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Sample records for reveals extended roles

  1. Longitudinal changes in extended roles in radiography

    International Nuclear Information System (INIS)

    Price, R.C.; Miller, L.R.; Mellor, F.

    2002-01-01

    Purpose: The purpose of this research was to identify the extent and scope of changes to radiography practice. Method: Questionnaires were sent to radiology managers at acute National Health Service (NHS) hospitals in 2000 as a follow-up to an initial survey conducted in 1998. Information was sought on region, teaching/non-teaching status, the nature of extended role tasks undertaken and the year in which these tasks were first undertaken. Results: Some 172 questionnaires were returned from a total of 253 dispatched (68%). In 161 hospitals radiographers administered intravenous injections and performed barium enemas in 119 hospitals, while a red-dot system was in operation in 141 hospitals. Reporting by radiographers had increased since 1998. Replies indicated that, at 124 hospitals, radiographers were reporting in ultrasound. Skeletal reporting was the second most-frequently-reported activity, with 63 hospitals indicating that radiographers reported in this modality, while barium enemas were reported by radiographers in 34 hospitals. Reporting was also undertaken by radiographers in mammography, nuclear medicine, paediatrics and chest radiography in a small minority of hospitals. There was no statistically significant difference between the adoption of tasks in non-teaching and teaching hospitals with the exception of radiographer-performed barium enemas (P=0.014) and red-dot systems (P=0.05). Radiographers were more likely to undertake both of these activities at non-teaching hospitals than in teaching hospitals. Regional differences were apparent in reporting, with a greater prevalence in the English regions than Northern Ireland, Scotland and Wales. Conclusion: The study provided data that demonstrated the extent to which hospitals are utilizing the developing skills of radiographers. This has important implications for future education and training needs for radiographers and for delivery of imaging services

  2. The role of extended family in diverse teens’ sexual health

    OpenAIRE

    Grossman, Jennifer M.; Tracy, Allison; Richer, Amanda; Erkut, Sumru

    2014-01-01

    Despite increasing extended family involvement in childrearing, particularly in minority families, few studies investigate their role in talking with teens about sex or how this relates to teens’ sexual behavior. This mixed methods study assesses extended family sexuality communication through a survey of 1492 diverse middle school students and interviews with 32 students. Logistic regression shows that participants who report having had sex are more likely to report talking with extended fam...

  3. Radiology Physician Extenders: A Literature Review of the History and Current Roles of Physician Extenders in Medical Imaging.

    Science.gov (United States)

    Sanders, Vicki L; Flanagan, Jennifer

    2015-01-01

    The purpose of the literature review was to assess the origins of radiology physician extenders and examine the current roles found in the literature of advanced practice physician extenders within medical imaging. Twenty-six articles relating to physician assistants (PAs), nurse practitioners (NPs), radiologist assistants (RAs), and nuclear medicine advanced associates (NMAAs) were reviewed to discern similarities and differences in history, scope of practice, and roles in the medical imaging field. The literature showed PAs and NPs are working mostly in interventional radiology. PAs, NPs, and RAs perform similar tasks in radiology, including history and physicals, evaluation and management, preprocedure work-up, obtaining informed consent, initial observations/reports, and post-procedure follow-up. NPs and PAs perform a variety of procedures but most commonly vascular access, paracentesis, and thoracentesis. RAs perform gastrointestinal, genitourinary, nonvascular invasive fluoroscopy procedures, and vascular access procedures. The review revealed NMAAs are working in an advanced role, but no specific performances of procedures was found in the literature, only suggested tasks and clinical competencies. PAs, NPs, and RAs are currently the three main midlevel providers used in medical imaging. These midlevel providers are being used in a variety of ways to increase the efficiency of the radiologist and provide diagnostic and therapeutic radiologic procedures to patients. NMAAs are being used in medical imaging but little literature is available on current roles in clinical practice. More research is needed to assess the exact procedures and duties being performed by these medical imaging physician extenders.

  4. Hazard identification by extended multilevel flow modelling with function roles

    DEFF Research Database (Denmark)

    Wu, Jing; Zhang, Laibin; Jørgensen, Sten Bay

    2014-01-01

    ) is extended with functi on roles to complete HAZOP studies in principle. A graphical MFM editor, which is combined with the reasoning engine (MFM Workbench) developed by DTU is applied to automate HAZOP studies. The method is proposed to suppor t the ‘brain-storming’ sessions in traditional HAZOP analysis...

  5. Social phenotype extended to communities: expanded multilevel social selection analysis reveals fitness consequences of interspecific interactions.

    Science.gov (United States)

    Campobello, Daniela; Hare, James F; Sarà, Maurizio

    2015-04-01

    In social species, fitness consequences are associated with both individual and social phenotypes. Social selection analysis has quantified the contribution of conspecific social traits to individual fitness. There has been no attempt, however, to apply a social selection approach to quantify the fitness implications of heterospecific social phenotypes. Here, we propose a novel social selection based approach integrating the role of all social interactions at the community level. We extended multilevel selection analysis by including a term accounting for the group phenotype of heterospecifics. We analyzed nest activity as a model social trait common to two species, the lesser kestrel (Falco naumanni) