent enzymes, namely tyrosinase and Cu-Zn superoxide dismutase, are decreased significantly following the consumption of AgNPs, despite the constant level of copper present in the tissue. Consequently, we propose a mechanism whereby consumption of excess AgNPs in association with membrane bound copper transporter proteins cause sequestration of copper, thus creating a condition that resembles copper starvation. This model also explains the cuticular demelanization effect resulting from AgNP since tyrosinase activity is essential for melanin biosynthesis. Finally, we claim that Drosophila, an established genetic model system, can be well utilized for further understanding of the biological effects of nanoparticles.

  15. Mechanism of Silver Nanoparticles Action on Insect Pigmentation Reveals Intervention of Copper Homeostasis

    Science.gov (United States)

    Armstrong, Najealicka; Ramamoorthy, Malaisamy; Lyon, Delina; Jones, Kimberly; Duttaroy, Atanu

    2013-01-01

    Silver nanoparticles (AgNPs), like almost all nanoparticles, are potentially toxic beyond a certain concentration because the survival of the organism is compromised due to scores of pathophysiological abnormalities past that concentration. However, the mechanism of AgNP toxicity remains undetermined. Instead of applying a toxic dose, we attempted to monitor the effects of AgNPs at a nonlethal concentration on wild type Drosophila melanogaster by exposing them throughout their development. All adult flies raised in AgNP doped food showed that up to 50 mg/L concentration AgNP has no negative influence on median survival; however, these flies appeared uniformly lighter in body color due to the loss of melanin pigments in their cuticle. Additionally, fertility and vertical movement ability were compromised due to AgNP feeding. Determination of the amount of free ionic silver (Ag+) led us to claim that the observed biological effects have resulted from the AgNPs and not from Ag+. Biochemical analysis suggests that the activity of copper dependent enzymes, namely tyrosinase and Cu-Zn superoxide dismutase, are decreased significantly following the consumption of AgNPs, despite the constant level of copper present in the tissue. Consequently, we propose a mechanism whereby consumption of excess AgNPs in association with membrane bound copper transporter proteins cause sequestration of copper, thus creating a condition that resembles copper starvation. This model also explains the cuticular demelanization effect resulting from AgNP since tyrosinase activity is essential for melanin biosynthesis. Finally, we claim that Drosophila, an established genetic model system, can be well utilized for further understanding of the biological effects of nanoparticles. PMID:23308159

  16. Mechanism of silver nanoparticles action on insect pigmentation reveals intervention of copper homeostasis.

    Science.gov (United States)

    Armstrong, Najealicka; Ramamoorthy, Malaisamy; Lyon, Delina; Jones, Kimberly; Duttaroy, Atanu

    2013-01-01

    Silver nanoparticles (AgNPs), like almost all nanoparticles, are potentially toxic beyond a certain concentration because the survival of the organism is compromised due to scores of pathophysiological abnormalities past that concentration. However, the mechanism of AgNP toxicity remains undetermined. Instead of applying a toxic dose, we attempted to monitor the effects of AgNPs at a nonlethal concentration on wild type Drosophila melanogaster by exposing them throughout their development. All adult flies raised in AgNP doped food showed that up to 50 mg/L concentration AgNP has no negative influence on median survival; however, these flies appeared uniformly lighter in body color due to the loss of melanin pigments in their cuticle. Additionally, fertility and vertical movement ability were compromised due to AgNP feeding. Determination of the amount of free ionic silver (Ag(+)) led us to claim that the observed biological effects have resulted from the AgNPs and not from Ag(+). Biochemical analysis suggests that the activity of copper dependent enzymes, namely tyrosinase and Cu-Zn superoxide dismutase, are decreased significantly following the consumption of AgNPs, despite the constant level of copper present in the tissue. Consequently, we propose a mechanism whereby consumption of excess AgNPs in association with membrane bound copper transporter proteins cause sequestration of copper, thus creating a condition that resembles copper starvation. This model also explains the cuticular demelanization effect resulting from AgNP since tyrosinase activity is essential for melanin biosynthesis. Finally, we claim that Drosophila, an established genetic model system, can be well utilized for further understanding of the biological effects of nanoparticles.

  17. Proteomic analysis reveals resistance mechanism against biofuel hexane in Synechocystis sp. PCC 6803

    Directory of Open Access Journals (Sweden)

    Liu Jie

    2012-09-01

    Full Text Available Abstract Background Recent studies have demonstrated that photosynthetic cyanobacteria could be an excellent cell factory to produce renewable biofuels and chemicals due to their capability to utilize solar energy and CO2 as the sole energy and carbon sources. Biosynthesis of carbon-neutral biofuel alkanes with good chemical and physical properties has been proposed. However, to make the process economically feasible, one major hurdle to improve the low cell tolerance to alkanes needed to be overcome. Results Towards the goal to develop robust and high-alkane-tolerant hosts, in this study, the responses of model cyanobacterial Synechocystis PCC 6803 to hexane, a representative of alkane, were investigated using a quantitative proteomics approach with iTRAQ - LC-MS/MS technologies. In total, 1,492 unique proteins were identified, representing about 42% of all predicted protein in the Synechocystis genome. Among all proteins identified, a total of 164 and 77 proteins were found up- and down-regulated, respectively. Functional annotation and KEGG pathway enrichment analyses showed that common stress responses were induced by hexane in Synechocystis. Notably, a large number of transporters and membrane-bound proteins, proteins against oxidative stress and proteins related to sulfur relay system and photosynthesis were induced, suggesting that they are possibly the major protection mechanisms against hexane toxicity. Conclusion The study provided the first comprehensive view of the complicated molecular mechanism employed by cyanobacterial model species, Synechocystis to defend against hexane stress. The study also provided a list of potential targets to engineer Synechocystis against hexane stress.

  18. Coupled electrophysiological recording and single cell transcriptome analyses revealed molecular mechanisms underlying neuronal maturation

    Directory of Open Access Journals (Sweden)

    Xiaoying Chen

    2016-02-01

    Full Text Available ABSTRACT The mammalian brain is heterogeneous, containing billions of neurons and trillions of synapses forming various neural circuitries, through which sense, movement, thought, and emotion arise. The cellular heterogeneity of the brain has made it difficult to study the molecular logic of neural circuitry wiring, pruning, activation, and plasticity, until recently, transcriptome analyses with single cell resolution makes decoding of gene regulatory networks underlying aforementioned circuitry properties possible. Here we report success in performing both electrophysiological and whole-genome transcriptome analyses on single human neurons in culture. Using Weighted Gene Coexpression Network Analyses (WGCNA, we identified gene clusters highly correlated with neuronal maturation judged by electrophysiological characteristics. A tight link between neuronal maturation and genes involved in ubiquitination and mitochondrial function was revealed. Moreover, we identified a list of candidate genes, which could potentially serve as biomarkers for neuronal maturation. Coupled electrophysiological recording and single cell transcriptome analysis will serve as powerful tools in the future to unveil molecular logics for neural circuitry functions.

  19. RNAseq revealed the important gene pathways controlling adaptive mechanisms under waterlogged stress in maize.

    Science.gov (United States)

    Arora, Kanika; Panda, Kusuma Kumari; Mittal, Shikha; Mallikarjuna, Mallana Gowdra; Rao, Atmakuri Ramakrishna; Dash, Prasanta Kumar; Thirunavukkarasu, Nepolean

    2017-09-08

    Waterlogging causes yield penalty in maize-growing countries of subtropical regions. Transcriptome analysis of the roots of a tolerant inbred HKI1105 using RNA sequencing revealed 21,364 differentially expressed genes (DEGs) under waterlogged stress condition. These 21,364 DEGs are known to regulate important pathways including energy-production, programmed cell death (PCD), aerenchyma formation, and ethylene responsiveness. High up-regulation of invertase (49-fold) and hexokinase (36-fold) in roots explained the ATP requirement in waterlogging condition. Also, high up-regulation of expansins (42-fold), plant aspartic protease A3 (19-fold), polygalacturonases (16-fold), respiratory burst oxidase homolog (12-fold), and hydrolases (11-fold) explained the PCD of root cortical cells followed by the formation of aerenchyma tissue during waterlogging stress. We hypothesized that the oxygen transfer in waterlogged roots is promoted by a cross-talk of fermentative, metabolic, and glycolytic pathways that generate ATPs for PCD and aerenchyma formation in root cortical cells. SNPs were mapped to the DEGs regulating aerenchyma formation (12), ethylene-responsive factors (11), and glycolysis (4) under stress. RNAseq derived SNPs can be used in selection approaches to breed tolerant hybrids. Overall, this investigation provided significant evidence of genes operating in the adaptive traits such as ethylene production and aerenchyma formation to cope-up the waterlogging stress.

  20. Structure of a Blm10 Complex Reveals Common Mechanisms for Proteasome Binding and Gate Opening

    Energy Technology Data Exchange (ETDEWEB)

    Sadre-Bazzaz, K.; Robinson, H.; Whitby, F. G.; Formosa, T.; Hill, C. P.

    2010-03-12

    The proteasome is an abundant protease that is critically important for numerous cellular pathways. Proteasomes are activated in vitro by three known classes of proteins/complexes, including Blm10/PA200. Here, we report a 3.4 {angstrom} resolution crystal structure of a proteasome-Blm10 complex, which reveals that Blm10 surrounds the proteasome entry pore in the 1.2 MDa complex to form a largely closed dome that is expected to restrict access of potential substrates. This architecture and the observation that Blm10 induces a disordered proteasome gate structure challenge the assumption that Blm10 functions as an activator of proteolysis in vivo. The Blm10 C terminus binds in the same manner as seen for 11S activators and inferred for 19S/PAN activators and indicates a unified model for gate opening. We also demonstrate that Blm10 acts to maintain mitochondrial function. Consistent with the structural data, the C-terminal residues of Blm10 are needed for this activity.

  1. Structure of Oxidized Alpha-Haemoglobin Bound to AHSP Reveals a Protective Mechanism for HAEM

    Energy Technology Data Exchange (ETDEWEB)

    Feng,L.; Zhou, S.; Gu, L.; Gell, D.; MacKay, J.; Weiss, M.; Gow, A.; Shi, Y.

    2005-01-01

    The synthesis of hemoglobin A (HbA) is exquisitely coordinated during erythrocyte development to prevent damaging effects from individual {alpha}- and {beta}-subunits. The {alpha}-hemoglobin-stabilizing protein (AHSP) binds {alpha}-hemoglobin ({alpha}Hb), inhibits the ability of {alpha}Hb to generate reactive oxygen species and prevents its precipitation on exposure to oxidant stress. The structure of AHSP bound to ferrous {alpha}Hb is thought to represent a transitional complex through which {alpha}Hb is converted to a non-reactive, hexacoordinate ferric form. Here we report the crystal structure of this ferric {alpha}Hb-AHSP complex at 2.4 Angstrom resolution. Our findings reveal a striking bis-histidyl configuration in which both the proximal and the distal histidines coordinate the haem iron atom. To attain this unusual conformation, segments of {alpha}Hb undergo drastic structural rearrangements, including the repositioning of several {alpha}-helices. Moreover, conversion to the ferric bis-histidine configuration strongly and specifically inhibits redox chemistry catalysis and haem loss from {alpha}Hb. The observed structural changes, which impair the chemical reactivity of haem iron, explain how AHSP stabilizes {alpha}Hb and prevents its damaging effects in cells.

  2. Mechanical models of sandfish locomotion reveal principles of high performance subsurface sand-swimming

    Science.gov (United States)

    Maladen, Ryan D.; Ding, Yang; Umbanhowar, Paul B.; Kamor, Adam; Goldman, Daniel I.

    2011-01-01

    We integrate biological experiment, empirical theory, numerical simulation and a physical model to reveal principles of undulatory locomotion in granular media. High-speed X-ray imaging of the sandfish lizard, Scincus scincus, in 3 mm glass particles shows that it swims within the medium without using its limbs by propagating a single-period travelling sinusoidal wave down its body, resulting in a wave efficiency, η, the ratio of its average forward speed to the wave speed, of approximately 0.5. A resistive force theory (RFT) that balances granular thrust and drag forces along the body predicts η close to the observed value. We test this prediction against two other more detailed modelling approaches: a numerical model of the sandfish coupled to a discrete particle simulation of the granular medium, and an undulatory robot that swims within granular media. Using these models and analytical solutions of the RFT, we vary the ratio of undulation amplitude to wavelength (A/λ) and demonstrate an optimal condition for sand-swimming, which for a given A results from the competition between η and λ. The RFT, in agreement with the simulated and physical models, predicts that for a single-period sinusoidal wave, maximal speed occurs for A/λ ≈ 0.2, the same kinematics used by the sandfish. PMID:21378020

  3. Ribosome•RelA structures reveal the mechanism of stringent response activation

    Science.gov (United States)

    Loveland, Anna B; Bah, Eugene; Madireddy, Rohini; Zhang, Ying; Brilot, Axel F; Grigorieff, Nikolaus; Korostelev, Andrei A

    2016-01-01

    Stringent response is a conserved bacterial stress response underlying virulence and antibiotic resistance. RelA/SpoT-homolog proteins synthesize transcriptional modulators (p)ppGpp, allowing bacteria to adapt to stress. RelA is activated during amino-acid starvation, when cognate deacyl-tRNA binds to the ribosomal A (aminoacyl-tRNA) site. We report four cryo-EM structures of E. coli RelA bound to the 70S ribosome, in the absence and presence of deacyl-tRNA accommodating in the 30S A site. The boomerang-shaped RelA with a wingspan of more than 100 Å wraps around the A/R (30S A-site/RelA-bound) tRNA. The CCA end of the A/R tRNA pins the central TGS domain against the 30S subunit, presenting the (p)ppGpp-synthetase domain near the 30S spur. The ribosome and A/R tRNA are captured in three conformations, revealing hitherto elusive states of tRNA engagement with the ribosomal decoding center. Decoding-center rearrangements are coupled with the step-wise 30S-subunit 'closure', providing insights into the dynamics of high-fidelity tRNA decoding. DOI: http://dx.doi.org/10.7554/eLife.17029.001 PMID:27434674

  4. Expression profiles of key phenylpropanoid genes during Vanilla planifolia pod development reveal a positive correlation between PAL gene expression and vanillin biosynthesis.

    Science.gov (United States)

    Fock-Bastide, Isabelle; Palama, Tony Lionel; Bory, Séverine; Lécolier, Aurélie; Noirot, Michel; Joët, Thierry

    2014-01-01

    In Vanilla planifolia pods, development of flavor precursors is dependent on the phenylpropanoid pathway. The distinctive vanilla aroma is produced by numerous phenolic compounds of which vanillin is the most important. Because of the economic importance of vanilla, vanillin biosynthetic pathways have been extensively studied but agreement has not yet been reached on the processes leading to its accumulation. In order to explore the transcriptional control exerted on these pathways, five key phenylpropanoid genes expressed during pod development were identified and their mRNA accumulation profiles were evaluated during pod development and maturation using quantitative real-time PCR. As a prerequisite for expression analysis using qRT-PCR, five potential reference genes were tested, and two genes encoding Actin and EF1 were shown to be the most stable reference genes for accurate normalization during pod development. For the first time, genes encoding a phenylalanine ammonia-lyase (VpPAL1) and a cinnamate 4-hydroxylase (VpC4H1) were identified in vanilla pods and studied during maturation. Among phenylpropanoid genes, differential regulation was observed from 3 to 8 months after pollination. VpPAL1 was gradually up-regulated, reaching the maximum expression level at maturity. In contrast, genes encoding 4HBS, C4H, OMT2 and OMT3 did not show significant increase in expression levels after the fourth month post-pollination. Expression profiling of these key phenylpropanoid genes is also discussed in light of accumulation patterns for key phenolic compounds. Interestingly, VpPAL1 gene expression was shown to be positively correlated to maturation and vanillin accumulation.

  5. Silicon oxide nanowire growth mechanisms revealed by real-time electron microscopy

    Science.gov (United States)

    Kolíbal, Miroslav; Novák, Libor; Shanley, Toby; Toth, Milos; Šikola, Tomáš

    2015-12-01

    Growth of one-dimensional materials is possible through numerous mechanisms that affect the nanowire structure and morphology. Here, we explain why a wide range of morphologies is observed when silicon oxide nanowires are grown on silicon substrates using liquid gallium catalyst droplets. We show that a gallium oxide overlayer is needed for nanowire nucleation at typical growth temperatures, and that it can decompose during growth and, hence, dramatically alter the nanowire morphology. Gallium oxide decomposition is attributed to etching caused by hydrogen that can be supplied by thermal dissociation of H2O (a common impurity). We show that H2O dissociation is catalyzed by silicon substrates at temperatures as low as 320 °C, identify the material supply pathways and processes that rate-limit nanowire growth under dry and wet atmospheres, and present a detailed growth model that explains contradictory results reported in prior studies. We also show that under wet atmospheres the Ga droplets can be mobile and promote nanowire growth as they traverse the silicon substrate.Growth of one-dimensional materials is possible through numerous mechanisms that affect the nanowire structure and morphology. Here, we explain why a wide range of morphologies is observed when silicon oxide nanowires are grown on silicon substrates using liquid gallium catalyst droplets. We show that a gallium oxide overlayer is needed for nanowire nucleation at typical growth temperatures, and that it can decompose during growth and, hence, dramatically alter the nanowire morphology. Gallium oxide decomposition is attributed to etching caused by hydrogen that can be supplied by thermal dissociation of H2O (a common impurity). We show that H2O dissociation is catalyzed by silicon substrates at temperatures as low as 320 °C, identify the material supply pathways and processes that rate-limit nanowire growth under dry and wet atmospheres, and present a detailed growth model that explains

  6. Force measurements reveal how small binders perturb the dissociation mechanisms of DNA duplex sequences

    Science.gov (United States)

    Burmistrova, Anastasia; Fresch, Barbara; Sluysmans, Damien; de Pauw, Edwin; Remacle, Françoise; Duwez, Anne-Sophie

    2016-06-01

    The force-driven separation of double-stranded DNA is crucial to the accomplishment of cellular processes like genome transactions. Ligands binding to short DNA sequences can have a local stabilizing or destabilizing effect and thus severely affect these processes. Although the design of ligands that bind to specific sequences is a field of intense research with promising biomedical applications, so far, their effect on the force-induced strand separation has remained elusive. Here, by means of AFM-based single molecule force spectroscopy, we show the co-existence of two different mechanisms for the separation of a short DNA duplex and demonstrate how they are perturbed by small binders. With the support of Molecular Dynamics simulations, we evidence that above a critical pulling rate one of the dissociation pathways becomes dominant, with a dramatic effect on the rupture forces. Around the critical threshold, we observe a drop of the most probable rupture forces for ligand-stabilized duplexes. Our results offer a deep understanding of how a stable DNA-ligand complex behaves under force-driven strand separation.The force-driven separation of double-stranded DNA is crucial to the accomplishment of cellular processes like genome transactions. Ligands binding to short DNA sequences can have a local stabilizing or destabilizing effect and thus severely affect these processes. Although the design of ligands that bind to specific sequences is a field of intense research with promising biomedical applications, so far, their effect on the force-induced strand separation has remained elusive. Here, by means of AFM-based single molecule force spectroscopy, we show the co-existence of two different mechanisms for the separation of a short DNA duplex and demonstrate how they are perturbed by small binders. With the support of Molecular Dynamics simulations, we evidence that above a critical pulling rate one of the dissociation pathways becomes dominant, with a dramatic effect

  7. Mechanisms of thermal adaptation revealed from the genomes of the Antarctic

    Energy Technology Data Exchange (ETDEWEB)

    Saunders, Neil F.W.; Thomas, Torsten; Curmi, Paul M.G.; Mattick, John S.; Kuczek, Elizabeth; Slade, Rob; Davis, John; Franzmann, Peter; Boone, David; Rusterholtz, Karl; Feldman, Robert; Gates, Chris; Bench, Shellie; Sowers, Kevin; Kadner, Kristen; Aerts, Andrea; Dehal, Paramvir; Detter, Chris; Glavina, Tijana; Lucas, Susan; Richardson, Paul; Larimer, Frank; Hauser , Frank; Hauser, Loren; Land, Miriam; Cavicchioli, Richard

    2003-03-01

    We generated draft genome sequences for two cold-adapted Archaea, Methanogenium frigidum and Methanococcoides burtonii, to identify genotypic characteristics that distinguish them from Archaea with a higher optimal growth temperature (OGT). Comparative genomics revealed trends in amino acid and tRNA composition, and structural features of proteins. Proteins from the cold-adapted Archaea are characterized by a higher content of non-charged polar amino acids, particularly Gln and Thr and a lower content of hydrophobic amino acids, particularly Leu. Sequence data from nine methanogen genomes (OGT 15-98 C) was used to generate 1 111 modeled protein structures. Analysis of the models from the cold-adapted Archaea showed a strong tendency in the solvent accessible area for more Gln, Thr an hydrophobic residues and fewer charged residues. A cold shock domain (CSD) protein (CspA homolog) was identified in M. frigidum, two hypothetical proteins with CSD-folds in M. burtonii, and a unique winged helix DNA-binding domain protein in M. burtonii. This suggests that these types of nucleic acid binding proteins have a critical role in cold-adapted Archaea. Structural analysis of tRNA sequences from the Archaea indicated that GC content is the major factor influencing tRNA stability in hyperthermophiles, but not in the psychrophiles, mesophiles or moderate thermophiles. Below an OGT of 60 C, the GC content in tRNA was largely unchanged, indicating that any requirement for flexibility of tRNA in psychrophiles is mediated by other means. This is the first time that comparisons have been performed with genome data from Archaea spanning the growth temperature extremes from psychrophiles to hyperthermophiles.

  8. Physiological and transcriptomic analyses reveal a response mechanism to cold stress in Santalum album L. leaves

    Science.gov (United States)

    Zhang, Xinhua; Teixeira da Silva, Jaime A.; Niu, Meiyun; Li, Mingzhi; He, Chunmei; Zhao, Jinhui; Zeng, Songjun; Duan, Jun; Ma, Guohua

    2017-01-01

    Santalum album L. (Indian sandalwood) is an economically important plant species because of its ability to produce highly valued perfume oils. Little is known about the mechanisms by which S. album adapts to low temperatures. In this study, we obtained 100,445,724 raw reads by paired-end sequencing from S. album leaves. Physiological and transcriptomic changes in sandalwood seedlings exposed to 4 °C for 0–48 h were characterized. Cold stress induced the accumulation of malondialdehyde, proline and soluble carbohydrates, and increased the levels of antioxidants. A total of 4,424 differentially expressed genes were responsive to cold, including 3,075 cold-induced and 1,349 cold-repressed genes. When cold stress was prolonged, there was an increase in the expression of cold-responsive genes coding for transporters, responses to stimuli and stress, regulation of defense response, as well as genes related to signal transduction of all phytohormones. Candidate genes in the terpenoid biosynthetic pathway were identified, eight of which were significantly involved in the cold stress response. Gene expression analyses using qRT-PCR showed a peak in the accumulation of SaCBF2 to 4, 50-fold more than control leaves and roots following 12 h and 24 h of cold stress, respectively. The CBF-dependent pathway may play a crucial role in increasing cold tolerance. PMID:28169358

  9. Quantum mechanical electronic structure calculation reveals orientation dependence of hydrogen bond energy in proteins.

    Science.gov (United States)

    Mondal, Abhisek; Datta, Saumen

    2017-06-01

    Hydrogen bond plays a unique role in governing macromolecular interactions with exquisite specificity. These interactions govern the fundamental biological processes like protein folding, enzymatic catalysis, molecular recognition. Despite extensive research work, till date there is no proper report available about the hydrogen bond's energy surface with respect to its geometric parameters, directly derived from proteins. Herein, we have deciphered the potential energy landscape of hydrogen bond directly from the macromolecular coordinates obtained from Protein Data Bank using quantum mechanical electronic structure calculations. The findings unravel the hydrogen bonding energies of proteins in parametric space. These data can be used to understand the energies of such directional interactions involved in biological molecules. Quantitative characterization has also been performed using Shannon entropic calculations for atoms participating in hydrogen bond. Collectively, our results constitute an improved way of understanding hydrogen bond energies in case of proteins and complement the knowledge-based potential. Proteins 2017; 85:1046-1055. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  10. Active mechanics reveal molecular-scale force kinetics in living oocytes

    CERN Document Server

    Ahmed, Wylie W; Almonacid, Maria; Bussonnier, Matthias; Verlhac, Marie-Helene; Gov, Nir S; Visco, Paolo; van Wijland, Frederic; Betz, Timo

    2015-01-01

    Unlike traditional materials, living cells actively generate forces at the molecular scale that change their structure and mechanical properties. This nonequilibrium activity is essential for cellular function, and drives processes such as cell division. Single molecule studies have uncovered the detailed force kinetics of isolated motor proteins in-vitro, however their behavior in-vivo has been elusive due to the complex environment inside the cell. Here, we quantify active force generation in living oocytes using in-vivo optical trapping and laser interferometry of endogenous vesicles. We integrate an experimental and theoretical framework to connect mesoscopic measurements of nonequilibrium properties to the underlying molecular-scale force kinetics. Our results show that force generation by myosin-V drives the cytoplasmic-skeleton out-of-equilibrium (at frequencies below 300 Hz) and actively softens the environment. In vivo myosin-V activity generates a force of $F \\sim 0.4$ pN, with a power-stroke of len...

  11. Molecular mechanism for preQ1-II riboswitch function revealed by molecular dynamics

    Science.gov (United States)

    Aytenfisu, Asaminew H.; Liberman, Joseph A.; Wedekind, Joseph E.; Mathews, David H.

    2015-01-01

    Riboswitches are RNA molecules that regulate gene expression using conformational change, affected by binding of small molecule ligands. A crystal structure of a ligand-bound class II preQ1 riboswitch has been determined in a previous structural study. To gain insight into the dynamics of this riboswitch in solution, eight total molecular dynamic simulations, four with and four without ligand, were performed using the Amber force field. In the presence of ligand, all four of the simulations demonstrated rearranged base pairs at the 3′ end, consistent with expected base-pairing from comparative sequence analysis in a prior bioinformatic analysis; this suggests the pairing in this region was altered by crystallization. Additionally, in the absence of ligand, three of the simulations demonstrated similar changes in base-pairing at the ligand binding site. Significantly, although most of the riboswitch architecture remained intact in the respective trajectories, the P3 stem was destabilized in the ligand-free simulations in a way that exposed the Shine–Dalgarno sequence. This work illustrates how destabilization of two major groove base triples can influence a nearby H-type pseudoknot and provides a mechanism for control of gene expression by a fold that is frequently found in bacterial riboswitches. PMID:26370581

  12. Molecular interaction of zp3 to zp3r reveals a cross-species fertilization mechanism

    Directory of Open Access Journals (Sweden)

    Reni Kurniati

    2017-05-01

    Full Text Available Objective: To evaluate the role of ZP3R in the species-specific fertilization mechanism. Methods: ZP3/ZP3R protein sequences of Mus musculus, Rattus norvegicus, and Cavia porcellus were downloaded from UNIPROT. Percentage of amino acids that was calculated by using the SIAS program. Protein sequences modeled was established by using the Modeller 9.14 program and glycosylation of the ZP3 using GlyProt program. Docking simulation of the ZP3R-ZP3 was performed between the same species and different species with PatchDock program. Results: Comparison of the ZP3R and ZP3 structure between species showed that ZP3 in these three species was more similar than ZP3R. Docking simulations of protein showed that changes in the pattern of the ZP3-ZP3R domain for interaction on cross-species compared to the same species. Changes in the pattern of binding ZP3R-ZP3 made spermegg binding was not functional and could inhibit cross-fertilization. Conclusions: ZP3R-ZP3 interaction is species-specific, and the role of ZP3R is greater than ZP3 in determining the species-specific recognition stage and sperm-egg binding.

  13. Computational modelling of cell chain migration reveals mechanisms that sustain follow-the-leader behaviour.

    Science.gov (United States)

    Wynn, Michelle L; Kulesa, Paul M; Schnell, Santiago

    2012-07-07

    Follow-the-leader chain migration is a striking cell migratory behaviour observed during vertebrate development, adult neurogenesis and cancer metastasis. Although cell-cell contact and extracellular matrix (ECM) cues have been proposed to promote this phenomenon, mechanisms that underlie chain migration persistence remain unclear. Here, we developed a quantitative agent-based modelling framework to test mechanistic hypotheses of chain migration persistence. We defined chain migration and its persistence based on evidence from the highly migratory neural crest model system, where cells within a chain extend and retract filopodia in short-lived cell contacts and move together as a collective. In our agent-based simulations, we began with a set of agents arranged as a chain and systematically probed the influence of model parameters to identify factors critical to the maintenance of the chain migration pattern. We discovered that chain migration persistence requires a high degree of directional bias in both lead and follower cells towards the target. Chain migration persistence was also promoted when lead cells maintained cell contact with followers, but not vice-versa. Finally, providing a path of least resistance in the ECM was not sufficient alone to drive chain persistence. Our results indicate that chain migration persistence depends on the interplay of directional cell movement and biased cell-cell contact.

  14. Aiptasia sp. larvae as a model to reveal mechanisms of symbiont selection in cnidarians

    KAUST Repository

    Wolfowicz, Iliona

    2016-09-01

    Symbiosis, defined as the persistent association between two distinct species, is an evolutionary and ecologically critical phenomenon facilitating survival of both partners in diverse habitats. The biodiversity of coral reef ecosystems depends on a functional symbiosis with photosynthetic dinoflagellates of the highly diverse genus Symbiodinium, which reside in coral host cells and continuously support their nutrition. The mechanisms underlying symbiont selection to establish a stable endosymbiosis in non-symbiotic juvenile corals are unclear. Here we show for the first time that symbiont selection patterns for larvae of two Acropora coral species and the model anemone Aiptasia are similar under controlled conditions. We find that Aiptasia larvae distinguish between compatible and incompatible symbionts during uptake into the gastric cavity and phagocytosis. Using RNA-Seq, we identify a set of candidate genes potentially involved in symbiosis establishment. Together, our data complement existing molecular resources to mechanistically dissect symbiont phagocytosis in cnidarians under controlled conditions, thereby strengthening the role of Aiptasia larvae as a powerful model for cnidarian endosymbiosis establishment.

  15. Transcriptomic analysis reveals the flooding tolerant mechanism in flooding tolerant line and abscisic acid treated soybean.

    Science.gov (United States)

    Yin, Xiaojian; Hiraga, Susumu; Hajika, Makita; Nishimura, Minoru; Komatsu, Setsuko

    2017-03-01

    Soybean is highly sensitive to flooding stress and exhibits markedly reduced plant growth and grain yield under flooding conditions. To explore the mechanisms underlying initial flooding tolerance in soybean, RNA sequencing-based transcriptomic analysis was performed using a flooding-tolerant line and ABA-treated soybean. A total of 31 genes included 12 genes that exhibited similar temporal patterns were commonly changed in these plant groups in response to flooding and they were mainly involved in RNA regulation and protein metabolism. The mRNA expression of matrix metalloproteinase, glucose-6-phosphate isomerase, ATPase family AAA domain-containing protein 1, and cytochrome P450 77A1 was up-regulated in wild-type soybean under flooding conditions; however, no changes were detected in the flooding-tolerant line or ABA-treated soybean. The mRNA expression of cytochrome P450 77A1 was specifically up-regulated in root tips by flooding stress, but returned to the level found in control plants following treatment with the P450 inhibitor uniconazole. The survival ratio and root fresh weight of plants were markedly improved by 3-h uniconazole treatment under flooding stress. Taken together, these results suggest that cytochrome P450 77A1 is suppressed by uniconazole treatment and that this inhibition may enhance soybean tolerance to flooding stress.

  16. Transcriptomic analysis of Ustilago maydis infecting Arabidopsis reveals important aspects of the fungus pathogenic mechanisms.

    Science.gov (United States)

    Martínez-Soto, Domingo; Robledo-Briones, Angélica M; Estrada-Luna, Andrés A; Ruiz-Herrera, José

    2013-08-01

    Transcriptomic and biochemical analyses of the experimental pathosystem constituted by Ustilago maydis and Arabidopsis thaliana were performed. Haploid or diploid strains of U. maydis inoculated in A. thaliana plantlets grew on the surface and within the plant tissues in the form of mycelium, inducing chlorosis, anthocyanin formation, malformations, necrosis and adventitious roots development, but not teliospores. Symptoms were more severe in plants inoculated with the haploid strain which grew more vigorously than the diploid strain. RNA extracted at different times post-infection was used for hybridization of one-channel microarrays that were analyzed focusing on the fungal genes involved in the general pathogenic process, biogenesis of the fungal cell wall and the secretome. In total, 3,537 and 3,299 genes were differentially expressed in the haploid and diploid strains, respectively. Differentially expressed genes were related to different functional categories and many of them showed a similar regulation occurring in U. maydis infecting maize. Our data suggest that the haploid strain behaves as a necrotrophic pathogen, whereas the diploid behaves as a biotrophic pathogen. The results obtained are evidence of the usefulness of the U. maydis-A. thaliana pathosystem for the analysis of the pathogenic mechanisms of U. maydis.

  17. Potassium starvation in yeast: mechanisms of homeostasis revealed by mathematical modeling.

    Directory of Open Access Journals (Sweden)

    Matthias Kahm

    Full Text Available The intrinsic ability of cells to adapt to a wide range of environmental conditions is a fundamental process required for survival. Potassium is the most abundant cation in living cells and is required for essential cellular processes, including the regulation of cell volume, pH and protein synthesis. Yeast cells can grow from low micromolar to molar potassium concentrations and utilize sophisticated control mechanisms to keep the internal potassium concentration in a viable range. We developed a mathematical model for Saccharomyces cerevisiae to explore the complex interplay between biophysical forces and molecular regulation facilitating potassium homeostasis. By using a novel inference method ("the reverse tracking algorithm" we predicted and then verified experimentally that the main regulators under conditions of potassium starvation are proton fluxes responding to changes of potassium concentrations. In contrast to the prevailing view, we show that regulation of the main potassium transport systems (Trk1,2 and Nha1 in the plasma membrane is not sufficient to achieve homeostasis.

  18. Potassium starvation in yeast: mechanisms of homeostasis revealed by mathematical modeling.

    Science.gov (United States)

    Kahm, Matthias; Navarrete, Clara; Llopis-Torregrosa, Vicent; Herrera, Rito; Barreto, Lina; Yenush, Lynne; Ariño, Joaquin; Ramos, Jose; Kschischo, Maik

    2012-01-01

    The intrinsic ability of cells to adapt to a wide range of environmental conditions is a fundamental process required for survival. Potassium is the most abundant cation in living cells and is required for essential cellular processes, including the regulation of cell volume, pH and protein synthesis. Yeast cells can grow from low micromolar to molar potassium concentrations and utilize sophisticated control mechanisms to keep the internal potassium concentration in a viable range. We developed a mathematical model for Saccharomyces cerevisiae to explore the complex interplay between biophysical forces and molecular regulation facilitating potassium homeostasis. By using a novel inference method ("the reverse tracking algorithm") we predicted and then verified experimentally that the main regulators under conditions of potassium starvation are proton fluxes responding to changes of potassium concentrations. In contrast to the prevailing view, we show that regulation of the main potassium transport systems (Trk1,2 and Nha1) in the plasma membrane is not sufficient to achieve homeostasis.

  19. Holographic Imaging Reveals the Mechanism of Wall Entrapment in Swimming Bacteria

    Science.gov (United States)

    Bianchi, Silvio; Saglimbeni, Filippo; Di Leonardo, Roberto

    2017-01-01

    Self-propelled particles, both biological and synthetic, are stably trapped by walls and develop high concentration peaks over bounding surfaces. In swimming bacteria, like E. coli, the physical mechanism behind wall entrapment is an intricate mixture of hydrodynamic and steric interactions with a strongly anisotropic character. The building of a clear physical picture of this phenomenon demands direct and full three-dimensional experimental observations of individual wall entrapment events. Here, we demonstrate that, by using a combination of three-axis holographic microscopy and optical tweezers, it is possible to obtain volumetric reconstructions of individual E. coli cells that are sequentially released at a controlled distance and angle from a flat solid wall. We find that hydrodynamic couplings can slow down the cell before collision, but reorientation only occurs while the cell is in constant contact with the wall. In the trapped state, all cells swim with the average body axis pointing into the surface. The amplitude of this pitch angle is anticorrelated to the amplitude of wobbling, thus indicating that entrapment is dominated by near-field couplings between the cell body and the wall. Our approach opens the way to three-dimensional quantitative studies of a broad range of fast dynamical processes in motile bacteria and eukaryotic cells.

  20. The structure of a thermostable mutant of pro-papain reveals its activation mechanism.

    Science.gov (United States)

    Roy, Sumana; Choudhury, Debi; Aich, Pulakesh; Dattagupta, Jiban K; Biswas, Sampa

    2012-12-01

    Papain is the archetype of a broad class of cysteine proteases (clan C1A) that contain a pro-peptide in the zymogen form which is required for correct folding and spatio-temporal regulation of proteolytic activity in the initial stages after expression. This study reports the X-ray structure of the zymogen of a thermostable mutant of papain at 2.6 Å resolution. The overall structure, in particular that of the mature part of the protease, is similar to those of other members of the family. The structure provides an explanation for the molecular basis of the maintenance of latency of the proteolytic activity of the zymogen by its pro-segment at neutral pH. The structural analysis, together with biochemical and biophysical studies, demonstrated that the pro-segment of the zymogen undergoes a rearrangement in the form of a structural loosening at acidic pH which triggers the proteolytic activation cascade. This study further explains the bimolecular stepwise autocatalytic activation mechanism by limited proteolysis of the zymogen of papain at the molecular level. The possible factors responsible for the higher thermal stability of the papain mutant have also been analyzed.

  1. Competing mechanisms for perfluoroalkyl acid accumulation in plants revealed using an Arabidopsis model system.

    Science.gov (United States)

    Müller, Claudia E; LeFevre, Gregory H; Timofte, Anca E; Hussain, Fatima A; Sattely, Elizabeth S; Luthy, Richard G

    2016-05-01

    Perfluoroalkyl acids (PFAAs) bioaccumulate in plants, presenting a human exposure route if present in irrigation water. Curiously, accumulation of PFAAs in plant tissues is greatest for both the short-chain and long-chain PFAAs, generating a U-shaped relationship with chain length. In the present study, the authors decouple competing mechanisms of PFAA accumulation using a hydroponic model plant system (Arabidopsis thaliana) exposed to a suite of 10 PFAAs to determine uptake, depuration, and translocation kinetics. Rapid saturation of root concentrations occurred for all PFAAs except perfluorobutanoate, the least-sorptive (shortest-chain) PFAA. Shoot concentrations increased continuously, indicating that PFAAs are efficiently transported and accumulate in shoots. Tissue concentrations of PFAAs during depuration rapidly declined in roots but remained constant in shoots, demonstrating irreversibility of the translocation process. Root and shoot concentration factors followed the U-shaped trend with perfluoroalkyl chain length; however, when normalized to dead-tissue sorption, this relationship linearized. The authors therefore introduce a novel term, the "sorption normalized concentration factor," to describe PFAA accumulation in plants; because of their hydrophobicity, sorption is the determining factor for long-chain PFAAs, whereas the shortest-chain PFAAs are most effectively transported in the plant. The present study provides a mechanistic explanation for previously unexplained PFAA accumulation trends in plants and suggests that shorter-chained PFAAs may bioaccumulate more readily in edible portions.

  2. Revealing the underlying absorption and emission mechanism of nitrogen doped graphene quantum dots.

    Science.gov (United States)

    Niu, Xianghong; Li, Yunhai; Shu, Huabing; Wang, Jinlan

    2016-11-24

    Nitrogen-doped graphene quantum dots (N-GQDs) hold promising application in electronics and optoelectronics because of their excellent photo-stability, tunable photoluminescence and high quantum yield. However, the absorption and emission mechanisms have been debated for years. Here, by employing time-dependent density functional theory, we demonstrate that the different N-doping types and positions give rise to different absorption and emission behaviors, which successfully addresses the inconsistency observed in different experiments. Specifically, center doping creates mid-states, rendering non-fluorescence, while edge N-doping modulates the energy levels of excited states and increases the radiation transition probability, thus enhancing fluorescence strength. More importantly, the even hybridization of frontier orbitals between edge N atoms and GQDs leads to a blue-shift of both absorption and emission spectra, while the uneven hybridization of frontier orbitals induces a red-shift. Solvent effects on N-GQDs are further explored by the conductor-like screening model and it is found that strong polarity of the solvent can cause a red-shift and enhance the intensity of both absorption and emission spectra.

  3. Mice deficient in MCT8 reveal a mechanism regulating thyroid hormone secretion.

    Science.gov (United States)

    Di Cosmo, Caterina; Liao, Xiao-Hui; Dumitrescu, Alexandra M; Philp, Nancy J; Weiss, Roy E; Refetoff, Samuel

    2010-09-01

    The mechanism of thyroid hormone (TH) secretion from the thyroid gland into blood is unknown. Humans and mice deficient in monocarboxylate transporter 8 (MCT8) have low serum thyroxine (T4) levels that cannot be fully explained by increased deiodination. Here, we have shown that Mct8 is localized at the basolateral membrane of thyrocytes and that the serum TH concentration is reduced in Mct8-KO mice early after being taken off a treatment that almost completely depleted the thyroid gland of TH. Thyroid glands in Mct8-KO mice contained more non-thyroglobulin-associated T4 and triiodothyronine than did those in wild-type mice, independent of deiodination. In addition, depletion of thyroidal TH content was slower during iodine deficiency. After administration of 125I, the rate of both its secretion from the thyroid gland and its appearance in the serum as trichloroacetic acid-precipitable radioactivity was greatly reduced in Mct8-KO mice. Similarly, the secretion of T4 induced by injection of thyrotropin was reduced in Mct8-KO in which endogenous TSH and T4 were suppressed by administration of triiodothyronine. To our knowledge, this study is the first to demonstrate that Mct8 is involved in the secretion of TH from the thyroid gland and contributes, in part, to the low serum T4 level observed in MCT8-deficient patients.

  4. Molecular mechanism for preQ1-II riboswitch function revealed by molecular dynamics.

    Science.gov (United States)

    Aytenfisu, Asaminew H; Liberman, Joseph A; Wedekind, Joseph E; Mathews, David H

    2015-11-01

    Riboswitches are RNA molecules that regulate gene expression using conformational change, affected by binding of small molecule ligands. A crystal structure of a ligand-bound class II preQ1 riboswitch has been determined in a previous structural study. To gain insight into the dynamics of this riboswitch in solution, eight total molecular dynamic simulations, four with and four without ligand, were performed using the Amber force field. In the presence of ligand, all four of the simulations demonstrated rearranged base pairs at the 3' end, consistent with expected base-pairing from comparative sequence analysis in a prior bioinformatic analysis; this suggests the pairing in this region was altered by crystallization. Additionally, in the absence of ligand, three of the simulations demonstrated similar changes in base-pairing at the ligand binding site. Significantly, although most of the riboswitch architecture remained intact in the respective trajectories, the P3 stem was destabilized in the ligand-free simulations in a way that exposed the Shine-Dalgarno sequence. This work illustrates how destabilization of two major groove base triples can influence a nearby H-type pseudoknot and provides a mechanism for control of gene expression by a fold that is frequently found in bacterial riboswitches.

  5. Distinct mechanisms for spiro-carbon formation reveal biosynthetic pathway crosstalk.

    Science.gov (United States)

    Tsunematsu, Yuta; Ishikawa, Noriyasu; Wakana, Daigo; Goda, Yukihiro; Noguchi, Hiroshi; Moriya, Hisao; Hotta, Kinya; Watanabe, Kenji

    2013-12-01

    Spirotryprostatins, an indole alkaloid class of nonribosomal peptides isolated from Aspergillus fumigatus, are known for their antimitotic activity in tumor cells. Because spirotryprostatins and many other chemically complex spiro-carbon-bearing natural products exhibit useful biological activities, identifying and understanding the mechanism of spiro-carbon biosynthesis is of great interest. Here we report a detailed study of spiro-ring formation in spirotryprostatins from tryprostatins derived from the fumitremorgin biosynthetic pathway, using reactants and products prepared with engineered yeast and fungal strains. Unexpectedly, FqzB, an FAD-dependent monooxygenase from the unrelated fumiquinazoline biosynthetic pathway, catalyzed spiro-carbon formation in spirotryprostatin A via an epoxidation route. Furthermore, FtmG, a cytochrome P450 from the fumitremorgin biosynthetic pathway, was determined to catalyze the spiro-ring formation in spirotryprostatin B. Our results highlight the versatile role of oxygenating enzymes in the biosynthesis of structurally complex natural products and indicate that cross-talk of different biosynthetic pathways allows product diversification in natural product biosynthesis.

  6. Mechanism of cisplatin proximal tubule toxicity revealed by integrating transcriptomics, proteomics, metabolomics and biokinetics.

    Science.gov (United States)

    Wilmes, Anja; Bielow, Chris; Ranninger, Christina; Bellwon, Patricia; Aschauer, Lydia; Limonciel, Alice; Chassaigne, Hubert; Kristl, Theresa; Aiche, Stephan; Huber, Christian G; Guillou, Claude; Hewitt, Philipp; Leonard, Martin O; Dekant, Wolfgang; Bois, Frederic; Jennings, Paul

    2015-12-25

    Cisplatin is one of the most widely used chemotherapeutic agents for the treatment of solid tumours. The major dose-limiting factor is nephrotoxicity, in particular in the proximal tubule. Here, we use an integrated omics approach, including transcriptomics, proteomics and metabolomics coupled to biokinetics to identify cell stress response pathways induced by cisplatin. The human renal proximal tubular cell line RPTEC/TERT1 was treated with sub-cytotoxic concentrations of cisplatin (0.5 and 2 μM) in a daily repeat dose treating regime for up to 14 days. Biokinetic analysis showed that cisplatin was taken up from the basolateral compartment, transported to the apical compartment, and accumulated in cells over time. This is in line with basolateral uptake of cisplatin via organic cation transporter 2 and bioactivation via gamma-glutamyl transpeptidase located on the apical side of proximal tubular cells. Cisplatin affected several pathways including, p53 signalling, Nrf2 mediated oxidative stress response, mitochondrial processes, mTOR and AMPK signalling. In addition, we identified novel pathways changed by cisplatin, including eIF2 signalling, actin nucleation via the ARP/WASP complex and regulation of cell polarization. In conclusion, using an integrated omic approach together with biokinetics we have identified both novel and established mechanisms of cisplatin toxicity. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Holographic Imaging Reveals the Mechanism of Wall Entrapment in Swimming Bacteria

    Directory of Open Access Journals (Sweden)

    Silvio Bianchi

    2017-01-01

    Full Text Available Self-propelled particles, both biological and synthetic, are stably trapped by walls and develop high concentration peaks over bounding surfaces. In swimming bacteria, like E. coli, the physical mechanism behind wall entrapment is an intricate mixture of hydrodynamic and steric interactions with a strongly anisotropic character. The building of a clear physical picture of this phenomenon demands direct and full three-dimensional experimental observations of individual wall entrapment events. Here, we demonstrate that, by using a combination of three-axis holographic microscopy and optical tweezers, it is possible to obtain volumetric reconstructions of individual E. coli cells that are sequentially released at a controlled distance and angle from a flat solid wall. We find that hydrodynamic couplings can slow down the cell before collision, but reorientation only occurs while the cell is in constant contact with the wall. In the trapped state, all cells swim with the average body axis pointing into the surface. The amplitude of this pitch angle is anticorrelated to the amplitude of wobbling, thus indicating that entrapment is dominated by near-field couplings between the cell body and the wall. Our approach opens the way to three-dimensional quantitative studies of a broad range of fast dynamical processes in motile bacteria and eukaryotic cells.

  8. Multiwavelength polarimetry and integrated MHD+Polarized Radiation simulation reveal the blazar flaring mechanism

    Science.gov (United States)

    Zhang, Haocheng; Li, Hui; Taylor, Gregory B.

    2017-08-01

    In addition to multiwavelength variability, blazar polarization signatures are highly variable. Optical polarimetry has shown two distinct features: first, in both quiescent and flaring states, blazar polarization degree generally stays around 10% to 30%; second, after major polarization variations, such as polarization angle swings, the polarization degree quickly restores to its initial state. We have performed integrated relativistic magnetohydrodynamic (MHD) + radiation and polarization simulations of the blazar emission region. Our approach evolves the magnetic fields and flows using the first principles, so we can calculate the spatial and temporal dependent polarization signatures and compare them with observations.Our results show that the above two observational trends indicate the blazar flaring region should be strongly magnetized with the magnetic energy density higher than the plasma rest mass energy density. In such an environment, the 3D kink instability may trigger magnetic reconnection to accelerate particles and give rise to flares. In view of future high-energy polarimetry, this integrated MHD+polarization simulation technique will deliver new constraints on jet’s physical conditions and particle acceleration mechanisms.

  9. The maize milkweed pod1 mutant reveals a mechanism to modify organ morphology.

    Science.gov (United States)

    Johnston, Robyn; Candela, Héctor; Hake, Sarah; Foster, Toshi

    2010-07-01

    Plant lateral organs, such as leaves, have three primary axes of growth-proximal-distal, medial--lateral and adaxial-abaxial (dorsal-ventral). Although most leaves are planar, modified leaf forms, such as the bikeeled grass prophyll, can be found in nature. A detailed examination of normal prophyll development indicates that polarity is established differently in the keels than in other parts of the prophyll. Analysis of the maize HD-ZIPIII gene rolled leaf1 (rld1) suggests that altered expression patterns are responsible for keel outgrowth. Recessive mutations in the maize (Zea mays) KANADI (KAN) gene milkweed pod1 (mwp1), which promotes abaxial cell identity, strongly affect development of the prophyll and silks (fused carpels). The prophyll is reduced to two unfused midribs and the silks are narrow and misshapen. Our data indicate that the prophyll and other fused organs are particularly sensitive to disruptions in adaxial-abaxial polarity. In addition, lateral and proximal-distal growth of most lateral organs is reduced in the mwp1-R mutant, supporting a role for the adaxial-abaxial boundary in promoting growth along both axes. We propose that the adaxial-abaxial patterning mechanism has been co-opted during evolution to generate diverse organ morphologies. (c) 2010 Wiley-Liss, Inc.

  10. Tracking planets and moons: mechanisms of object tracking revealed with a new paradigm.

    Science.gov (United States)

    Tombu, Michael; Seiffert, Adriane E

    2011-04-01

    People can attend to and track multiple moving objects over time. Cognitive theories of this ability emphasize location information and differ on the importance of motion information. Results from several experiments have shown that increasing object speed impairs performance, although speed was confounded with other properties such as proximity of objects to one another. Here, we introduce a new paradigm to study multiple object tracking in which object speed and object proximity were manipulated independently. Like the motion of a planet and moon, each target-distractor pair rotated about both a common local point as well as the center of the screen. Tracking performance was strongly affected by object speed even when proximity was controlled. Additional results suggest that two different mechanisms are used in object tracking--one sensitive to speed and proximity and the other sensitive to the number of distractors. These observations support models of object tracking that include information about object motion and reject models that use location alone.

  11. Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation.

    Science.gov (United States)

    Staus, Dean P; Strachan, Ryan T; Manglik, Aashish; Pani, Biswaranjan; Kahsai, Alem W; Kim, Tae Hun; Wingler, Laura M; Ahn, Seungkirl; Chatterjee, Arnab; Masoudi, Ali; Kruse, Andrew C; Pardon, Els; Steyaert, Jan; Weis, William I; Prosser, R Scott; Kobilka, Brian K; Costa, Tommaso; Lefkowitz, Robert J

    2016-07-21

    G-protein-coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signalling effectors such as G proteins and β-arrestins. It is well known that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (‘efficacy’). Furthermore, increasing biophysical evidence, primarily using the β2-adrenergic receptor (β2AR) as a model system, supports the existence of multiple active and inactive conformational states. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct β2AR conformations using single domain camelid antibodies (nanobodies)—a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60). We show that Nb60 stabilizes a previously unappreciated low-affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoprenaline has a 15,000-fold higher affinity for β2AR in the presence of Nb80 compared to the affinity of isoprenaline for β2AR in the presence of Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the β2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of

  12. How Cannabis Causes Paranoia: Using the Intravenous Administration of ∆9-Tetrahydrocannabinol (THC) to Identify Key Cognitive Mechanisms Leading to Paranoia

    OpenAIRE

    Freeman, Daniel; Dunn, Graham; Murray, Robin M.; Evans, Nicole; Lister, Rachel; Antley, Angus; Slater, Mel; Godlewska, Beata; Cornish, Robert; Williams, Jonathan; Di Simplicio, Martina; Igoumenou, Artemis; Brenneisen, Rudolf; Tunbridge, Elizabeth M.; Harrison, Paul J

    2014-01-01

    Paranoia is receiving increasing attention in its own right, since it is a central experience of psychotic disorders and a marker of the health of a society. Paranoia is associated with use of the most commonly taken illicit drug, cannabis. The objective was to determine whether the principal psychoactive ingredient of cannabis—∆9-tetrahydrocannabinol (THC)—causes paranoia and to use the drug as a probe to identify key cognitive mechanisms underlying paranoia. A randomized, placebo-controlled...

  13. Introduction to the China Jellyfish Project——The Key Processes, Mechanism and Ecological Consequences of Jellyfish Bloom in China Coastal Waters

    Institute of Scientific and Technical Information of China (English)

    SUN Xiaoxia; WANG Shiwei; SUN Song

    2011-01-01

    @@ As a National Basic Research Program (973 Program) of China, the Key Processes, Mechanism and Ecological Consequences of Jellyfish Bloom in China Coastal Waters led by Prof.SUN Song from Institute of Oceanology, Chinese Academy of Sciences (IOCAS) was endorsed by the Ministry of Science and Technology.Recently, the kick-off meeting of this five-year (2011-2015) program was held on January 15-16 in Qingdao,China.More than 120 participants attended the meeting.

  14. Genomic imprinting: potential function and mechanisms revealed by the Prader-Willi and Angelman syndromes.

    Science.gov (United States)

    Glenn, C C; Driscoll, D J; Yang, T P; Nicholls, R D

    1997-04-01

    The Prader-Willi (PWS) and Angelman (AS) syndromes are two clinically distinct syndromes which result from lack of expression of imprinted genes within chromosome 15q11-q13. These two syndromes result from 15q11-q13 deletions, chromosome 15 uniparental disomy (UPD), imprinting centre mutations and, for AS, probable mutations in a single gene. The differential phenotype results from a paternal genetic deficiency in PWS patients and a maternal genetic deficiency in AS patients. Within 15q11-q13, four genes (SNRPN, IPW, ZNF127, FNZ127) and two expressed sequence tags (PAR1 and PAR5) have been found to be expressed only from the paternally inherited chromosome, and therefore all must be considered candidate genes involved in the pathogenesis of PWS. A candidate AS gene (UBE3A) has very recently been identified. The mechanisms of imprinted gene expression are not yet understood, but it is clear that DNA methylation is involved in both somatic cell expression and inheritance of the imprint. The presence of DNA methylation imprints that distinguish the paternally and maternally inherited alleles is a common characteristic of all known imprinted genes which have been studied extensively, including SNRPN and ZNF127. Recently, several PWS and AS patients have been found that have microdeletions in a region upstream of the SNRPN gene referred to as the imprinting centre, or IC. Paternal IC deletions in PWS patients and maternal IC deletions in AS patients result in uniparental DNA methylation and uniparental gene expression at biparentally inherited loci. The IC is a novel genetic element which controls initial resetting of the parental imprint in the germline for all imprinted gene expression over a 1.5-2.5 Mb region within chromosome 15q11-q13.

  15. Pathway analysis reveals common pro-survival mechanisms of metyrapone and carbenoxolone after traumatic brain injury.

    Directory of Open Access Journals (Sweden)

    Helen L Hellmich

    Full Text Available Developing new pharmacotherapies for traumatic brain injury (TBI requires elucidation of the neuroprotective mechanisms of many structurally and functionally diverse compounds. To test our hypothesis that diverse neuroprotective drugs similarly affect common gene targets after TBI, we compared the effects of two drugs, metyrapone (MT and carbenoxolone (CB, which, though used clinically for noncognitive conditions, improved learning and memory in rats and humans. Although structurally different, both MT and CB inhibit a common molecular target, 11β hydroxysteroid dehydrogenase type 1, which converts inactive cortisone to cortisol, thereby effectively reducing glucocorticoid levels. We examined injury-induced signaling pathways to determine how the effects of these two compounds correlate with pro-survival effects in surviving neurons of the injured rat hippocampus. We found that treatment of TBI rats with MT or CB acutely induced in hippocampal neurons transcriptional profiles that were remarkably similar (i.e., a coordinated attenuation of gene expression across multiple injury-induced cell signaling networks. We also found, to a lesser extent, a coordinated increase in cell survival signals. Analysis of injury-induced gene expression altered by MT and CB provided additional insight into the protective effects of each. Both drugs attenuated expression of genes in the apoptosis, death receptor and stress signaling pathways, as well as multiple genes in the oxidative phosphorylation pathway such as subunits of NADH dehydrogenase (Complex1, cytochrome c oxidase (Complex IV and ATP synthase (Complex V. This suggests an overall inhibition of mitochondrial function. Complex 1 is the primary source of reactive oxygen species in the mitochondrial oxidative phosphorylation pathway, thus linking the protective effects of these drugs to a reduction in oxidative stress. The net effect of the drug-induced transcriptional changes observed here indicates that

  16. Mechanisms of radiation strengthening in Fe–Cr alloys as revealed by atomistic studies

    Energy Technology Data Exchange (ETDEWEB)

    Terentyev, D.; Bonny, G. [Structural Materials Modelling and Microstructure Unit, SMA/NMS, Studiecentrum voor Kernenergie, Centre d’Etudes de l’Energie Nucléaire (SCK-CEN), Boeretang 200, 2400 Mol (Belgium); Domain, C.; Monnet, G. [EDF-R and D, Département MMC, Les Renardières, 77818 Moret sur Loing Cedex (France); Malerba, L., E-mail: lmalerba@sckcen.be [Structural Materials Modelling and Microstructure Unit, SMA/NMS, Studiecentrum voor Kernenergie, Centre d’Etudes de l’Energie Nucléaire (SCK-CEN), Boeretang 200, 2400 Mol (Belgium)

    2013-11-15

    A review of experimental results shows that the dependence on Cr content of radiation-induced strengthening in Fe–Cr alloys and ferritic/martensitic steels is peculiar, exhibiting an increase as soon as Cr is added, followed by a local maximum and then a local minimum. This dependence is to date unexplained. In this paper we try to rationalise it, by reviewing recent (published and unpublished) molecular dynamics simulations work, devoted to the investigation of several possible mechanisms of radiation strengthening in Fe–Cr. In particular, the following questions are addressed quantitatively: (i) Does Cr influence the glide of dislocations? If so, how? (ii) Does Cr influence the interaction between dislocations and radiation-produced defects? If so, why? The latter question involves also a study of the interaction of moving dislocations with experimentally observed Cr-enriched loops. We find that the fact of shifting from a loop-absorption (pure Fe) to a loop-non-absorption (Fe–Cr) regime, because of the Cr–enrichment of loops, contributes to explaining why Fe–Cr alloys harden more under irradiation than Fe. If, in addition, the existence of a large density of invisible and Cr-enriched loops is postulated, the origin of the effect becomes even more clear. Moreover, the different strength of 〈1 1 1〉 and 〈1 0 0〉 loops as obstacles to dislocations movement, depending on whether or not loop absorption can occur, might explain why radiation strengthening decreases between 2% and 9%Cr. The formation of α′ precipitates, on the other hand, explains why radiation strengthening increases again above 9%Cr. Altogether, these effects might explain the origin of the minimum of radiation-induced embrittlement at 9%Cr, as correlated to strengthening.

  17. Cognitive control mechanisms revealed by ERP and fMRI: evidence from repeated task-switching.

    Science.gov (United States)

    Swainson, R; Cunnington, R; Jackson, G M; Rorden, C; Peters, A M; Morris, P G; Jackson, S R

    2003-08-15

    We investigated the extent to which a common neural mechanism is involved in task set-switching and response withholding, factors that are frequently confounded in task-switching and go/no-go paradigms. Subjects' brain activity was measured using event-related electrical potentials (ERPs) and event-related functional MRI (fMRI) neuroimaging in separate studies using the same cognitive paradigm. Subjects made compatible left/right keypress responses to left/right arrow stimuli of 1000 msec duration; they switched every two trials between responding at stimulus onset (GO task-green arrows) and stimulus offset (WAIT task-red arrows). With-holding an immediate response (WAIT vs. GO) elicited an enhancement of the frontal N2 ERP and lateral PFC activation of the right hemisphere, both previously associated with the "no-go" response, but only on switch trials. Task-switching (switch vs. nonswitch) was associated with frontal N2 amplification and right hemisphere ventrolateral PFC activation, but only for the WAIT task. The anterior cingulate cortex (ACC) was the only brain region to be activated for both types of task switch, but this activation was located more rostrally for the WAIT than for the GO switch trials. We conclude that the frontal N2 ERP and lateral PFC activation are not markers for withholding an immediate response or switching tasks per se, but are associated with switching into a response-suppression mode. Different regions within the ACC may be involved in two processes integral to task-switching: processing response conflict (rostral ACC) and overcoming prior response suppression (caudal ACC).

  18. MicroRNA and messenger RNA profiling reveals new biomarkers and mechanisms for RDX induced neurotoxicity.

    Science.gov (United States)

    Deng, Youping; Ai, Junmei; Guan, Xin; Wang, Zhaohui; Yan, Bin; Zhang, Daqin; Liu, Chang; Wilbanks, Mitch S; Escalon, Barbara Lynn; Meyers, Sharon A; Yang, Mary Qu; Perkins, Edward J

    2014-01-01

    RDX is a well-known pollutant to induce neurotoxicity. MicroRNAs (miRNA) and messenger RNA (mRNA) profiles are useful tools for toxicogenomics studies. It is worthy to integrate MiRNA and mRNA expression data to understand RDX-induced neurotoxicity. Rats were treated with or without RDX for 48 h. Both miRNA and mRNA profiles were conducted using brain tissues. Nine miRNAs were significantly regulated by RDX. Of these, 6 and 3 miRNAs were up- and down-regulated respectively. The putative target genes of RDX-regulated miRNAs were highly nervous system function genes and pathways enriched. Fifteen differentially genes altered by RDX from mRNA profiles were the putative targets of regulated miRNAs. The induction of miR-71, miR-27ab, miR-98, and miR-135a expression by RDX, could reduce the expression of the genes POLE4, C5ORF13, SULF1 and ROCK2, and eventually induce neurotoxicity. Over-expression of miR-27ab, or reduction of the expression of unknown miRNAs by RDX, could up-regulate HMGCR expression and contribute to neurotoxicity. RDX regulated immune and inflammation response miRNAs and genes could contribute to RDX- induced neurotoxicity and other toxicities as well as animal defending reaction response to RDX exposure. Our results demonstrate that integrating miRNA and mRNA profiles is valuable to indentify novel biomarkers and molecular mechanisms for RDX-induced neurological disorder and neurotoxicity.

  19. Whole-genome sequencing reveals complex mechanisms of intrinsic resistance to BRAF inhibition.

    Science.gov (United States)

    Turajlic, S; Furney, S J; Stamp, G; Rana, S; Ricken, G; Oduko, Y; Saturno, G; Springer, C; Hayes, A; Gore, M; Larkin, J; Marais, R

    2014-05-01

    BRAF is mutated in ∼42% of human melanomas (COSMIC. http://www.sanger.ac.uk/genetics/CGP/cosmic/) and pharmacological BRAF inhibitors such as vemurafenib and dabrafenib achieve dramatic responses in patients whose tumours harbour BRAF(V600) mutations. Objective responses occur in ∼50% of patients and disease stabilisation in a further ∼30%, but ∼20% of patients present primary or innate resistance and do not respond. Here, we investigated the underlying cause of treatment failure in a patient with BRAF mutant melanoma who presented primary resistance. We carried out whole-genome sequencing and single nucleotide polymorphism (SNP) array analysis of five metastatic tumours from the patient. We validated mechanisms of resistance in a cell line derived from the patient's tumour. We observed that the majority of the single-nucleotide variants identified were shared across all tumour sites, but also saw site-specific copy-number alterations in discrete cell populations at different sites. We found that two ubiquitous mutations mediated resistance to BRAF inhibition in these tumours. A mutation in GNAQ sustained mitogen-activated protein kinase (MAPK) signalling, whereas a mutation in PTEN activated the PI3 K/AKT pathway. Inhibition of both pathways synergised to block the growth of the cells. Our analyses show that the five metastases arose from a common progenitor and acquired additional alterations after disease dissemination. We demonstrate that a distinct combination of mutations mediated primary resistance to BRAF inhibition in this patient. These mutations were present in all five tumours and in a tumour sample taken before BRAF inhibitor treatment was administered. Inhibition of both pathways was required to block tumour cell growth, suggesting that combined targeting of these pathways could have been a valid therapeutic approach for this patient.

  20. A channelopathy mechanism revealed by direct calmodulin activation of TrpV4.

    Science.gov (United States)

    Loukin, Stephen H; Teng, Jinfeng; Kung, Ching

    2015-07-28

    Ca(2+)-calmodulin (CaM) regulates varieties of ion channels, including Transient Receptor Potential vanilloid subtype 4 (TrpV4). It has previously been proposed that internal Ca(2+) increases TrpV4 activity through Ca(2+)-CaM binding to a C-terminal Ca(2+)-CaM binding domain (CBD). We confirmed this model by directly presenting Ca(2+)-CaM protein to membrane patches excised from TrpV4-expressing oocytes. Over 50 TRPV4 mutations are now known to cause heritable skeletal dysplasia (SD) and other diseases in human. We have previously examined 14 SD alleles and found them to all have gain-of-function effects, with the gain of constitutive open probability paralleling disease severity. Among the 14 SD alleles examined, E797K and P799L are located immediate upstream of the CBD. They not only have increase basal activity, but, unlike the wild-type or other SD-mutant channels examined, they were greatly reduced in their response to Ca(2+)-CaM. Deleting a 10-residue upstream peptide (Δ795-804) that covers the two SD mutant sites resulted in strong constitutive activity and the complete lack of Ca(2+)-CaM response. We propose that the region immediately upstream of CBD is an autoinhibitory domain that maintains the closed state through electrostatic interactions, and adjacent detachable Ca(2+)-CaM binding to CBD sterically interferes with this autoinhibition. This work further supports the notion that TrpV4 mutations cause SD by constitutive leakage. However, the closed conformation is likely destabilized by various mutations by different mechanisms, including the permanent removal of an autoinhibition documented here.

  1. Large-scale Models Reveal the Two-component Mechanics of Striated Muscle

    Directory of Open Access Journals (Sweden)

    Robert Jarosch

    2008-12-01

    Full Text Available This paper provides a comprehensive explanation of striated muscle mechanics and contraction on the basis of filament rotations. Helical proteins, particularly the coiled-coils of tropomyosin, myosin and α-actinin, shorten their H-bonds cooperatively and produce torque and filament rotations when the Coulombic net-charge repulsion of their highly charged side-chains is diminished by interaction with ions. The classical “two-component model” of active muscle differentiated a “contractile component” which stretches the “series elastic component” during force production. The contractile components are the helically shaped thin filaments of muscle that shorten the sarcomeres by clockwise drilling into the myosin cross-bridges with torque decrease (= force-deficit. Muscle stretch means drawing out the thin filament helices off the cross-bridges under passive counterclockwise rotation with torque increase (= stretch activation. Since each thin filament is anchored by four elastic α-actinin Z-filaments (provided with forceregulating sites for Ca2+ binding, the thin filament rotations change the torsional twist of the four Z-filaments as the “series elastic components”. Large scale models simulate the changes of structure and force in the Z-band by the different Z-filament twisting stages A, B, C, D, E, F and G. Stage D corresponds to the isometric state. The basic phenomena of muscle physiology, i. e. latency relaxation, Fenn-effect, the force-velocity relation, the length-tension relation, unexplained energy, shortening heat, the Huxley-Simmons phases, etc. are explained and interpreted with the help of the model experiments.

  2. Mechanical influences on morphogenesis of the knee joint revealed through morphological, molecular and computational analysis of immobilised embryos.

    Science.gov (United States)

    Roddy, Karen A; Prendergast, Patrick J; Murphy, Paula

    2011-02-28

    Very little is known about the regulation of morphogenesis in synovial joints. Mechanical forces generated from muscle contractions are required for normal development of several aspects of normal skeletogenesis. Here we show that biophysical stimuli generated by muscle contractions impact multiple events during chick knee joint morphogenesis influencing differential growth of the skeletal rudiment epiphyses and patterning of the emerging tissues in the joint interzone. Immobilisation of chick embryos was achieved through treatment with the neuromuscular blocking agent Decamethonium Bromide. The effects on development of the knee joint were examined using a combination of computational modelling to predict alterations in biophysical stimuli, detailed morphometric analysis of 3D digital representations, cell proliferation assays and in situ hybridisation to examine the expression of a selected panel of genes known to regulate joint development. This work revealed the precise changes to shape, particularly in the distal femur, that occur in an altered mechanical environment, corresponding to predicted changes in the spatial and dynamic patterns of mechanical stimuli and region specific changes in cell proliferation rates. In addition, we show altered patterning of the emerging tissues of the joint interzone with the loss of clearly defined and organised cell territories revealed by loss of characteristic interzone gene expression and abnormal expression of cartilage markers. This work shows that local dynamic patterns of biophysical stimuli generated from muscle contractions in the embryo act as a source of positional information guiding patterning and morphogenesis of the developing knee joint.

  3. The plasma membrane aquaporin NtAQP1 is a key component of the leaf unfolding mechanism in tobacco

    NARCIS (Netherlands)

    Siefritz, F.; Otto, B.; Beinert, G.P.; Krol, van der A.R.; Kaldenhoff, R.

    2004-01-01

    Epinastic leaf movement of tobacco is based on differential growth of the upper and lower leaf surface and is distinct from the motor organ-driven mechanism of nyctinastic leaf movement of, for example, mimosa species. The epinastic leaf movement of tobacco is observed not only under diurnal light r

  4. 基于生物特征认证的用户密钥保护机制%User Key Protection Mechanism Based on Biometrics Authentication

    Institute of Scientific and Technical Information of China (English)

    王志刚; 罗云锋

    2015-01-01

    As the cryptographic technology is used extensively in information security ,it’s an important problem to pro‐tect the user key in security .A solution for user key protection mechanism based on fingerprint authentication technology is proposed .The architecture of the user key protection system and the process of acquiring the user key are described .This scheme guarantees the security and usability of user key .%密码技术广泛应用于信息安全领域,如何安全地保存密钥都是需要解决的关键问题。论文提出了一种基于生物特征认证的用户密钥保护机制,描述了基于指纹认证的用户密钥保护系统组成及用户密钥获取流程。该方法保证了用户密钥的安全,同时也便于用户密钥的使用。

  5. Nitrogen as a key regulator of flowering in Fagus crenata: understanding the physiological mechanism of masting by gene expression analysis.

    Science.gov (United States)

    Miyazaki, Yuko; Maruyama, Yosuke; Chiba, Yukako; Kobayashi, Masaki J; Joseph, Benesh; Shimizu, Kentaro K; Mochida, Keiichi; Hiura, Tsutom; Kon, Hirokazu; Satake, Akiko

    2014-10-01

    The role of resource availability in determining the incidence of masting has been widely studied, but how floral transition and initiation are regulated by the resource level is unclear. We tested the hypothesis that floral transition is stimulated by high resource availabiltiy in Fagus crenata based on a new technique, the expression analyses of flowering genes. We isolated F. crenata orthologues of FLOWERING LOCUS T, LEAFY and APETALA1, and confirmed their functions using transgenic Arabidopsis thaliana. We monitored the gene expression levels for 5 years and detected a cycle of on and off years, which was correlated with fluctuations of the shoot-nitrogen concentration. Nitrogen fertilisation resulted in the significantly higher expression of flowering genes than the control, where all of the fertilised trees flowered, whereas the control did not. Our findings identified nitrogen as a key regulator of mast flowering, thereby providing new empirical evidence to support the resource budget model.

  6. Developmental trajectories of gene expression reveal candidates for diapause termination: a key life-history transition in the apple maggot fly Rhagoletis pomonella.

    Science.gov (United States)

    Ragland, Gregory J; Egan, Scott P; Feder, Jeffrey L; Berlocher, Stewart H; Hahn, Daniel A

    2011-12-01

    The timing of dormancy is a rapidly evolving life-history trait playing a crucial role in the synchronization of seasonal life cycles and adaptation to environmental change. But the physiological mechanisms regulating dormancy in animals remain poorly understood. In insects, dormancy (diapause) is a developmentally dynamic state, and the mechanisms that control diapause transitions affect seasonal timing. Here we used microarrays to examine patterns of gene expression during dormancy termination: a crucial life-history transition in the apple maggot fly Rhagoletis pomonella (Walsh). This species is a model system for host race formation and ecological speciation via changes in diapause regulation of seasonality. Our goal was to pinpoint the timing of the transition from diapause to post-diapause development and to identify candidate genes and pathways for regulation of diapause termination. Samples were taken at six metabolically defined developmental landmarks, and time-series analysis suggests that release from metabolic depression coincides with preparation for or resumption of active cell cycling and morphogenesis, defining the 'end' of diapause. However, marked changes in expression, including members of pathways such as Wnt and TOR signaling, also occur prior to the metabolic rate increase, electing these pathways as candidates for early regulation of diapause termination. We discuss these results with respect to generalities in insect diapause physiology and to our long-term goal of identifying mechanisms of diapause adaptation in the Rhagoletis system.

  7. OCTOPUS-LIKE 2, a novel player in Arabidopsis root and vascular development, reveals a key role for OCTOPUS family genes in root metaphloem sieve tube differentiation.

    Science.gov (United States)

    Ruiz Sola, M Aguila; Coiro, Mario; Crivelli, Simona; Zeeman, Samuel C; Schmidt Kjølner Hansen, Signe; Truernit, Elisabeth

    2017-09-06

    Protophloem and metaphloem sieve tubes are essential for transporting carbohydrates and signalling molecules towards sink tissues. OCTOPUS (OPS) was previously identified as an important regulator of protophloem differentiation in Arabidopsis roots. Here, we investigated the role of OCTOPUS-LIKE 2 (OPL2), a gene homologous to OPS. OPL2 expression patterns were analysed, and functional equivalence of OPS and OPL2 was tested. Mutant and double mutant phenotypes were investigated. OPS and OPL2 displayed overlapping expression patterns and a high degree of functional overlap. A mutation in OPL2 revealed redundant functions of OPS and OPL2 in developmental processes in which OPS was known to play a role, notably cotyledon vascular patterning and protophloem development. Moreover, we also uncovered redundant roles for OPS and OPL2 in leaf vascular patterning and, most interestingly, metaphloem sieve tube differentiation. Our results reveal a novel OPS-like protein that, together with OPS, is an important regulator of vascular patterning, root growth and phloem development. OPS and OPL2 are the first genes identified that play a role in metaphloem sieve tube differentiation. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  8. RNA sequencing of Populus x canadensis roots identifies key molecular mechanisms underlying physiological adaption to excess zinc.

    Science.gov (United States)

    Ariani, Andrea; Di Baccio, Daniela; Romeo, Stefania; Lombardi, Lara; Andreucci, Andrea; Lux, Alexander; Horner, David Stephen; Sebastiani, Luca

    2015-01-01

    Populus x canadensis clone I-214 exhibits a general indicator phenotype in response to excess Zn, and a higher metal uptake in roots than in shoots with a reduced translocation to aerial parts under hydroponic conditions. This physiological adaptation seems mainly regulated by roots, although the molecular mechanisms that underlie these processes are still poorly understood. Here, differential expression analysis using RNA-sequencing technology was used to identify the molecular mechanisms involved in the response to excess Zn in root. In order to maximize specificity of detection of differentially expressed (DE) genes, we consider the intersection of genes identified by three distinct statistical approaches (61 up- and 19 down-regulated) and validate them by RT-qPCR, yielding an agreement of 93% between the two experimental techniques. Gene Ontology (GO) terms related to oxidation-reduction processes, transport and cellular iron ion homeostasis were enriched among DE genes, highlighting the importance of metal homeostasis in adaptation to excess Zn by P. x canadensis clone I-214. We identified the up-regulation of two Populus metal transporters (ZIP2 and NRAMP1) probably involved in metal uptake, and the down-regulation of a NAS4 gene involved in metal translocation. We identified also four Fe-homeostasis transcription factors (two bHLH38 genes, FIT and BTS) that were differentially expressed, probably for reducing Zn-induced Fe-deficiency. In particular, we suggest that the down-regulation of FIT transcription factor could be a mechanism to cope with Zn-induced Fe-deficiency in Populus. These results provide insight into the molecular mechanisms involved in adaption to excess Zn in Populus spp., but could also constitute a starting point for the identification and characterization of molecular markers or biotechnological targets for possible improvement of phytoremediation performances of poplar trees.

  9. RNA sequencing of Populus x canadensis roots identifies key molecular mechanisms underlying physiological adaption to excess zinc.

    Directory of Open Access Journals (Sweden)

    Andrea Ariani

    Full Text Available Populus x canadensis clone I-214 exhibits a general indicator phenotype in response to excess Zn, and a higher metal uptake in roots than in shoots with a reduced translocation to aerial parts under hydroponic conditions. This physiological adaptation seems mainly regulated by roots, although the molecular mechanisms that underlie these processes are still poorly understood. Here, differential expression analysis using RNA-sequencing technology was used to identify the molecular mechanisms involved in the response to excess Zn in root. In order to maximize specificity of detection of differentially expressed (DE genes, we consider the intersection of genes identified by three distinct statistical approaches (61 up- and 19 down-regulated and validate them by RT-qPCR, yielding an agreement of 93% between the two experimental techniques. Gene Ontology (GO terms related to oxidation-reduction processes, transport and cellular iron ion homeostasis were enriched among DE genes, highlighting the importance of metal homeostasis in adaptation to excess Zn by P. x canadensis clone I-214. We identified the up-regulation of two Populus metal transporters (ZIP2 and NRAMP1 probably involved in metal uptake, and the down-regulation of a NAS4 gene involved in metal translocation. We identified also four Fe-homeostasis transcription factors (two bHLH38 genes, FIT and BTS that were differentially expressed, probably for reducing Zn-induced Fe-deficiency. In particular, we suggest that the down-regulation of FIT transcription factor could be a mechanism to cope with Zn-induced Fe-deficiency in Populus. These results provide insight into the molecular mechanisms involved in adaption to excess Zn in Populus spp., but could also constitute a starting point for the identification and characterization of molecular markers or biotechnological targets for possible improvement of phytoremediation performances of poplar trees.

  10. Two Antagonistic MALT1 Auto-Cleavage Mechanisms Reveal a Role for TRAF6 to Unleash MALT1 Activation

    OpenAIRE

    Ginster, Stefanie; Bardet, Maureen; Unterreiner, Adeline; Malinverni, Claire; Renner, Florian; Lam, Stephen; Freuler, Felix; Gerrits, Bertran; Voshol, Johannes; Calzascia, Thomas; Régnier, Catherine H.; Renatus, Martin; Nikolay, Rainer; Israël, Laura; Bornancin, Frédéric

    2017-01-01

    The paracaspase MALT1 has arginine-directed proteolytic activity triggered by engagement of immune receptors. Recruitment of MALT1 into activation complexes is required for MALT1 proteolytic function. Here, co-expression of MALT1 in HEK293 cells, either with activated CARD11 and BCL10 or with TRAF6, was used to explore the mechanism of MALT1 activation at the molecular level. This work identified a prominent self-cleavage site of MALT1 isoform A (MALT1A) at R781 (R770 in MALT1B) and revealed ...

  11. Formal Definitions of Unbounded Evolution and Innovation Reveal Universal Mechanisms for Open-Ended Evolution in Dynamical Systems.

    Science.gov (United States)

    Adams, Alyssa; Zenil, Hector; Davies, Paul C W; Walker, Sara Imari

    2017-04-20

    Open-ended evolution (OEE) is relevant to a variety of biological, artificial and technological systems, but has been challenging to reproduce in silico. Most theoretical efforts focus on key aspects of open-ended evolution as it appears in biology. We recast the problem as a more general one in dynamical systems theory, providing simple criteria for open-ended evolution based on two hallmark features: unbounded evolution and innovation. We define unbounded evolution as patterns that are non-repeating within the expected Poincare recurrence time of an isolated system, and innovation as trajectories not observed in isolated systems. As a case study, we implement novel variants of cellular automata (CA) where the update rules are allowed to vary with time in three alternative ways. Each is capable of generating conditions for open-ended evolution, but vary in their ability to do so. We find that state-dependent dynamics, regarded as a hallmark of life, statistically out-performs other candidate mechanisms, and is the only mechanism to produce open-ended evolution in a scalable manner, essential to the notion of ongoing evolution. This analysis suggests a new framework for unifying mechanisms for generating OEE with features distinctive to life and its artifacts, with broad applicability to biological and artificial systems.

  12. Plasma metabolomic profiling to reveal antipyretic mechanism of Shuang-huang-lian injection on yeast-induced pyrexia rats.

    Directory of Open Access Journals (Sweden)

    Xiaoyan Gao

    Full Text Available Shuang-huang-lian injection (SHLI is a famous Chinese patent medicine, which has been wildly used in clinic for the treatment of acute respiratory tract infection, pneumonia, influenza, etc. The existing randomized controlled trial (RCT studies suggested that SHLI could afford a certain anti-febrile action. However, seldom does research concern the pharmacological mechanisms of SHLI. In the current study, we explored plasma metabolomic profiling technique and selected potential metabolic markers to reveal the antipyretic mechanism of SHLI on yeast-induced pyrexia rat model using UPLC-Q-TOF/MS coupled with multivariate statistical analysis and pattern recognition techniques. We discovered a significant perturbance of metabolic profile in the plasma of fever rats and obvious reversion in SHLI-administered rats. Eight potential biomarkers, i.e. 1 3-hydeoxybutyric acid, 2 leucine, 3 16:0 LPC, 4 allocholic acid, 5 vitamin B2, 6 Cys-Lys-His, 7 18:2 LPC, and 8 3-hydroxychola-7, 22-dien-24-oic acid, were screened out by OPLS-DA approach. Five potential perturbed metabolic pathways, i.e. 1 valine, leucine, and isoleucine biosynthesis, 2 glycerophospholipid metabolism, 3 ketone bodies synthesis and degradation, 4 bile acid biosynthesis, and 5 riboflavin metabolism, were revealed to relate to the antipyretic mechanisms of SHLI. Overall, we investigated antipyretic mechanisms of SHLI at metabolomic level for the first time, and the obtained results highlights the necessity of adopting metabolomics as a reliable tool for understanding the holism and synergism of Chinese patent drug.

  13. In Vivo Loss of Function Screening Reveals Carbonic Anhydrase IX as a Key Modulator of Tumor Initiating Potential in Primary Pancreatic Tumors

    Directory of Open Access Journals (Sweden)

    Nabendu Pore

    2015-06-01

    Full Text Available Reprogramming of energy metabolism is one of the emerging hallmarks of cancer. Up-regulation of energy metabolism pathways fuels cell growth and division, a key characteristic of neoplastic disease, and can lead to dependency on specific metabolic pathways. Thus, targeting energy metabolism pathways might offer the opportunity for novel therapeutics. Here, we describe the application of a novel in vivo screening approach for the identification of genes involved in cancer metabolism using a patient-derived pancreatic xenograft model. Lentiviruses expressing short hairpin RNAs (shRNAs targeting 12 different cell surface protein transporters were separately transduced into the primary pancreatic tumor cells. Transduced cells were pooled and implanted into mice. Tumors were harvested at different times, and the frequency of each shRNA was determined as a measure of which ones prevented tumor growth. Several targets including carbonic anhydrase IX (CAIX, monocarboxylate transporter 4, and anionic amino acid transporter light chain, xc- system (xCT were identified in these studies and shown to be required for tumor initiation and growth. Interestingly, CAIX was overexpressed in the tumor initiating cell population. CAIX expression alone correlated with a highly tumorigenic subpopulation of cells. Furthermore, CAIX expression was essential for tumor initiation because shRNA knockdown eliminated the ability of cells to grow in vivo. To the best of our knowledge, this is the first parallel in vivo assessment of multiple novel oncology target genes using a patient-derived pancreatic tumor model.

  14. Analysis of natural variants of the hepatitis C virus internal ribosome entry site reveals that primary sequence plays a key role in cap-independent translation

    Science.gov (United States)

    Barría, María Inés; González, Angel; Vera-Otarola, Jorge; León, Ursula; Vollrath, Valeska; Marsac, Delphine; Monasterio, Octavio; Pérez-Acle, Tomás; Soza, Alejandro; López-Lastra, Marcelo

    2009-01-01

    The HCV internal ribosome entry site (IRES) spans a region of ∼340 nt that encompasses most of the 5′ untranslated region (5′UTR) of the viral mRNA and the first 24–40 nt of the core-coding region. To investigate the implication of altering the primary sequence of the 5′UTR on IRES activity, naturally occurring variants of the 5′UTR were isolated from clinical samples and analyzed. The impact of the identified mutations on translation was evaluated in the context of RLuc/FLuc bicistronic RNAs. Results show that depending on their location within the RNA structure, these naturally occurring mutations cause a range of effects on IRES activity. However, mutations within subdomain IIId hinder HCV IRES-mediated translation. In an attempt to explain these data, the dynamic behavior of the subdomain IIId was analyzed by means of molecular dynamics (MD) simulations. Despite the loss of function, MD simulations predicted that mutant G266A/G268U possesses a structure similar to the wt-RNA. This prediction was validated by analyzing the secondary structure of the isolated IIId RNAs by circular dichroism spectroscopy in the presence or absence of Mg2+ ions. These data strongly suggest that the primary sequence of subdomain IIId plays a key role in HCV IRES-mediated translation. PMID:19106142

  15. Structure of Rhodococcus equi virulence-associated protein B (VapB) reveals an eight-stranded antiparallel β-barrel consisting of two Greek-key motifs.

    Science.gov (United States)

    Geerds, Christina; Wohlmann, Jens; Haas, Albert; Niemann, Hartmut H

    2014-07-01

    Members of the virulence-associated protein (Vap) family from the pathogen Rhodococcus equi regulate virulence in an unknown manner. They do not share recognizable sequence homology with any protein of known structure. VapB and VapA are normally associated with isolates from pigs and horses, respectively. To contribute to a molecular understanding of Vap function, the crystal structure of a protease-resistant VapB fragment was determined at 1.4 Å resolution. The structure was solved by SAD phasing employing the anomalous signal of one endogenous S atom and two bound Co ions with low occupancy. VapB is an eight-stranded antiparallel β-barrel with a single helix. Structural similarity to avidins suggests a potential binding function. Unlike other eight- or ten-stranded β-barrels found in avidins, bacterial outer membrane proteins, fatty-acid-binding proteins and lysozyme inhibitors, Vaps do not have a next-neighbour arrangement but consist of two Greek-key motifs with strand order 41238567, suggesting an unusual or even unique topology.

  16. Structure of Yeast OSBP-Related Protein Osh1 Reveals Key Determinants for Lipid Transport and Protein Targeting at the Nucleus-Vacuole Junction.

    Science.gov (United States)

    Manik, Mohammad Kawsar; Yang, Huiseon; Tong, Junsen; Im, Young Jun

    2017-03-10

    Yeast Osh1 belongs to the oxysterol-binding protein (OSBP) family of proteins and contains multiple targeting modules optimized for lipid transport at the nucleus-vacuole junction (NVJ). The key determinants for NVJ targeting and the role of Osh1 at NVJs have remained elusive because of unknown lipid specificities. In this study, we determined the structures of the ankyrin repeat domain (ANK), and OSBP-related domain (ORD) of Osh1, in complex with Nvj1 and ergosterol, respectively. The Osh1 ANK forms a unique bi-lobed structure that recognizes a cytosolic helical segment of Nvj1. We discovered that Osh1 ORD binds ergosterol and phosphatidylinositol 4-phosphate PI(4)P in a competitive manner, suggesting counter-transport function of the two lipids. Ergosterol is bound to the hydrophobic pocket in a head-down orientation, and the structure of the PI(4)P-binding site in Osh1 is well conserved. Our results suggest that Osh1 performs non-vesicular transport of ergosterol and PI(4)P at the NVJ.

  17. The mechanism of heterogeneous beta-lactam resistance in MRSA: key role of the stringent stress response.

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    Choonkeun Kim

    Full Text Available All methicillin resistant S. aureus (MRSA strains carry an acquired genetic determinant--mecA or mecC--which encode for a low affinity penicillin binding protein -PBP2A or PBP2A'--that can continue the catalysis of peptidoglycan transpeptidation in the presence of high concentrations of beta-lactam antibiotics which would inhibit the native PBPs normally involved with the synthesis of staphylococcal cell wall peptidoglycan. In contrast to this common genetic and biochemical mechanism carried by all MRSA strains, the level of beta-lactam antibiotic resistance shows a very wide strain to strain variation, the mechanism of which has remained poorly understood. The overwhelming majority of MRSA strains produce a unique--heterogeneous--phenotype in which the great majority of the bacteria exhibit very poor resistance often close to the MIC value of susceptible S. aureus strains. However, cultures of such heterogeneously resistant MRSA strains also contain subpopulations of bacteria with extremely high beta-lactam MIC values and the resistance level and frequency of the highly resistant cells in such strain is a characteristic of the particular MRSA clone. In the study described in this communication, we used a variety of experimental models to understand the mechanism of heterogeneous beta-lactam resistance. Methicillin-susceptible S. aureus (MSSA that received the mecA determinant in the laboratory either on a plasmid or in the form of a chromosomal SCCmec cassette, generated heterogeneously resistant cultures and the highly resistant subpopulations that emerged in these models had increased levels of PBP2A and were composed of bacteria in which the stringent stress response was induced. Each of the major heterogeneously resistant clones of MRSA clinical isolates could be converted to express high level and homogeneous resistance if the growth medium contained an inducer of the stringent stress response.

  18. Technique Key Points of Deep Tillage Land Preparation Mechanization Operation%深松整地机械化作业技术要领

    Institute of Scientific and Technical Information of China (English)

    吕斌

    2015-01-01

    The technique of deep tillage land preparation mechanization is expanded each year, and is becoming one of main agricultural mechanization techniques in Liaoning province and even nationwide. In the article, it introduced implementing effect and operation mode of deep tillage land preparation mechanization technique, set up implementation key points and using points, in order to provide technical guidance for applying deep tillage technique for farmers.%深松整地机械化技术的推广应用面积逐年扩大,成为辽宁乃至全国范围内主推的农机化技术之一. 介绍深松整地机械化技术的实施效果和作业方式,制定实施要点和使用要领,以期为农户科学使用深松作业技术提供指导.

  19. Mn²⁺-deficiency reveals a key role for the Pleurotus ostreatus versatile peroxidase (VP4) in oxidation of aromatic compounds.

    Science.gov (United States)

    Knop, Doriv; Ben-Ari, Julius; Salame, Tomer M; Levinson, Dana; Yarden, Oded; Hadar, Yitzhak

    2014-08-01

    The manganese peroxidase gene family (mnps) is a part of the ligninolytic system of Pleurotus ostreatus. This gene family is comprised of nine members, mnp1-9, encoding short manganese peroxidases (short-MnPs) or versatile peroxidases (VPs). We show that unlike in Mn(2+)-amended glucose-peptone (GP) medium, where redundancy among mnps was reported, in Mn(2+)-deficient GP medium mnp4 [encoding versatile peroxidase isoenzyme 4 (VP4)] has a key and nonredundant function. The abundance of mnps transcripts at time points corresponding to the tropophase (active growth), early idiophase, and idiophase indicates that mnp4 is the predominantly expressed mnp gene and that its relative predominance is dependent on the age of the culture. In this medium, azo dye, Orange II (OII) decolorization occurs only during the idiophase and a Δmnp4 strain showed a drastic reduction in this decolorization. Three degradation metabolites were identified by liquid chromatography-mass spectroscopy (LC-MS), indicating both asymmetric and symmetric enzymatic cleavage of the azo-bond. In addition, the culture filtrate of Δmnp4 showed negligible values of oxidation capability of four typical VP substrates: Mn(2+), 2,6-dimethoxyphenol, phenol red, and Reactive Black 5 (RB5), compared to the wild-type strain PC9. We concluded that under Mn(2+)-deficient GP culture, VP4 (encoded by mnp4) is the main active ligninolytic enzyme able to oxidize Mn(2+) as well as high and low redox potential aromatic substrate, including dyes. Furthermore, other VPs/MnPs do not compensate for the lack of VP4 activity.

  20. TRAIL-Based High Throughput Screening Reveals a Link between TRAIL-Mediated Apoptosis and Glutathione Reductase, a Key Component of Oxidative Stress Response.

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    Dmitri Rozanov

    Full Text Available A high throughput screen for compounds that induce TRAIL-mediated apoptosis identified ML100 as an active chemical probe, which potentiated TRAIL activity in prostate carcinoma PPC-1 and melanoma MDA-MB-435 cells. Follow-up in silico modeling and profiling in cell-based assays allowed us to identify NSC130362, pharmacophore analog of ML100 that induced 65-95% cytotoxicity in cancer cells and did not affect the viability of human primary hepatocytes. In agreement with the activation of the apoptotic pathway, both ML100 and NSC130362 synergistically with TRAIL induced caspase-3/7 activity in MDA-MB-435 cells. Subsequent affinity chromatography and inhibition studies convincingly demonstrated that glutathione reductase (GSR, a key component of the oxidative stress response, is a target of NSC130362. In accordance with the role of GSR in the TRAIL pathway, GSR gene silencing potentiated TRAIL activity in MDA-MB-435 cells but not in human hepatocytes. Inhibition of GSR activity resulted in the induction of oxidative stress, as was evidenced by an increase in intracellular reactive oxygen species (ROS and peroxidation of mitochondrial membrane after NSC130362 treatment in MDA-MB-435 cells but not in human hepatocytes. The antioxidant reduced glutathione (GSH fully protected MDA-MB-435 cells from cell lysis induced by NSC130362 and TRAIL, thereby further confirming the interplay between GSR and TRAIL. As a consequence of activation of oxidative stress, combined treatment of different oxidative stress inducers and NSC130362 promoted cell death in a variety of cancer cells but not in hepatocytes in cell-based assays and in in vivo, in a mouse tumor xenograft model.

  1. Structure of transmembrane domain of lysosome-associated membrane protein type 2a (LAMP-2A) reveals key features for substrate specificity in chaperone-mediated autophagy.

    Science.gov (United States)

    Rout, Ashok K; Strub, Marie-Paule; Piszczek, Grzegorz; Tjandra, Nico

    2014-12-19

    Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation.

  2. Functional Characterization of the GATA Transcription Factors GNC and CGA1 Reveals Their Key Role in Chloroplast Development, Growth, and Division in Arabidopsis1[W][OA

    Science.gov (United States)

    Chiang, Yi-Hsuan; Zubo, Yan O.; Tapken, Wiebke; Kim, Hyo Jung; Lavanway, Ann M.; Howard, Louisa; Pilon, Marinus; Kieber, Joseph J.; Schaller, G. Eric

    2012-01-01

    Chloroplasts develop from proplastids in a process that requires the interplay of nuclear and chloroplast genomes, but key steps in this developmental process have yet to be elucidated. Here, we show that the nucleus-localized transcription factors GATA NITRATE-INDUCIBLE CARBON-METABOLISM-INVOLVED (GNC) and CYTOKININ-RESPONSIVE GATA1 (CGA1) regulate chloroplast development, growth, and division in Arabidopsis (Arabidopsis thaliana). GNC and CGA1 are highly expressed in green tissues, and the phytohormone cytokinin regulates their expression. A gnc cga1 mutant exhibits a reduction in overall chlorophyll levels as well as in chloroplast size in the hypocotyl. Ectopic overexpression of either GNC or CGA1 promotes chloroplast biogenesis in hypocotyl cortex and root pericycle cells, based on increases in the number and size of the chloroplasts, and also results in expanded zones of chloroplast production into the epidermis of hypocotyls and cotyledons and into the cortex of roots. Ectopic overexpression also promotes the development of etioplasts from proplastids in dark-grown seedlings, subsequently enhancing the deetiolation process. Inducible expression of GNC demonstrates that GNC-mediated chloroplast biogenesis can be regulated postembryonically, notably so for chloroplast production in cotyledon epidermal cells. Analysis of the gnc cga1 loss-of-function and overexpression lines supports a role for these transcription factors in regulating the effects of cytokinin on chloroplast division. These data support a model in which GNC and CGA1 serve as two of the master transcriptional regulators of chloroplast biogenesis, acting downstream of cytokinin and mediating the development of chloroplasts from proplastids and enhancing chloroplast growth and division in specific tissues. PMID:22811435

  3. Structure, mechanics, and binding mode heterogeneity of LEDGF/p75-DNA nucleoprotein complexes revealed by scanning force microscopy

    Science.gov (United States)

    Vanderlinden, Willem; Lipfert, Jan; Demeulemeester, Jonas; Debyser, Zeger; de Feyter, Steven

    2014-04-01

    LEDGF/p75 is a transcriptional coactivator implicated in the pathogenesis of AIDS and leukemia. In these contexts, LEDGF/p75 acts as a cofactor by tethering protein cargo to transcriptionally active regions in the human genome. Our study - based on scanning force microscopy (SFM) imaging - is the first to provide structural information on the interaction of LEDGF/p75 with DNA. Two novel approaches that allow obtaining insights into the DNA conformation inside nucleoprotein complexes revealed (1) that LEDGF/p75 can bind at least in three different binding modes, (2) how DNA topology and protein dimerization affect these binding modes, and (3) geometrical and mechanical aspects of the nucleoprotein complexes. These structural and mechanical details will help us to better understand the cellular mechanisms of LEDGF/p75 as a transcriptional coactivator and as a cofactor in disease.LEDGF/p75 is a transcriptional coactivator implicated in the pathogenesis of AIDS and leukemia. In these contexts, LEDGF/p75 acts as a cofactor by tethering protein cargo to transcriptionally active regions in the human genome. Our study - based on scanning force microscopy (SFM) imaging - is the first to provide structural information on the interaction of LEDGF/p75 with DNA. Two novel approaches that allow obtaining insights into the DNA conformation inside nucleoprotein complexes revealed (1) that LEDGF/p75 can bind at least in three different binding modes, (2) how DNA topology and protein dimerization affect these binding modes, and (3) geometrical and mechanical aspects of the nucleoprotein complexes. These structural and mechanical details will help us to better understand the cellular mechanisms of LEDGF/p75 as a transcriptional coactivator and as a cofactor in disease. Electronic supplementary information (ESI) available: SFM topographs of phage lambda DNA in situ, in the absence and presence of LEDGF/p75; model-independent tests for DNA chain equilibration in 2D; SFM topographs of

  4. High-Throughput siRNA Screening to Reveal GATA-2 Upstream Transcriptional Mechanisms in Hematopoietic Cells.

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    Yo Saito

    Full Text Available Hematopoietic stem cells can self-renew and differentiate into all blood cell types. The transcription factor GATA-2 is expressed in both hematopoietic stem and progenitor cells and is essential for cell proliferation, survival, and differentiation. Recently, evidence from studies of aplastic anemia, MonoMAC syndrome, and lung cancer has demonstrated a mechanistic link between GATA-2 and human pathophysiology. GATA-2-dependent disease processes have been extensively analyzed; however, the transcriptional mechanisms upstream of GATA-2 remain less understood. Here, we conducted high-throughput small-interfering-RNA (siRNA library screening and showed that YN-1, a human erythroleukemia cell line, expressed high levels of GATA-2 following the activation of the hematopoietic-specific 1S promoter. As transient luciferase reporter assay in YN-1 cells revealed the highest promoter activity in the 1S promoter fused with GATA-2 intronic enhancer (+9.9 kb/1S; therefore, we established a cell line capable of stably expressing +9.9 kb/1S-Luciferase. Subsequently, we screened 995 transcription factor genes and revealed that CITED2 acts as a GATA-2 activator in human hematopoietic cells. These results provide novel insights into and further identify the regulatory mechanism of GATA-2.

  5. Multiscale models of skeletal muscle reveal the complex effects of muscular dystrophy on tissue mechanics and damage susceptibility.

    Science.gov (United States)

    Virgilio, Kelley M; Martin, Kyle S; Peirce, Shayn M; Blemker, Silvia S

    2015-04-06

    Computational models have been increasingly used to study the tissue-level constitutive properties of muscle microstructure; however, these models were not created to study or incorporate the influence of disease-associated modifications in muscle. The purpose of this paper was to develop a novel multiscale muscle modelling framework to elucidate the relationship between microstructural disease adaptations and modifications in both mechanical properties of muscle and strain in the cell membrane. We used an agent-based model to randomly generate new muscle fibre geometries and mapped them into a finite-element model representing a cross section of a muscle fascicle. The framework enabled us to explore variability in the shape and arrangement of fibres, as well as to incorporate disease-related changes. We applied this method to reveal the trade-offs between mechanical properties and damage susceptibility in Duchenne muscular dystrophy (DMD). DMD is a fatal genetic disease caused by a lack of the transmembrane protein dystrophin, leading to muscle wasting and death due to cardiac or pulmonary complications. The most prevalent microstructural variations in DMD include: lack of transmembrane proteins, fibrosis, fatty infiltration and variation in fibre cross-sectional area. A parameter analysis of these variations and case study of DMD revealed that the nature of fibrosis and density of transmembrane proteins strongly affected the stiffness of the muscle and susceptibility to membrane damage.

  6. Crystal Structures and Inhibition Kinetics Reveal a Two-Stage Catalytic Mechanism with Drug Design Implications for Rhomboid Proteolysis.

    Science.gov (United States)

    Cho, Sangwoo; Dickey, Seth W; Urban, Siniša

    2016-02-04

    Intramembrane proteases signal by releasing proteins from the membrane, but despite their importance, their enzymatic mechanisms remain obscure. We probed rhomboid proteases with reversible, mechanism-based inhibitors that allow precise kinetic analysis and faithfully mimic the transition state structurally. Unexpectedly, inhibition by peptide aldehydes is non-competitive, revealing that in the Michaelis complex, substrate does not contact the catalytic center. Structural analysis in a membrane revealed that all extracellular loops of rhomboid make stabilizing interactions with substrate, but mainly through backbone interactions, explaining rhomboid's broad sequence selectivity. At the catalytic site, the tetrahedral intermediate lies covalently attached to the catalytic serine alone, with the oxyanion stabilized by unusual tripartite interactions with the side chains of H150, N154, and the backbone of S201. We also visualized unexpected substrate-enzyme interactions at the non-essential P2/P3 residues. These "extra" interactions foster potent rhomboid inhibition in living cells, thereby opening avenues for rational design of selective rhomboid inhibitors. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Transcriptomic analysis reveals the molecular mechanisms of drought-stress-induced decreases in Camellia sinensis leaf quality

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    Weidong eWang

    2016-03-01

    Full Text Available The tea plant [Camellia sinensis (L. O. Kuntze] is an important commercial crop rich in bioactive ingredients, especially catechins, caffeine, theanine and other free amino acids, which the quality of tea leaves depends on. Drought is the most important environmental stress affecting the yield and quality of this plant. In this study, the effects of drought stress on the phenotype, physiological characteristics and major bioactive ingredients accumulation of C. sinensis leaves were examined, and the results indicated that drought stress resulted in dehydration and wilt of the leaves, and significant decrease in the total polyphenols and free amino acids and increase in the total flavonoids. In addition, HPLC analysis showed that the catechins, caffeine, theanine and some free amino acids in C. sinensis leaves were significantly reduced in response to drought stress, implying that drought stress severely decreased the quality of C. sinensis leaves. Furthermore, differentially expressed genes (DEGs related to amino acid metabolism and secondary metabolism were identified and quantified in C. sinensis leaves under drought stress using high-throughput Illumina RNA-Seq technology, especially the key regulatory genes of the catechins, caffeine and theanine biosynthesis pathways. The expression levels of key regulatory genes were consistent with the results from the HPLC analysis, which indicate a potential molecular mechanism for the above results. Taken together, these data provide further insights into the mechanisms underlying the change in the quality of C. sinensis leaves under environmental stress, which involve changes in the accumulation of major bioactive ingredients, especially catechins, caffeine, theanine and other free amino acids.

  8. Genetic manipulation of a transcription-regulating sequence of porcine reproductive and respiratory syndrome virus reveals key nucleotides determining its activity.

    Science.gov (United States)

    Zheng, Haihong; Zhang, Keyu; Zhu, Xing-Quan; Liu, Changlong; Lu, Jiaqi; Gao, Fei; Zhou, Yan; Zheng, Hao; Lin, Tao; Li, Liwei; Tong, Guangzhi; Wei, Zuzhang; Yuan, Shishan

    2014-08-01

    The factors that determine the transcription-regulating sequence (TRS) activity of porcine reproductive and respiratory syndrome virus (PRRSV) remain largely unclear. In this study, the effect of mutagenesis of conserved C nucleotides at positions 5 and 6 in the leader TRS (TRS-L) and/or canonical body TRS7 (TRS-B7) on the synthesis of subgenomic (sg) mRNA and virus infectivity was investigated in the context of a type 2 PRRSV infectious cDNA clone. The results showed that a double C mutation in the leader TRS completely abolished sg mRNAs synthesis and virus infectivity, but a single C mutation did not. A single C or double C mutation in TRS-B7.1 or/and TRS-B7.2 impaired or abolished the corresponding sg mRNA synthesis. Introduction of identical mutations in the leader and body TRSs partially restored sg mRNA7.1 and/or sg mRNA7.2 transcription, indicating that the base-pairing interaction between sense TRS-L and cTRS-B is a crucial factor influencing sg mRNA synthesis. Analysis of the mRNA leader-body junctions of mutants provided evidence for a mechanism of discontinuous minus-strand transcription. This study also showed that mutational inactivation of TRS-B7.1 or TRS-B7.2 did not affect the production of infectious progeny virus, and the sg mRNA formed from each of them could express N protein. However, TRS-B7.1 plays more important roles than TRS-B7.2 in maintaining the growth characteristic of type 2 PRRSV. These results provide more insight into the molecular mechanism of genome expression and subgenomic mRNA transcription of PRRSV.

  9. "Fluctuograms" reveal the intermittent intra-protein communication in subtilisin Carlsberg and correlate mechanical coupling with co-evolution.

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    Jordi Silvestre-Ryan

    2011-03-01

    Full Text Available The mechanism of intra-protein communication and allosteric coupling is key to understanding the structure-property relationship of protein function. For subtilisin Carlsberg, the Ca²+-binding loop is distal to substrate-binding and active sites, yet the serine protease function depends on Ca²+ binding. The atomic molecular dynamics (MD simulations of apo and Ca²+-bound subtilisin show similar structures and there is no direct evidence that subtilisin has alternative conformations. To model the intra-protein communication due to Ca²+ binding, we transform the sequential segments of an atomic MD trajectory into separate elastic network models to represent anharmonicity and nonlinearity effectively as the temporal and spatial variation of the mechanical coupling network. In analogy to the spectrogram of sound waves, this transformation is termed the "fluctuogram" of protein dynamics. We illustrate that the Ca²+-bound and apo states of subtilisin have different fluctuograms and that intra-protein communication proceeds intermittently both in space and in time. We found that residues with large mechanical coupling variation due to Ca²+ binding correlate with the reported mutation sites selected by directed evolution for improving the stability of subtilisin and its activity in a non-aqueous environment. Furthermore, we utilize the fluctuograms calculated from MD to capture the highly correlated residues in a multiple sequence alignment. We show that in addition to the magnitude, the variance of coupling strength is also an indicative property for the sequence correlation observed in a statistical coupling analysis. The results of this work illustrate that the mechanical coupling networks calculated from atomic details can be used to correlate with functionally important mutation sites and co-evolution.

  10. Albino T-DNA tomato mutant reveals a key function of 1-deoxy-D-xylulose-5-phosphate synthase (DXS1) in plant development and survival

    Science.gov (United States)

    García-Alcázar, Manuel; Giménez, Estela; Pineda, Benito; Capel, Carmen; García-Sogo, Begoña; Sánchez, Sibilla; Yuste-Lisbona, Fernando J.; Angosto, Trinidad; Capel, Juan; Moreno, Vicente; Lozano, Rafael

    2017-01-01

    Photosynthetic activity is indispensable for plant growth and survival and it depends on the synthesis of plastidial isoprenoids as chlorophylls and carotenoids. In the non-mevalonate pathway (MEP), the 1-deoxy-D-xylulose-5-phosphate synthase 1 (DXS1) enzyme has been postulated to catalyze the rate-limiting step in the formation of plastidial isoprenoids. In tomato, the function of DXS1 has only been studied in fruits, and hence its functional relevance during plant development remains unknown. Here we report the characterization of the wls-2297 tomato mutant, whose severe deficiency in chlorophylls and carotenoids promotes an albino phenotype. Additionally, growth of mutant seedlings was arrested without developing vegetative organs, which resulted in premature lethality. Gene cloning and silencing experiments revealed that the phenotype of wls-2297 mutant was caused by 38.6 kb-deletion promoted by a single T-DNA insertion affecting the DXS1 gene. This was corroborated by in vivo and molecular complementation assays, which allowed the rescue of mutant phenotype. Further characterization of tomato plants overexpressing DXS1 and comparative expression analysis indicate that DXS1 may play other important roles besides to that proposed during fruit carotenoid biosynthesis. Taken together, these results demonstrate that DXS1 is essentially required for the development and survival of tomato plants. PMID:28350010

  11. The structural pathway of interleukin 1 (IL-1 initiated signaling reveals mechanisms of oncogenic mutations and SNPs in inflammation and cancer.

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    Saliha Ece Acuner Ozbabacan

    2014-02-01

    Full Text Available Interleukin-1 (IL-1 is a large cytokine family closely related to innate immunity and inflammation. IL-1 proteins are key players in signaling pathways such as apoptosis, TLR, MAPK, NLR and NF-κB. The IL-1 pathway is also associated with cancer, and chronic inflammation increases the risk of tumor development via oncogenic mutations. Here we illustrate that the structures of interfaces between proteins in this pathway bearing the mutations may reveal how. Proteins are frequently regulated via their interactions, which can turn them ON or OFF. We show that oncogenic mutations are significantly at or adjoining interface regions, and can abolish (or enhance the protein-protein interaction, making the protein constitutively active (or inactive, if it is a repressor. We combine known structures of protein-protein complexes and those that we have predicted for the IL-1 pathway, and integrate them with literature information. In the reconstructed pathway there are 104 interactions between proteins whose three dimensional structures are experimentally identified; only 15 have experimentally-determined structures of the interacting complexes. By predicting the protein-protein complexes throughout the pathway via the PRISM algorithm, the structural coverage increases from 15% to 71%. In silico mutagenesis and comparison of the predicted binding energies reveal the mechanisms of how oncogenic and single nucleotide polymorphism (SNP mutations can abrogate the interactions or increase the binding affinity of the mutant to the native partner. Computational mapping of mutations on the interface of the predicted complexes may constitute a powerful strategy to explain the mechanisms of activation/inhibition. It can also help explain how an oncogenic mutation or SNP works.

  12. Celastrol's Anticancer Mechanism Revealed

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    @@ The Thunder of God Vine (Tripterygium wilfordii Hook F.), or Lei Gong Teng in Chinese, is a plant that has been used for ages by traditional Chinese medicine to treat inflammatory conditions. In recent years it has aroused attention of scientists around the world because of its proved efficacies in treating such diseases as rheumatoid arthritis.

  13. The complete genome and proteome of Laribacter hongkongensis reveal potential mechanisms for adaptations to different temperatures and habitats.

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    Patrick C Y Woo

    2009-03-01

    Full Text Available Laribacter hongkongensis is a newly discovered Gram-negative bacillus of the Neisseriaceae family associated with freshwater fish-borne gastroenteritis and traveler's diarrhea. The complete genome sequence of L. hongkongensis HLHK9, recovered from an immunocompetent patient with severe gastroenteritis, consists of a 3,169-kb chromosome with G+C content of 62.35%. Genome analysis reveals different mechanisms potentially important for its adaptation to diverse habitats of human and freshwater fish intestines and freshwater environments. The gene contents support its phenotypic properties and suggest that amino acids and fatty acids can be used as carbon sources. The extensive variety of transporters, including multidrug efflux and heavy metal transporters as well as genes involved in chemotaxis, may enable L. hongkongensis to survive in different environmental niches. Genes encoding urease, bile salts efflux pump, adhesin, catalase, superoxide dismutase, and other putative virulence factors-such as hemolysins, RTX toxins, patatin-like proteins, phospholipase A1, and collagenases-are present. Proteomes of L. hongkongensis HLHK9 cultured at 37 degrees C (human body temperature and 20 degrees C (freshwater habitat temperature showed differential gene expression, including two homologous copies of argB, argB-20, and argB-37, which encode two isoenzymes of N-acetyl-L-glutamate kinase (NAGK-NAGK-20 and NAGK-37-in the arginine biosynthesis pathway. NAGK-20 showed higher expression at 20 degrees C, whereas NAGK-37 showed higher expression at 37 degrees C. NAGK-20 also had a lower optimal temperature for enzymatic activities and was inhibited by arginine probably as negative-feedback control. Similar duplicated copies of argB are also observed in bacteria from hot springs such as Thermus thermophilus, Deinococcus geothermalis, Deinococcus radiodurans, and Roseiflexus castenholzii, suggesting that similar mechanisms for temperature adaptation may be

  14. Structure reveals regulatory mechanisms of a MaoC-like hydratase from Phytophthora capsici involved in biosynthesis of polyhydroxyalkanoates (PHAs.

    Directory of Open Access Journals (Sweden)

    Huizheng Wang

    Full Text Available BACKGROUND: Polyhydroxyalkanoates (PHAs have attracted increasing attention as "green plastic" due to their biodegradable, biocompatible, thermoplastic, and mechanical properties, and considerable research has been undertaken to develop low cost/high efficiency processes for the production of PHAs. MaoC-like hydratase (MaoC, which belongs to (R-hydratase involved in linking the β-oxidation and the PHA biosynthetic pathways, has been identified recently. Understanding the regulatory mechanisms of (R-hydratase catalysis is critical for efficient production of PHAs that promise synthesis an environment-friendly plastic. METHODOLOGY/PRINCIPAL FINDINGS: We have determined the crystal structure of a new MaoC recognized from Phytophthora capsici. The crystal structure of the enzyme was solved at 2.00 Å resolution. The structure shows that MaoC has a canonical (R-hydratase fold with an N-domain and a C-domain. Supporting its dimerization observed in structure, MaoC forms a stable homodimer in solution. Mutations that disrupt the dimeric MaoC result in a complete loss of activity toward crotonyl-CoA, indicating that dimerization is required for the enzymatic activity of MaoC. Importantly, structure comparison reveals that a loop unique to MaoC interacts with an α-helix that harbors the catalytic residues of MaoC. Deletion of the loop enhances the enzymatic activity of MaoC, suggesting its inhibitory role in regulating the activity of MaoC. CONCLUSIONS/SIGNIFICANCE: The data in our study reveal the regulatory mechanism of an (R-hydratase, providing information on enzyme engineering to produce low cost PHAs.

  15. De novo Sequencing and Transcriptome Analysis Reveal Key Genes Regulating Steroid Metabolism in Leaves, Roots, Adventitious Roots and Calli of Periploca sepium Bunge

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    Xinglin Li

    2017-04-01

    Full Text Available Periploca sepium Bunge is a traditional medicinal plant, whose root bark is important for Chinese herbal medicine. Its major bioactive compounds are C21 steroids and periplocin, a kind of cardiac glycoside, which are derived from the steroid synthesis pathway. However, research on P. sepium genome or transcriptomes and their related genes has been lacking for a long time. In this study we estimated this species nuclear genome size at 170 Mb (using flow cytometry. Then, RNA sequencing of four different tissue samples of P. sepium (leaves, roots, adventitious roots, and calli was done using the sequencing platform Illumina/Solexa Hiseq 2,500. After de novo assembly and quantitative assessment, 90,375 all-transcripts and 71,629 all-unigenes were finally generated. Annotation efforts that used a number of public databases resulted in detailed annotation information for the transcripts. In addition, differentially expressed genes (DEGs were identified by using digital gene profiling based on the reads per kilobase of transcript per million reads mapped (RPKM values. Compared with the leaf samples (L, up-regulated genes and down-regulated genes were eventually obtained. To deepen our understanding of these DEGs, we performed two enrichment analyses: gene ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG. Here, the analysis focused upon the expression characteristics of those genes involved in the terpene metabolic pathway and the steroid biosynthesis pathway, to better elucidate the molecular mechanism of bioactive steroid synthesis in P. sepium. The bioinformatics analysis enabled us to find many genes that are involved in bioactive steroid biosynthesis. These genes encoded acetyl-CoA acetyltransferase (ACAT, HMG-CoA synthase (HMGS, HMG-CoA reductase (HMGR, mevalonate kinase (MK, phosphomevalonate kinase (PMK, mevalonate diphosphate decarboxylase (MDD, isopentenylpyrophosphate isomerase (IPPI, farnesyl pyrophosphate synthase (FPS

  16. The Motivational Foundations of Prosocial Behavior From A Developmental Perspective-Evolutionary Roots and Key Psychological Mechanisms: Introduction to the Special Section.

    Science.gov (United States)

    Davidov, Maayan; Vaish, Amrisha; Knafo-Noam, Ariel; Hastings, Paul D

    2016-11-01

    Prosocial behavior is versatile, multifaceted, and complex. This special section seeks to advance coherent, integrative understanding of prosocial development by addressing this topic through the prism of motivations. This conceptual Introduction presents key ideas that provide a framework for thinking about motivation for prosocial behavior and its development. It outlines the evolutionary roots of prosocial behavior, underscoring the interdependent roles of nature and nurture. This is followed by a discussion of several key psychological mechanisms reflecting different motivations for prosocial action (empathy for a distressed other, concern about another's goal, desire to act in accordance with internalized prosocial norms, and guilt). We discuss the critical components of each motivation and highlight pertinent contributions of the special section articles. © 2016 The Authors. Child Development © 2016 Society for Research in Child Development, Inc.

  17. A Handover Security Mechanism Employing the Diffie-Hellman Key Exchange Approach for the IEEE802.16e Wireless Networks

    Directory of Open Access Journals (Sweden)

    Yi-Fu Ciou

    2011-01-01

    Full Text Available In this paper, we propose a handover authentication mechanism, called the handover key management and authentication scheme (HaKMA for short, which as a three-layer authentication architecture is a new version of our previous work, the Diffie-Hellman-PKDS-based authentication method (DiHam for short improving its key generation flow and adding a handover authentication scheme to respectively speed up the handover process and increase the security level for mobile stations (MSs. AAA server supported authentication is also enhanced by invoking an improved extensible authentication protocol (EAP. According to the analyses of this study the HaKMA can effectively and efficiently provide user authentication and balance data security and system performance during handover.

  18. Comparative genome analysis of three eukaryotic parasites with differing abilities to transform leukocytes reveals key mediators of theileria-induced leukocyte transformation

    KAUST Repository

    Hayashida, Kyoko

    2012-09-04

    We sequenced the genome of Theileria orientalis, a tick-borne apicomplexan protozoan parasite of cattle. The focus of this study was a comparative genome analysis of T. orientalis relative to other highly pathogenic Theileria species, T. parva and T. annulata. T. parva and T. annulata induce transformation of infected cells of lymphocyte or macrophage/monocyte lineages; in contrast, T. orientalis does not induce uncontrolled proliferation of infected leukocytes and multiplies predominantly within infected erythrocytes. While synteny across homologous chromosomes of the three Theileria species was found to be well conserved overall, subtelomeric structures were found to differ substantially, as T. orientalis lacks the large tandemly arrayed subtelomere-encoded variable secreted protein-encoding gene family. Moreover, expansion of particular gene families by gene duplication was found in the genomes of the two transforming Theileria species, most notably, the TashAT/TpHN and Tar/Tpr gene families. Gene families that are present only in T. parva and T. annulata and not in T. orientalis, Babesia bovis, or Plasmo-dium were also identified. Identification of differences between the genome sequences of Theileria species with different abilities to transform and immortalize bovine leukocytes will provide insight into proteins and mechanisms that have evolved to induce and regulate this process. The T. orientalis genome database is available at http://totdb.czc.hokudai.ac.jp/. 2012 Hayashida et al. T.

  19. Comparative genome analysis of three eukaryotic parasites with differing abilities to transform leukocytes reveals key mediators of Theileria-induced leukocyte transformation.

    Science.gov (United States)

    Hayashida, Kyoko; Hara, Yuichiro; Abe, Takashi; Yamasaki, Chisato; Toyoda, Atsushi; Kosuge, Takehide; Suzuki, Yutaka; Sato, Yoshiharu; Kawashima, Shuichi; Katayama, Toshiaki; Wakaguri, Hiroyuki; Inoue, Noboru; Homma, Keiichi; Tada-Umezaki, Masahito; Yagi, Yukio; Fujii, Yasuyuki; Habara, Takuya; Kanehisa, Minoru; Watanabe, Hidemi; Ito, Kimihito; Gojobori, Takashi; Sugawara, Hideaki; Imanishi, Tadashi; Weir, William; Gardner, Malcolm; Pain, Arnab; Shiels, Brian; Hattori, Masahira; Nene, Vishvanath; Sugimoto, Chihiro

    2012-01-01

    We sequenced the genome of Theileria orientalis, a tick-borne apicomplexan protozoan parasite of cattle. The focus of this study was a comparative genome analysis of T. orientalis relative to other highly pathogenic Theileria species, T. parva and T. annulata. T. parva and T. annulata induce transformation of infected cells of lymphocyte or macrophage/monocyte lineages; in contrast, T. orientalis does not induce uncontrolled proliferation of infected leukocytes and multiplies predominantly within infected erythrocytes. While synteny across homologous chromosomes of the three Theileria species was found to be well conserved overall, subtelomeric structures were found to differ substantially, as T. orientalis lacks the large tandemly arrayed subtelomere-encoded variable secreted protein-encoding gene family. Moreover, expansion of particular gene families by gene duplication was found in the genomes of the two transforming Theileria species, most notably, the TashAT/TpHN and Tar/Tpr gene families. Gene families that are present only in T. parva and T. annulata and not in T. orientalis, Babesia bovis, or Plasmodium were also identified. Identification of differences between the genome sequences of Theileria species with different abilities to transform and immortalize bovine leukocytes will provide insight into proteins and mechanisms that have evolved to induce and regulate this process. The T. orientalis genome database is available at http://totdb.czc.hokudai.ac.jp/.

  20. Localizing gene regulation reveals a staggered wood decay mechanism for the brown rot fungus Postia placenta

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jiwei; Presley, Gerald N.; Hammel, Kenneth E.; Ryu, Jae-San; Menke, Jon R.; Figueroa, Melania; Hu, Dehong; Orr, Galya; Schilling, Jonathan S.

    2016-09-12

    The fungi that cause brown rot of wood are essential contributors to biomass recycling in forest ecosystems. Their highly efficient cellulolytic systems, which may have practical applications, apparently depend on a combination of two mechanisms: nonselective oxidation of the lignocellulose by reactive oxygen species (ROS) coupled with hydrolysis of the polysaccharide components by a limited set of glycoside hydrolases (GHs). Since the production of strongly oxidizing ROS appears incompatible with the operation of GHs, it has been proposed that the fungi regulate ROS production by maintaining concentration gradients of the chelated metal ions they use to generate extracellular oxidants. However, calculations have indicated that this protective mechanism is physically infeasible. We examined a different hypothesis, that expression of ROS and GH components is temporally staggered by brown rot fungi in wood. We sectioned thin wafers of spruce and aspen that had been colonized directionally by Postia placenta and measured expression of relevant genes and some of the encoded enzymes, thus using the spatial distribution of fungal hyphae to resolve a fine-scale temporal sequence. Hierarchical clustering of gene expression for eight oxidoreductases thought to have a role in ROS production and of eight GHs revealed a zone of oxidoreductase upregulation at the hyphal front that persisted about 48 h before upregulation of the GHs. Additional evidence for differential expression was provided by localization of endoglucanase, xylanase, mannanase, and laccase activities in the colonized wood. Our results support a two-step mechanism for brown rot, in which substrate oxidation precedes enzymatic hydrolysis.

  1. Phosphoproteome Analysis Reveals the Molecular Mechanisms Underlying Deoxynivalenol-Induced Intestinal Toxicity in IPEC-J2 Cells

    Science.gov (United States)

    Zhang, Zhi-Qi; Wang, Song-Bo; Wang, Rui-Guo; Zhang, Wei; Wang, Pei-Long; Su, Xiao-Ou

    2016-01-01

    Deoxynivalenol (DON) is a widespread trichothecene mycotoxin that commonly contaminates cereal crops and has various toxic effects in animals and humans. DON primarily targets the gastrointestinal tract, the first barrier against ingested food contaminants. In this study, an isobaric tag for relative and absolute quantitation (iTRAQ)-based phosphoproteomic approach was employed to elucidate the molecular mechanisms underlying DON-mediated intestinal toxicity in porcine epithelial cells (IPEC-J2) exposed to 20 μM DON for 60 min. There were 4153 unique phosphopeptides, representing 389 phosphorylation sites, detected in 1821 phosphoproteins. We found that 289 phosphopeptides corresponding to 255 phosphoproteins were differentially phosphorylated in response to DON. Comprehensive Gene Ontology (GO) analysis combined with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment revealed that, in addition to previously well-characterized mitogen-activated protein kinase (MAPK) signaling, DON exposure altered phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and Janus kinase/signal transducer, and activator of transcription (JAK/STAT) pathways. These pathways are involved in a wide range of biological processes, including apoptosis, the intestinal barrier, intestinal inflammation, and the intestinal absorption of glucose. DON-induced changes are likely to contribute to the intestinal dysfunction. Overall, identification of relevant signaling pathways yielded new insights into the molecular mechanisms underlying DON-induced intestinal toxicity, and might help in the development of improved mechanism-based risk assessments in animals and humans. PMID:27669298

  2. Phosphoproteome Analysis Reveals the Molecular Mechanisms Underlying Deoxynivalenol-Induced Intestinal Toxicity in IPEC-J2 Cells

    Directory of Open Access Journals (Sweden)

    Zhi-Qi Zhang

    2016-09-01

    Full Text Available Deoxynivalenol (DON is a widespread trichothecene mycotoxin that commonly contaminates cereal crops and has various toxic effects in animals and humans. DON primarily targets the gastrointestinal tract, the first barrier against ingested food contaminants. In this study, an isobaric tag for relative and absolute quantitation (iTRAQ-based phosphoproteomic approach was employed to elucidate the molecular mechanisms underlying DON-mediated intestinal toxicity in porcine epithelial cells (IPEC-J2 exposed to 20 μM DON for 60 min. There were 4153 unique phosphopeptides, representing 389 phosphorylation sites, detected in 1821 phosphoproteins. We found that 289 phosphopeptides corresponding to 255 phosphoproteins were differentially phosphorylated in response to DON. Comprehensive Gene Ontology (GO analysis combined with Kyoto Encyclopedia of Genes and Genomes (KEGG pathway enrichment revealed that, in addition to previously well-characterized mitogen-activated protein kinase (MAPK signaling, DON exposure altered phosphatidylinositol 3-kinase/Akt (PI3K/Akt and Janus kinase/signal transducer, and activator of transcription (JAK/STAT pathways. These pathways are involved in a wide range of biological processes, including apoptosis, the intestinal barrier, intestinal inflammation, and the intestinal absorption of glucose. DON-induced changes are likely to contribute to the intestinal dysfunction. Overall, identification of relevant signaling pathways yielded new insights into the molecular mechanisms underlying DON-induced intestinal toxicity, and might help in the development of improved mechanism-based risk assessments in animals and humans.

  3. Biochemical Analysis Reveals the Multifactorial Mechanism of Histone H3 Clipping by Chicken Liver Histone H3 Protease

    KAUST Repository

    Chauhan, Sakshi

    2016-09-02

    Proteolytic clipping of histone H3 has been identified in many organisms. Despite several studies, the mechanism of clipping, the substrate specificity, and the significance of this poorly understood epigenetic mechanism are not clear. We have previously reported histone H3 specific proteolytic clipping and a protein inhibitor in chicken liver. However, the sites of clipping are still not known very well. In this study, we attempt to identify clipping sites in histone H3 and to determine the mechanism of inhibition by stefin B protein, a cysteine protease inhibitor. By employing site-directed mutagenesis and in vitro biochemical assays, we have identified three distinct clipping sites in recombinant human histone H3 and its variants (H3.1, H3.3, and H3t). However, post-translationally modified histones isolated from chicken liver and Saccharomyces cerevisiae wild-type cells showed different clipping patterns. Clipping of histone H3 N-terminal tail at three sites occurs in a sequential manner. We have further observed that clipping sites are regulated by the structure of the N-terminal tail as well as the globular domain of histone H3. We also have identified the QVVAG region of stefin B protein to be very crucial for inhibition of the protease activity. Altogether, our comprehensive biochemical studies have revealed three distinct clipping sites in histone H3 and their regulation by the structure of histone H3, histone modifications marks, and stefin B.

  4. De-novo assembly of mango fruit peel transcriptome reveals mechanisms of mango response to hot water treatment.

    Science.gov (United States)

    Luria, Neta; Sela, Noa; Yaari, Mor; Feygenberg, Oleg; Kobiler, Ilana; Lers, Amnon; Prusky, Dov

    2014-11-05

    The mango belongs to the genus Mangifera, consisting of numerous tropical fruiting trees in the flowering plant family, Anacardiaceae. Postharvest treatment by hot water brushing (HWB) for 15-20 s was introduced commercially to improve fruit quality and reduce postharvest disease. This treatment enabled successful storage for 3-4 weeks at 12°C, with improved color and reduced disease development, but it enhanced lenticel discoloration on the fruit peel. We investigated global gene expression induced in fruit peel by HWB treatment, and identified key genes involved in mechanisms potentially associated with fruit resistance to pathogens, peel color improvement, and development of lenticel discoloration; this might explain the fruit's phenotypic responses. The mango transcriptome assembly was created and characterized by application of RNA-seq to fruit-peel samples. RNA-seq-based gene-expression profiling identified three main groups of genes associated with HWB treatment: 1) genes involved with biotic and abiotic stress responses and pathogen-defense mechanisms, which were highly expressed; 2) genes associated with chlorophyll degradation and photosynthesis, which showed transient and low expression; and 3) genes involved with sugar and flavonoid metabolism, which were highly expressed. We describe a new transcriptome of mango fruit peel of cultivar Shelly. The existence of three main groups of genes that were differentially expressed following HWB treatment suggests a molecular basis for the biochemical and physiological consequences of the postharvest HWB treatment, including resistance to pathogens, improved color development, and occurrence of lenticel discoloration.

  5. Common mechanisms involved in Alzheimer's disease and type 2 diabetes: a key role of chronic bacterial infection and inflammation.

    Science.gov (United States)

    Miklossy, Judith; McGeer, Patrick L

    2016-04-01

    Strong epidemiologic evidence and common molecular mechanisms support an association between Alzheimer's disease (AD) and type 2-diabetes. Local inflammation and amyloidosis occur in both diseases and are associated with periodontitis and various infectious agents. This article reviews the evidence for the presence of local inflammation and bacteria in type 2 diabetes and discusses host pathogen interactions in chronic inflammatory disorders. Chlamydophyla pneumoniae, Helicobacter pylori and spirochetes are demonstrated in association with dementia and brain lesions in AD and islet lesions in type 2 diabetes. The presence of pathogens in host tissues activates immune responses through Toll-like receptor signaling pathways. Evasion of pathogens from complement-mediated attack results in persistent infection, inflammation and amyloidosis. Amyloid beta and the pancreatic amyloid called amylin bind to lipid bilayers and produce Ca(2+) influx and bacteriolysis. Similarly to AD, accumulation of amylin deposits in type 2 diabetes may result from an innate immune response to chronic bacterial infections, which are known to be associated with amyloidosis. Further research based on an infectious origin of both AD and type 2 diabetes may lead to novel treatment strategies.

  6. Disturbed Laminar Blood Flow Vastly Augments Lipoprotein Retention in the Artery Wall: A Key Mechanism Distinguishing Susceptible From Resistant Sites.

    Science.gov (United States)

    Steffensen, Lasse Bach; Mortensen, Martin Bødtker; Kjolby, Mads; Hagensen, Mette Kallestrup; Oxvig, Claus; Bentzon, Jacob Fog

    2015-09-01

    Atherosclerosis develops initially at branch points and in areas of high vessel curvature. Moreover, experiments in hypercholesterolemic mice have shown that the introduction of disturbed flow in straight, atherosclerosis-resistant arterial segments turns them highly atherosclerosis susceptible. Several biomechanical mechanisms have been proposed, but none has been demonstrated. In the present study, we examined whether a causal link exists between disturbed laminar flow and the ability of the arterial wall to retain lipoproteins. Lipoprotein retention was detected at natural predilection sites of the murine thoracic aorta 18 hours after infusion of fluorescently labeled low-density lipoprotein. To test for causality between blood flow and the ability of these areas to retain lipoproteins, we manipulated blood flow in the straight segment of the common carotid artery using a constrictive collar. Disturbed laminar flow did not affect low-density lipoprotein influx, but increased the ability of the artery wall to bind low-density lipoprotein. Concordantly, disturbed laminar flow led to differential expression of genes associated with phenotypic modulation of vascular smooth muscle cells, increased expression of proteoglycan core proteins associated with lipoprotein retention, and of enzymes responsible for chondroitin sulfate glycosaminoglycan synthesis and sulfation. Blood flow regulates genes associated with vascular smooth muscle cell phenotypic modulation, as well as the expression and post-translational modification of lipoprotein-binding proteoglycan core proteins, and the introduction of disturbed laminar flow vastly augments the ability of a previously resistant, straight arterial segment to retain lipoproteins. © 2015 American Heart Association, Inc.

  7. The Key Role of Ball Milling Time in the Microstructure and Mechanical Property of Ni-TiCNP Composites

    Science.gov (United States)

    Zhou, Xiaoling; Huang, Hefei; Xie, Ruobing; Yang, Chao; Li, Zhijun; Jiang, Li; Ye, Xiangxi; Xu, Hongjie

    2016-12-01

    Titanium carbide nanoparticle-reinforced nickel-based alloys (Ni-TiCNP composites) with ball milling time ranging from 8 to 72 h were prepared by ball milling and spark plasma sintering. Transmission electron microscopy (TEM) and scanning electron microscopy equipped with electron backscatter diffraction were used to characterize the microstructures. Their hardness and tensile properties were measured using the Vickers pyramid method and tensile tests. TEM results showed that a slight coarsening of TiCNP occurred during the ball milling process. The grain sizes of the Ni-TiCNP composites with various ball milling times were different, but they were all much smaller than those of the pure Ni. In all cases, the Ni-TiCNP composites showed higher strengths and hardness values than the unreinforced pure nickel. Furthermore, the strength of the Ni-TiCNP composites increased initially and then decreased as a function of ball milling time. The maximum strengths occurred in the 24-h ball milling sample, which presented the lowest average grain size. The Hall-Petch strengthening was suggested to be the main reason responsible for such variations in mechanical properties. Additionally, the elongation percentage of the Ni-TiCNP composites decreased gradually with ball milling time. This may be caused by the change of microvoids in the composite as the ball milling time varies, which is also related to their fracture behavior.

  8. Key issues in the microchemical systems-based methanol fuel processor: Energy density, thermal integration, and heat loss mechanisms

    Science.gov (United States)

    Shah, Keyur; Besser, R. S.

    Microreactor technology is a promising approach in harnessing the high energy density of hydrocarbons and is being used to produce hydrogen-rich gases by reforming of methanol and other liquid hydrocarbons. However, on-demand H 2 generation for miniature proton exchange membrane fuel cell (PEMFC) systems has been a bottleneck problem, which has limited the development and demonstration of the PEMFC for high-performance portable power. A number of crucial challenges exist for the realization of practical portable fuel processors. Among these, the management of heat in a compact format is perhaps the most crucial challenge for portable fuel processors. In this study, a silicon microreactor-based catalytic methanol steam reforming reactor was designed, fabricated, and demonstrated in the context of complete thermal integration to understand this critical issue and develop a knowledge base required to rationally design and integrate the microchemical components of a fuel processor. Detailed thermal and reaction experiments were carried out to demonstrate the potential of microreactor-based on-demand H 2 generation. Based on thermal characterization experiments, the heat loss mechanisms and effective convective heat coefficients from the planar microreactor structure were determined and suggestions were made for scale up and implementation of packaging schemes to reduce different modes of heat losses.

  9. In vivo approaches reveal a key role for DCs in CD4+ T cell activation and parasite clearance during the acute phase of experimental blood-stage malaria.

    Directory of Open Access Journals (Sweden)

    Henrique Borges da Silva

    2015-02-01

    Full Text Available Dendritic cells (DCs are phagocytes that are highly specialized for antigen presentation. Heterogeneous populations of macrophages and DCs form a phagocyte network inside the red pulp (RP of the spleen, which is a major site for the control of blood-borne infections such as malaria. However, the dynamics of splenic DCs during Plasmodium infections are poorly understood, limiting our knowledge regarding their protective role in malaria. Here, we used in vivo experimental approaches that enabled us to deplete or visualize DCs in order to clarify these issues. To elucidate the roles of DCs and marginal zone macrophages in the protection against blood-stage malaria, we infected DTx (diphtheria toxin-treated C57BL/6.CD11c-DTR mice, as well as C57BL/6 mice treated with low doses of clodronate liposomes (ClLip, with Plasmodium chabaudi AS (Pc parasites. The first evidence suggesting that DCs could contribute directly to parasite clearance was an early effect of the DTx treatment, but not of the ClLip treatment, in parasitemia control. DCs were also required for CD4+ T cell responses during infection. The phagocytosis of infected red blood cells (iRBCs by splenic DCs was analyzed by confocal intravital microscopy, as well as by flow cytometry and immunofluorescence, at three distinct phases of Pc malaria: at the first encounter, at pre-crisis concomitant with parasitemia growth and at crisis when the parasitemia decline coincides with spleen closure. In vivo and ex vivo imaging of the spleen revealed that DCs actively phagocytize iRBCs and interact with CD4+ T cells both in T cell-rich areas and in the RP. Subcapsular RP DCs were highly efficient in the recognition and capture of iRBCs during pre-crisis, while complete DC maturation was only achieved during crisis. These findings indicate that, beyond their classical role in antigen presentation, DCs also contribute to the direct elimination of iRBCs during acute Plasmodium infection.

  10. Mutagenesis of p38alpha MAP Kinase Establishes Key Roles of Phe169 in Function and Structural Dynamics and Reveals a Novel DFG-OUT State

    Energy Technology Data Exchange (ETDEWEB)

    Bukhtiyarova,M.; Karpusas, M.; Northrup, K.; Namboodiri, H.; Springman, E.

    2007-01-01

    In order to study the role of Phe169 in p38{alpha} MAP kinase structure and function, wild-type p38{alpha} and five p38{alpha} DFG motif mutants were examined in vitro for phosphorylation by MKK6, kinase activity toward ATF2 substrate, thermal stability, and X-ray crystal structure. All six p38{alpha} variants were efficiently phosphorylated by MKK6. However, only one activated p38{alpha} mutant (F169Y) possessed measurable kinase activity (1% compared to wild-type). The loss of kinase activity among the DFG mutants may result from an inability to correctly position Asp168 in the activated form of p38{alpha}. Two mutations significantly increased the thermal stability of p38{alpha} (F169A {Delta}T{sub m} = 1.3 {sup o}C and D168G {Delta}T{sub m} = 3.8 {sup o}C), and two mutations significantly decreased the stability of p38{alpha} (F169R {Delta}T{sub m} = -3.2 {sup o}C and F169G {Delta}T{sub m} = -4.7 {sup o}C). Interestingly, X-ray crystal structures of two thermally destabilized p38{alpha}-F169R and p38{alpha}-F169G mutants revealed a DFG-OUT conformation in the absence of an inhibitor molecule. This DFG-OUT conformation, termed {alpha}-DFG-OUT, is different from the ones previously identified in p38{alpha} crystal structures with bound inhibitors and postulated from high-temperature molecular dynamics simulations. Taken together, these results indicate that Phe169 is optimized for p38{alpha} functional activity and structural dynamics, rather than for structural stability. The {alpha}-DFG-OUT conformation observed for p38{alpha}-F169R and p38{alpha}-F169G may represent a naturally occurring intermediate state of p38{alpha} that provides access for binding of allosteric inhibitors. A model of the local forces driving the DFG IN-OUT transition in p38{alpha} is proposed.

  11. In Vivo Approaches Reveal a Key Role for DCs in CD4+ T Cell Activation and Parasite Clearance during the Acute Phase of Experimental Blood-Stage Malaria

    Science.gov (United States)

    Borges da Silva, Henrique; Fonseca, Raíssa; Cassado, Alexandra dos Anjos; Machado de Salles, Érika; de Menezes, Maria Nogueira; Langhorne, Jean; Perez, Katia Regina; Cuccovia, Iolanda Midea; Ryffel, Bernhard; Barreto, Vasco M.; Marinho, Cláudio Romero Farias; Boscardin, Silvia Beatriz; Álvarez, José Maria; D’Império-Lima, Maria Regina; Tadokoro, Carlos Eduardo

    2015-01-01

    Dendritic cells (DCs) are phagocytes that are highly specialized for antigen presentation. Heterogeneous populations of macrophages and DCs form a phagocyte network inside the red pulp (RP) of the spleen, which is a major site for the control of blood-borne infections such as malaria. However, the dynamics of splenic DCs during Plasmodium infections are poorly understood, limiting our knowledge regarding their protective role in malaria. Here, we used in vivo experimental approaches that enabled us to deplete or visualize DCs in order to clarify these issues. To elucidate the roles of DCs and marginal zone macrophages in the protection against blood-stage malaria, we infected DTx (diphtheria toxin)-treated C57BL/6.CD11c-DTR mice, as well as C57BL/6 mice treated with low doses of clodronate liposomes (ClLip), with Plasmodium chabaudi AS (Pc) parasites. The first evidence suggesting that DCs could contribute directly to parasite clearance was an early effect of the DTx treatment, but not of the ClLip treatment, in parasitemia control. DCs were also required for CD4+ T cell responses during infection. The phagocytosis of infected red blood cells (iRBCs) by splenic DCs was analyzed by confocal intravital microscopy, as well as by flow cytometry and immunofluorescence, at three distinct phases of Pc malaria: at the first encounter, at pre-crisis concomitant with parasitemia growth and at crisis when the parasitemia decline coincides with spleen closure. In vivo and ex vivo imaging of the spleen revealed that DCs actively phagocytize iRBCs and interact with CD4+ T cells both in T cell-rich areas and in the RP. Subcapsular RP DCs were highly efficient in the recognition and capture of iRBCs during pre-crisis, while complete DC maturation was only achieved during crisis. These findings indicate that, beyond their classical role in antigen presentation, DCs also contribute to the direct elimination of iRBCs during acute Plasmodium infection. PMID:25658925

  12. Genome-wide analysis of brain and gonad transcripts reveals changes of key sex reversal-related genes expression and signaling pathways in three stages of Monopterus albus

    Science.gov (United States)

    Hu, Qing; Guo, Wei; Li, Dapeng

    2017-01-01

    Background The natural sex reversal severely affects the sex ratio and thus decreases the productivity of the rice field eel (Monopterus albus). How to understand and manipulate this process is one of the major issues for the rice field eel stocking. So far the genomics and transcriptomics data available for this species are still scarce. Here we provide a comprehensive study of transcriptomes of brain and gonad tissue in three sex stages (female, intersex and male) from the rice field eel to investigate changes in transcriptional level during the sex reversal process. Results Approximately 195 thousand unigenes were generated and over 44.4 thousand were functionally annotated. Comparative study between stages provided multiple differentially expressed genes in brain and gonad tissue. Overall 4668 genes were found to be of unequal abundance between gonad tissues, far more than that of the brain tissues (59 genes). These genes were enriched in several different signaling pathways. A number of 231 genes were found with different levels in gonad in each stage, with several reproduction-related genes included. A total of 19 candidate genes that could be most related to sex reversal were screened out, part of these genes’ expression patterns were validated by RT-qPCR. The expression of spef2, maats1, spag6 and dmc1 were abundant in testis, but was barely detected in females, while the 17β-hsd12, zpsbp3, gal3 and foxn5 were only expressed in ovary. Conclusion This study investigated the complexity of brain and gonad transcriptomes in three sex stages of the rice field eel. Integrated analysis of different gene expression and changes in signaling pathways, such as PI3K-Akt pathway, provided crucial data for further study of sex transformation mechanisms. PMID:28319194

  13. Noncoding RNA mediated traffic of foreign mRNA into chloroplasts reveals a novel signaling mechanism in plants.

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    Gustavo Gómez

    Full Text Available Communication between chloroplasts and the nucleus is one of the milestones of the evolution of plants on earth. Proteins encoded by ancestral chloroplast-endogenous genes were transferred to the nucleus during the endosymbiotic evolution and originated this communication, which is mainly dependent on specific transit-peptides. However, the identification of nuclear-encoded proteins targeted to the chloroplast lacking these canonical signals suggests the existence of an alternative cellular pathway tuning this metabolic crosstalk. Non-coding RNAS (NcRNAs are increasingly recognized as regulators of gene expression as they play roles previously believed to correspond to proteins. Avsunviroidae family viroids are the only noncoding functional RNAs that have been reported to traffic inside the chloroplasts. Elucidating mechanisms used by these pathogens to enter this organelle will unearth novel transport pathways in plant cells. Here we show that a viroid-derived NcRNA acting as a 5'UTR-end mediates the functional import of Green Fluorescent Protein (GFP mRNA into chloroplast. This claim is supported by the observation at confocal microscopy of a selective accumulation of GFP in the chloroplast of the leaves expressing the chimeric vd-5'UTR/GFP and by the detection of the GFP mRNA in chloroplasts isolated from cells expressing this construct. These results support the existence of an alternative signaling mechanism in plants between the host cell and chloroplasts, where an ncRNA functions as a key regulatory molecule to control the accumulation of nuclear-encoded proteins in this organelle. In addition, our findings provide a conceptual framework to develop new biotechnological tools in systems using plant chloroplast as bioreactors. Finally, viroids of the family Avsunviroidae have probably evolved to subvert this signaling mechanism to regulate their differential traffic into the chloroplast of infected cells.

  14. Structures of human cytosolic NADP-dependent isocitrate dehydrogenase reveal a novel self-regulatory mechanism of activity.

    Science.gov (United States)

    Xu, Xiang; Zhao, Jingyue; Xu, Zhen; Peng, Baozhen; Huang, Qiuhua; Arnold, Eddy; Ding, Jianping

    2004-08-06

    Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitrate to alpha-ketoglutarate, and regulation of the enzymatic activity of IDHs is crucial for their biological functions. Bacterial IDHs are reversibly regulated by phosphorylation of a strictly conserved serine residue at the active site. Eukaryotic NADP-dependent IDHs (NADP-IDHs) have been shown to have diverse important biological functions; however, their regulatory mechanism remains unclear. Structural studies of human cytosolic NADP-IDH (HcIDH) in complex with NADP and in complex with NADP, isocitrate, and Ca2+ reveal three biologically relevant conformational states of the enzyme that differ substantially in the structure of the active site and in the overall structure. A structural segment at the active site that forms a conserved alpha-helix in all known NADP-IDH structures assumes a loop conformation in the open, inactive form of HcIDH; a partially unraveled alpha-helix in the semi-open, intermediate form; and an alpha-helix in the closed, active form. The side chain of Asp279 of this segment occupies the isocitrate-binding site and forms hydrogen bonds with Ser94 (the equivalent of the phosphorylation site in bacterial IDHs) in the inactive form and chelates the metal ion in the active form. The structural data led us to propose a novel self-regulatory mechanism for HcIDH that mimics the phosphorylation mechanism used by the bacterial homologs, consistent with biochemical and biological data. This mechanism might be applicable to other eukaryotic NADP-IDHs. The results also provide insights into the recognition and specificity of substrate and cofactor by eukaryotic NADP-IDHs.

  15. Proteome and Computational Analyses Reveal New Insights into the Mechanisms of Hepatitis C Virus Mediated Liver Disease Post-Transplantation

    Science.gov (United States)

    Diamond, Deborah L.; Krasnoselsky, Alexei L.; Burnum, Kristin E.; Monroe, Matthew E.; Webb-Robertson, Bobbie-Jo; McDermott, Jason E.; Yeh, Matthew M.; Dzib, Jose Felipe Golib; Susnow, Nathan; Strom, Susan; Proll, Sean C.; Belisle, Sarah E.; Purdy, David E.; Rasmussen, Angela L.; Walters, Kathie-Anne; Jacobs, Jon M.; Gritsenko, Marina A.; Camp, David G.; Bhattacharya, Renuka; Perkins, James D.; Carithers, Robert L.; Liou, Iris W.; Larson, Anne M.; Benecke, Arndt; Waters, Katrina M.; Smith, Richard D.; Katze, Michael G.

    2012-01-01

    Liver transplant tissues offer the unique opportunity to model the longitudinal protein abundance changes occurring during hepatitis C virus (HCV)-associated liver disease progression in vivo. In this study, our goal was to identify molecular signatures, and potential key regulatory proteins, representative of the processes influencing early progression to fibrosis. We performed global protein profiling analyses on 24 liver biopsy specimens obtained from 15 HCV+ liver transplant recipients at 6 and/or 12 months post-transplantation. Differentially regulated proteins associated with early progression to fibrosis were identified by analysis of the area under the receiver operating characteristic curve (AUC). Analysis of serum metabolites was performed on samples obtained from an independent cohort of 60 HCV+ liver transplant patients. Computational modeling approaches were applied to identify potential key regulatory proteins of liver fibrogenesis. Among 4,324 proteins identified, 250 exhibited significant differential regulation in patients with rapidly progressive fibrosis. Patients with rapid fibrosis progression exhibited enrichment in differentially regulated proteins associated with various immune, hepatoprotective, and fibrogenic processes. The observed increase in pro-inflammatory activity and impairment in anti-oxidant defenses suggests that patients who develop significant liver injury experience elevated oxidative stresses. This was supported by an independent study demonstrating the altered abundance of oxidative stress associated serum metabolites in patients who develop severe liver injury. Computational modeling approaches further highlight a potentially important link between HCV-associated oxidative stress and epigenetic regulatory mechanisms impacting on liver fibrogenesis. In conclusion, our proteome and metabolome analyses provide new insights into the role for increased oxidative stress in the rapid fibrosis progression observed in HCV+ liver

  16. Crystal structure of P58(IPK) TPR fragment reveals the mechanism for its molecular chaperone activity in UPR.

    Science.gov (United States)

    Tao, Jiahui; Petrova, Kseniya; Ron, David; Sha, Bingdong

    2010-04-16

    P58(IPK) might function as an endoplasmic reticulum molecular chaperone to maintain protein folding homeostasis during unfolded protein responses. P58(IPK) contains nine tetratricopeptide repeat (TPR) motifs and a C-terminal J-domain within its primary sequence. To investigate the mechanism by which P58(IPK) functions to promote protein folding within the endoplasmic reticulum, we have determined the crystal structure of P58(IPK) TPR fragment to 2.5 A resolution by the SAD method. The crystal structure of P58(IPK) revealed three domains (I-III) with similar folds and each domain contains three TPR motifs. An ELISA assay indicated that P58(IPK) acts as a molecular chaperone by interacting with misfolded proteins such as luciferase and rhodanese. The P58(IPK) structure reveals a conserved hydrophobic patch located in domain I that might be involved in binding the misfolded polypeptides. Structure-based mutagenesis for the conserved hydrophobic residues located in domain I significantly reduced the molecular chaperone activity of P58(IPK).

  17. Crystal structures of human Ero1α reveal the mechanisms of regulated and targeted oxidation of PDI.

    Science.gov (United States)

    Inaba, Kenji; Masui, Shoji; Iida, Hiroka; Vavassori, Stefano; Sitia, Roberto; Suzuki, Mamoru

    2010-10-06

    In the endoplasmic reticulum (ER) of eukaryotic cells, Ero1 flavoenzymes promote oxidative protein folding through protein disulphide isomerase (PDI), generating reactive oxygen species (hydrogen peroxide) as byproducts. Therefore, Ero1 activity must be strictly regulated to avoid futile oxidation cycles in the ER. Although regulatory mechanisms restraining Ero1α activity ensure that not all PDIs are oxidized, its specificity towards PDI could allow other resident oxidoreductases to remain reduced and competent to carry out isomerization and reduction of protein substrates. In this study, crystal structures of human Ero1α were solved in its hyperactive and inactive forms. Our findings reveal that human Ero1α modulates its oxidative activity by properly positioning regulatory cysteines within an intrinsically flexible loop, and by fine-tuning the electron shuttle ability of the loop through disulphide rearrangements. Specific PDI targeting is guaranteed by electrostatic and hydrophobic interactions of Ero1α with the PDI b'-domain through its substrate-binding pocket. These results reveal the molecular basis of the regulation and specificity of protein disulphide formation in human cells.

  18. Interactions between Casein kinase Iepsilon (CKIepsilon and two substrates from disparate signaling pathways reveal mechanisms for substrate-kinase specificity.

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    Caroline Lund Dahlberg

    Full Text Available BACKGROUND: Members of the Casein Kinase I (CKI family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIepsilon and two substrates from different signaling pathways. METHODOLOGY/PRINCIPAL FINDINGS: CKIepsilon, but not CKIalpha, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIalpha's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIepsilon does not determine Dishevelled's and Period's preference for CKIepsilon nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIepsilon with its substrates. We demonstrate that autophosphorylation of CKIepsilon's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding. CONCLUSIONS/SIGNIFICANCE: The biochemical interactions between CKIepsilon and Disheveled, Period, and its own C-terminus lead to models that explain CKIepsilon's specificity and regulation.

  19. Transcriptomic analysis of tail regeneration in the lizard Anolis carolinensis reveals activation of conserved vertebrate developmental and repair mechanisms.

    Directory of Open Access Journals (Sweden)

    Elizabeth D Hutchins

    Full Text Available Lizards, which are amniote vertebrates like humans, are able to lose and regenerate a functional tail. Understanding the molecular basis of this process would advance regenerative approaches in amniotes, including humans. We have carried out the first transcriptomic analysis of tail regeneration in a lizard, the green anole Anolis carolinensis, which revealed 326 differentially expressed genes activating multiple developmental and repair mechanisms. Specifically, genes involved in wound response, hormonal regulation, musculoskeletal development, and the Wnt and MAPK/FGF pathways were differentially expressed along the regenerating tail axis. Furthermore, we identified 2 microRNA precursor families, 22 unclassified non-coding RNAs, and 3 novel protein-coding genes significantly enriched in the regenerating tail. However, high levels of progenitor/stem cell markers were not observed in any region of the regenerating tail. Furthermore, we observed multiple tissue-type specific clusters of proliferating cells along the regenerating tail, not localized to the tail tip. These findings predict a different mechanism of regeneration in the lizard than the blastema model described in the salamander and the zebrafish, which are anamniote vertebrates. Thus, lizard tail regrowth involves the activation of conserved developmental and wound response pathways, which are potential targets for regenerative medical therapies.

  20. Mechanically functional amyloid fibrils in the adhesive of a marine invertebrate as revealed by Raman spectroscopy and atomic force microscopy.

    Science.gov (United States)

    S Mostaert, Anika; Crockett, Rowena; Kearn, Graham; Cherny, Izhack; Gazit, Ehud; C Serpell, Louise; P Jarvis, Suzanne

    2009-01-01

    Amyloid fibrils are primarily known in a pathogenic context for their association with a wide range of debilitating human diseases. Here we show a marine invertebrate (Entobdella soleae) utilizes functional amyloid fibrils comparable to those of a unicellular prokaryote (Escherichia coli). Thioflavin-T binding and Raman spectroscopy provided evidence for the presence of amyloid in the adhesive of Entobdella soleae. We elucidated that for these two very different organisms, amyloid fibrils provide adhesive and cohesive strength to their natural adhesives. Comparing the nanoscale mechanical responses of these fibrils with those of pathogenic amyloid by atomic force microscopy revealed that the molecular level origin of the cohesive strength was associated with the generic intermolecular β-sheet structure of amyloid fibrils. Functional adhesive residues were found only in the case of the functional amyloid. Atomic force microscopy provided a useful means to characterize the internal structural forces within individual amyloid fibrils and how these relate to the mechanical performance of both functional and pathogenic amyloid. The mechanistic link of amyloid-based cohesive and adhesive strength could be widespread amongst natural adhesives, irrespective of environment, providing a new strategy for biomimicry and a new source of materials for understanding the formation and stability of amyloid fibrils more generally.

  1. A combined cryo-EM and molecular dynamics approach reveals the mechanism of ErmBL-mediated translation arrest

    Science.gov (United States)

    Arenz, Stefan; Bock, Lars V.; Graf, Michael; Innis, C. Axel; Beckmann, Roland; Grubmüller, Helmut; Vaiana, Andrea C.; Wilson, Daniel N.

    2016-07-01

    Nascent polypeptides can induce ribosome stalling, regulating downstream genes. Stalling of ErmBL peptide translation in the presence of the macrolide antibiotic erythromycin leads to resistance in Streptococcus sanguis. To reveal this stalling mechanism we obtained 3.6-Å-resolution cryo-EM structures of ErmBL-stalled ribosomes with erythromycin. The nascent peptide adopts an unusual conformation with the C-terminal Asp10 side chain in a previously unseen rotated position. Together with molecular dynamics simulations, the structures indicate that peptide-bond formation is inhibited by displacement of the peptidyl-tRNA A76 ribose from its canonical position, and by non-productive interactions of the A-tRNA Lys11 side chain with the A-site crevice. These two effects combine to perturb peptide-bond formation by increasing the distance between the attacking Lys11 amine and the Asp10 carbonyl carbon. The interplay between drug, peptide and ribosome uncovered here also provides insight into the fundamental mechanism of peptide-bond formation.

  2. Long-range correlations in the mechanics of small DNA circles under topological stress revealed by multi-scale simulation.

    Science.gov (United States)

    Sutthibutpong, Thana; Matek, Christian; Benham, Craig; Slade, Gabriel G; Noy, Agnes; Laughton, Charles; K Doye, Jonathan P; Louis, Ard A; Harris, Sarah A

    2016-11-02

    It is well established that gene regulation can be achieved through activator and repressor proteins that bind to DNA and switch particular genes on or off, and that complex metabolic networks determine the levels of transcription of a given gene at a given time. Using three complementary computational techniques to study the sequence-dependence of DNA denaturation within DNA minicircles, we have observed that whenever the ends of the DNA are constrained, information can be transferred over long distances directly by the transmission of mechanical stress through the DNA itself, without any requirement for external signalling factors. Our models combine atomistic molecular dynamics (MD) with coarse-grained simulations and statistical mechanical calculations to span three distinct spatial resolutions and timescale regimes. While they give a consensus view of the non-locality of sequence-dependent denaturation in highly bent and supercoiled DNA loops, each also reveals a unique aspect of long-range informational transfer that occurs as a result of restraining the DNA within the closed loop of the minicircles. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Potential antidepressant and resilience mechanism revealed by metabolomic study on peripheral blood mononuclear cells of stress resilient rats.

    Science.gov (United States)

    Li, Juan; Zhang, Shu-Xiao; Wang, Wei; Cheng, Ke; Guo, Hua; Rao, Cheng-Long; Yang, De-Yu; He, Yong; Zou, De-Zhi; Han, Yu; Zhao, Li-Bo; Li, Peng-Fei; Xie, Peng

    2017-03-01

    Resilience is an active coping response to stress, which plays a very important role in major depressive disorder study. The molecular mechanisms underlying such resilience are poorly understood. Peripheral blood mononuclear cells (PBMCs) were promising objects in unveiling the underlying pathogenesis of resilience. Hereby we carried out successive study on PBMCs metabolomics in resilient rats of chronic unpredictable mild stress (CUMS) model. A gas chromatography-mass spectrometry (GC-MS) metabolomic approach coupled with principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) was used to detect differential metabolites in PBMCs of resilient rats. Ingenuity Pathways Analysis (IPA) was applied for pathway analysis. A set of differential metabolites including Malic acid, Ornithine, l-Lysine, Stigmasterol, Oleic acid, γ-Tocopherol, Adenosine and N-acetyl-d-glucosamine were significantly altered in resilient rats, meanwhile promoting antidepressant research. As revealed by IPA that aberrant energy metabolism, HIFα signaling, neurotransmitter, O-GlcNAcylation and cAMP signaling cascade in peripheral might be evolved in the pathogenesis of coping mechanism. The GC-MS based metabolomics may contribute to better understanding of resilience, as well as shedding light on antidepressant discovery. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Atomistic MD simulation reveals the mechanism by which CETP penetrates into HDL enabling lipid transfer from HDL to CETP

    Science.gov (United States)

    Cilpa-Karhu, Geraldine; Jauhiainen, Matti; Riekkola, Marja-Liisa

    2015-01-01

    Inhibition of cholesterol ester transfer protein (CETP), a protein mediating transfer of neutral lipids between lipoproteins, has been proposed as a means to elevate atheroprotective HDL subpopulations and thereby reduce atherosclerosis. However, off-target and adverse effects of the inhibition have raised doubts about the molecular mechanism of CETP-HDL interaction. Recent experimental findings have demonstrated the penetration of CETP into HDL. However, atomic level resolution of CETP penetration into HDL, a prerequisite for a better understanding of CETP functionality and HDL atheroprotection, is missing. We constructed an HDL particle that mimics the actual human HDL mass composition and investigated for the first time, by large-scale atomistic molecular dynamics, the interaction of an upright CETP with a human HDL-mimicking model. The results demonstrated how CETP can penetrate the HDL particle surface, with the formation of an opening in the N barrel domain end of CETP, put in evidence the major anchoring role of a tryptophan-rich region of this domain, and unveiled the presence of a phenylalanine barrier controlling further access of HDL-derived lipids to the tunnel of CETP. The findings reveal novel atomistic details of the CETP-HDL interaction mechanism and can provide new insight into therapeutic strategies. PMID:25424006

  5. Transcriptomic analysis of tail regeneration in the lizard Anolis carolinensis reveals activation of conserved vertebrate developmental and repair mechanisms.

    Science.gov (United States)

    Hutchins, Elizabeth D; Markov, Glenn J; Eckalbar, Walter L; George, Rajani M; King, Jesse M; Tokuyama, Minami A; Geiger, Lauren A; Emmert, Nataliya; Ammar, Michael J; Allen, April N; Siniard, Ashley L; Corneveaux, Jason J; Fisher, Rebecca E; Wade, Juli; DeNardo, Dale F; Rawls, J Alan; Huentelman, Matthew J; Wilson-Rawls, Jeanne; Kusumi, Kenro

    2014-01-01

    Lizards, which are amniote vertebrates like humans, are able to lose and regenerate a functional tail. Understanding the molecular basis of this process would advance regenerative approaches in amniotes, including humans. We have carried out the first transcriptomic analysis of tail regeneration in a lizard, the green anole Anolis carolinensis, which revealed 326 differentially expressed genes activating multiple developmental and repair mechanisms. Specifically, genes involved in wound response, hormonal regulation, musculoskeletal development, and the Wnt and MAPK/FGF pathways were differentially expressed along the regenerating tail axis. Furthermore, we identified 2 microRNA precursor families, 22 unclassified non-coding RNAs, and 3 novel protein-coding genes significantly enriched in the regenerating tail. However, high levels of progenitor/stem cell markers were not observed in any region of the regenerating tail. Furthermore, we observed multiple tissue-type specific clusters of proliferating cells along the regenerating tail, not localized to the tail tip. These findings predict a different mechanism of regeneration in the lizard than the blastema model described in the salamander and the zebrafish, which are anamniote vertebrates. Thus, lizard tail regrowth involves the activation of conserved developmental and wound response pathways, which are potential targets for regenerative medical therapies.

  6. Periodicity in Attachment Organelle Revealed by Electron Cryotomography Suggests Conformational Changes in Gliding Mechanism of Mycoplasma pneumoniae

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    Akihiro Kawamoto

    2016-04-01

    Full Text Available Mycoplasma pneumoniae, a pathogenic bacterium, glides on host surfaces using a unique mechanism. It forms an attachment organelle at a cell pole as a protrusion comprised of knoblike surface structures and an internal core. Here, we analyzed the three-dimensional structure of the organelle in detail by electron cryotomography. On the surface, knoblike particles formed a two-dimensional array, albeit with limited regularity. Analyses using a nonbinding mutant and an antibody showed that the knoblike particles correspond to a naplike structure that has been observed by negative-staining electron microscopy and is likely to be formed as a complex of P1 adhesin, the key protein for binding and gliding. The paired thin and thick plates feature a rigid hexagonal lattice and striations with highly variable repeat distances, respectively. The combination of variable and invariant structures in the internal core and the P1 adhesin array on the surface suggest a model in which axial extension and compression of the thick plate along a rigid thin plate is coupled with attachment to and detachment from the substrate during gliding.

  7. Stuck at work? Quantitative proteomics of environmental wine yeast strains reveals the natural mechanism of overcoming stuck fermentation.

    Science.gov (United States)

    Szopinska, Aleksandra; Christ, Eva; Planchon, Sebastien; König, Helmut; Evers, Daniele; Renaut, Jenny

    2016-02-01

    During fermentation oenological yeast cells are subjected to a number of different stress conditions and must respond rapidly to the continuously changing environment of this harsh ecological niche. In this study we gained more insights into the cell adaptation mechanisms by linking proteome monitoring with knowledge on physiological behaviour of different strains during fermentation under model winemaking conditions. We used 2D-DIGE technology to monitor the proteome evolution of two newly discovered environmental yeast strains Saccharomyces bayanus and triple hybrid Saccharomyces cerevisiae × Saccharomyces kudriavzevii × S. bayanus and compared them to data obtained for the commercially available S. cerevisiae strain. All strains examined showed (i) different fermentative behaviour, (ii) stress resistance as well as (iii) susceptibility to stuck fermentation which was reflected in significant differences in protein expression levels. During our research we identified differentially expressed proteins in 155 gel spots which correspond to 70 different protein functions. Differences of expression between strains were observed mainly among proteins involved in stress response, proteins degradation pathways, cell redox homeostasis and amino acids biosynthesis. Interestingly, the newly discovered triple hybrid S. cerevisiae × S. kudriavzevii × S. bayanus strain which has the ability to naturally restart stuck fermentation showed a very strong induction of expression of two proteolytic enzymes: Pep4 and Prc1 that appear as numerous isoforms on the gel image and which may be the key to its unique properties. This study is an important step towards the better understanding of wine fermentations at a molecular level.

  8. Transcriptome profiling reveals the regulatory mechanism underlying pollination dependent and parthenocarpic fruit set mainly mediated by auxin and gibberellin.

    Science.gov (United States)

    Tang, Ning; Deng, Wei; Hu, Guojian; Hu, Nan; Li, Zhengguo

    2015-01-01

    Fruit set is a key process for crop production in tomato which occurs after successful pollination and fertilization naturally. However, parthenocarpic fruit development can be uncoupled from fertilization triggered by exogenous auxin or gibberellins (GAs). Global transcriptome knowledge during fruit initiation would help to characterize the molecular mechanisms by which these two hormones regulate pollination-dependent and -independent fruit set. In this work, digital gene expression tag profiling (DGE) technology was applied to compare the transcriptomes from pollinated and 2, 4-D/GA3-treated ovaries. Activation of carbohydrate metabolism, cell division and expansion as well as the down-regulation of MADS-box is a comprehensive regulatory pathway during pollination-dependent and parthenocarpic fruit set. The signaling cascades of auxin and GA are significantly modulated. The feedback regulations of Aux/IAAs and DELLA genes which functioned to fine-tune auxin and GA response respectively play fundamental roles in triggering fruit initiation. In addition, auxin regulates GA synthesis via up-regulation of GA20ox1 and down-regulation of KNOX. Accordingly, the effect of auxin on fruit set is mediated by GA via ARF2 and IAA9 down-regulation, suggesting that both pollination-dependent and parthenocarpic fruit set depend on the crosstalk between auxin and GA. This study characterizes the transcriptomic features of ovary development and more importantly unravels the integral roles of auxin and GA on pollination-dependent and parthenocarpic fruit set.

  9. Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche

    Energy Technology Data Exchange (ETDEWEB)

    Morin, Emmanuelle; Kohler, Annegret; Baker, Adam R.; Foulongne-Oriol, Marie; Lombard, Vincent; Nagy, Laszlo G.; Ohm, Robin A.; Patyshakuliyeva, Aleksandrina; Brun, Annick; Aerts, Andrea L.; Bailey, Andrew M.; Billette, Christophe; Coutinho, Pedro M.; Deakin, Greg; Doddapaneni, Harshavardhan; Floudas, Dimitrios; Grimwood, Jane; Hilden, Kristiina; Kues, Ursula; LaButti, Kurt M.; Lapidus, Alla; Lindquist, Erika A.; Lucas, Susan M.; Murat, Claude; Riley, Robert W.; Salamov, Asaf A.; Schmutz, Jeremy; Subramanian, Venkataramanan; Wosten, Han A. B.; Xu, Jianping; Eastwood, Daniel C.; Foster, Gary D.; Sonnenberg, Anton S. M.; Cullen, Dan; de Vries, Ronald P.; Lundell, Taina; Hibbett, David S.; Henrissat, Bernard; Burton, Kerry S.; Kerrigan, Richard W.; Challen, Michael P.; Grigoriev, Igor V.; Martin, Francis

    2012-04-27

    Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the button mushroom forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost and during mushroom formation. The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation are more highly expressed in compost. The striking expansion of heme-thiolate peroxidases and etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics.

  10. Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche.

    Science.gov (United States)

    Morin, Emmanuelle; Kohler, Annegret; Baker, Adam R; Foulongne-Oriol, Marie; Lombard, Vincent; Nagy, Laszlo G; Ohm, Robin A; Patyshakuliyeva, Aleksandrina; Brun, Annick; Aerts, Andrea L; Bailey, Andrew M; Billette, Christophe; Coutinho, Pedro M; Deakin, Greg; Doddapaneni, Harshavardhan; Floudas, Dimitrios; Grimwood, Jane; Hildén, Kristiina; Kües, Ursula; Labutti, Kurt M; Lapidus, Alla; Lindquist, Erika A; Lucas, Susan M; Murat, Claude; Riley, Robert W; Salamov, Asaf A; Schmutz, Jeremy; Subramanian, Venkataramanan; Wösten, Han A B; Xu, Jianping; Eastwood, Daniel C; Foster, Gary D; Sonnenberg, Anton S M; Cullen, Dan; de Vries, Ronald P; Lundell, Taina; Hibbett, David S; Henrissat, Bernard; Burton, Kerry S; Kerrigan, Richard W; Challen, Michael P; Grigoriev, Igor V; Martin, Francis

    2012-10-23

    Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the "button mushroom" forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost and during mushroom formation. The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation are more highly expressed in compost. The striking expansion of heme-thiolate peroxidases and β-etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics.

  11. Structural and Biochemical Characterization of a Cyanobacterial PP2C Phosphatase Reveals Insights into Catalytic Mechanism and Substrate Recognition

    Directory of Open Access Journals (Sweden)

    Yunlong Si

    2016-04-01

    Full Text Available PP2C-type phosphatases play roles in signal transduction pathways related to abiotic stress. The cyanobacterial PP2C-type phosphatase tPphA specifically dephosphorylates the PII protein, which is a key regulator in cyanobacteria adapting to nitrogen-deficient environments. Previous studies have shown that residue His39 of tPphA is critical for the enzyme’s recognition of the PII protein; however, the manner in which this residue determines tPphA substrate specificity is unknown. Here, we solved the crystal structure of H39A, a tPphA variant. The structure revealed that the mutation of residue His39 to alanine changes the conformation and the flexibility of the loop in which residue His39 is located, and these changes affect the substrate specificity of tPphA. Moreover, previous studies have assumed that the FLAP subdomain and the third metal (M3 of tPphA could mutually influence each other to regulate PP2C catalytic activity and substrate specificity. However, despite the variable conformations adopted by the FLAP subdomain, the position of M3 was consistent in the tPphA structure. These results indicate that the FLAP subdomain does not influence M3 and vice versa. In addition, a small screen of tPphA inhibitors was performed. Sanguinarine and Ni2+ were found to be the most effective inhibitors among the assayed chemicals. Finally, the dimeric form of tPphA was stabilized by cross-linkers and still exhibited catalytic activity towards p-nitrophenyl phosphate.

  12. Structure of the CaMKIIdelta/calmodulin complex reveals the molecular mechanism of CaMKII kinase activation.

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    Peter Rellos

    Full Text Available UNLABELLED: Long-term potentiation (LTP, a long-lasting enhancement in communication between neurons, is considered to be the major cellular mechanism underlying learning and memory. LTP triggers high-frequency calcium pulses that result in the activation of Calcium/Calmodulin (CaM-dependent kinase II (CaMKII. CaMKII acts as a molecular switch because it remains active for a long time after the return to basal calcium levels, which is a unique property required for CaMKII function. Here we describe the crystal structure of the human CaMKIIdelta/Ca2+/CaM complex, structures of all four human CaMKII catalytic domains in their autoinhibited states, as well as structures of human CaMKII oligomerization domains in their tetradecameric and physiological dodecameric states. All four autoinhibited human CaMKIIs were monomeric in the determined crystal structures but associated weakly in solution. In the CaMKIIdelta/Ca2+/CaM complex, the inhibitory region adopted an extended conformation and interacted with an adjacent catalytic domain positioning T287 into the active site of the interacting protomer. Comparisons with autoinhibited CaMKII structures showed that binding of calmodulin leads to the rearrangement of residues in the active site to a conformation suitable for ATP binding and to the closure of the binding groove for the autoinhibitory helix by helix alphaD. The structural data, together with biophysical interaction studies, reveals the mechanism of CaMKII activation by calmodulin and explains many of the unique regulatory properties of these two essential signaling molecules. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3-D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the Web plugin are available in Text S1.

  13. Mechanism of porcine liver xanthine oxidoreductase mediated N-oxide reduction of cyadox as revealed by docking and mutagenesis studies.

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    Chigang Chen

    Full Text Available Xanthine oxidoreductase (XOR is a cytoplasmic molybdenum-containing oxidoreductase, catalyzing both endogenous purines and exogenous compounds. It is suggested that XOR in porcine hepatocytes catalyzes the N-oxide reduction of quinoxaline 1,4-di-N-oxides (QdNOs. To elucidate the molecular mechanism underlying this metabolism, the cDNA of porcine XOR was cloned and heterologously expressed in Spodoptera frugiperda insect cells. The bovine XOR, showing sequence identity of 91% to porcine XOR, was employed as template for homology modeling. By docking cyadox, a representative compound of QdNOs, into porcine XOR model, eight amino acid residues, Gly47, Asn352, Ser360, Arg427, Asp430, Asp431, Ser1227 and Lys1230, were located at distances of less than 4Å to cyadox. Site-directed mutagenesis was performed to analyze their catalytic functions. Compared with wild type porcine XOR, G47A, S360P, D431A, S1227A, and K1230A displayed altered kinetic parameters in cyadox reduction, similarly to that in xanthine oxidation, indicating these mutations influenced electron-donating process of xanthine before subsequent electron transfer to cyadox to fulfill the N-oxide reduction. Differently, R427E and D430H, both located in the 424-434 loop, exhibited a much lower K(m and a decreased V(max respectively in cyadox reduction. Arg427 may be related to the substrate binding of porcine XOR to cyadox, and Asp430 is suggested to be involved in the transfer of electron to cyadox. This study initially reveals the possible catalytic mechanism of porcine XOR in cyadox metabolism, providing with novel insights into the structure-function relationship of XOR in the reduction of exogenous di-N-oxides.

  14. A mechanism of gap junction docking revealed by functional rescue of a human-disease-linked connexin mutant.

    Science.gov (United States)

    Gong, Xiang-Qun; Nakagawa, So; Tsukihara, Tomitake; Bai, Donglin

    2013-07-15

    Gap junctions are unique intercellular channels formed by the proper docking of two hemichannels from adjacent cells. Each hemichannel is a hexamer of connexins (Cxs) - the gap junction subunits, which are encoded by 21 homologous genes in the human genome. The docking of two hemichannels to form a functional gap junction channel is only possible between compatible Cxs, but the underlying molecular mechanism is unclear. On the basis of the crystal structure of the Cx26 gap junction, we developed homology models for homotypic and heterotypic channels from Cx32 and/or Cx26; these models predict six hydrogen bonds at the docking interface of each pair of the second extracellular domain (E2). A Cx32 mutation N175H and a human-disease-linked mutant N175D were predicted to lose the majority of the hydrogen bonds at the E2 docking-interface; experimentally both mutations failed to form morphological and functional gap junctions. To restore the lost hydrogen bonds, two complementary Cx26 mutants - K168V and K168A were designed to pair with the Cx32 mutants. When docked with Cx26K168V or K168A, the Cx32N175H mutant was successfully rescued morphologically and functionally in forming gap junction channels, but not Cx32 mutant N175Y. By testing more homotypic and heterotypic Cx32 and/or Cx26 mutant combinations, it is revealed that a minimum of four hydrogen bonds at each E2-docking interface are required for proper docking and functional channel formation between Cx26 and Cx32 hemichannels. Interestingly, the disease-linked Cx32N175D could be rescued by Cx26D179N, which restored five hydrogen bonds at the E2-docking interface. Our findings not only provide a mechanism for gap junction docking for Cx26 and Cx32 hemichannels, but also a potential therapeutic strategy for gap junction channelopathies.

  15. Teaching assistants' performance at identifying common introductory student difficulties in mechanics revealed by the Force Concept Inventory

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    Maries, Alexandru; Singh, Chandralekha

    2016-06-01

    The Force Concept Inventory (FCI) has been widely used to assess student understanding of introductory mechanics concepts by a variety of educators and physics education researchers. One reason for this extensive use is that many of the items on the FCI have strong distractor choices which correspond to students' alternate conceptions in mechanics. Instruction is unlikely to be effective if instructors do not know the common alternate conceptions of introductory physics students and explicitly take into account students' initial knowledge states in their instructional design. Here, we discuss research involving the FCI to evaluate one aspect of the pedagogical content knowledge of teaching assistants (TAs): knowledge of introductory student alternate conceptions in mechanics as revealed by the FCI. For each item on the FCI, the TAs were asked to identify the most common incorrect answer choice of introductory physics students. This exercise was followed by a class discussion with the TAs related to this task, including the importance of knowing student difficulties in teaching and learning. Then, we used FCI pretest and post-test data from a large population (˜900 ) of introductory physics students to assess the extent to which TAs were able to identify alternate conceptions of introductory students related to force and motion. In addition, we carried out think-aloud interviews with graduate students who had more than two semesters of teaching experience in recitations to examine how they reason about the task. We find that while the TAs, on average, performed better than random guessing at identifying introductory students' difficulties with FCI content, they did not identify many common difficulties that introductory physics students have after traditional instruction. We discuss specific alternate conceptions, the extent to which TAs are able to identify them, and results from the think-aloud interviews that provided valuable information about why TAs sometimes

  16. Teaching assistants’ performance at identifying common introductory student difficulties in mechanics revealed by the Force Concept Inventory

    Directory of Open Access Journals (Sweden)

    Alexandru Maries

    2016-05-01

    Full Text Available The Force Concept Inventory (FCI has been widely used to assess student understanding of introductory mechanics concepts by a variety of educators and physics education researchers. One reason for this extensive use is that many of the items on the FCI have strong distractor choices which correspond to students’ alternate conceptions in mechanics. Instruction is unlikely to be effective if instructors do not know the common alternate conceptions of introductory physics students and explicitly take into account students’ initial knowledge states in their instructional design. Here, we discuss research involving the FCI to evaluate one aspect of the pedagogical content knowledge of teaching assistants (TAs: knowledge of introductory student alternate conceptions in mechanics as revealed by the FCI. For each item on the FCI, the TAs were asked to identify the most common incorrect answer choice of introductory physics students. This exercise was followed by a class discussion with the TAs related to this task, including the importance of knowing student difficulties in teaching and learning. Then, we used FCI pretest and post-test data from a large population (∼900 of introductory physics stud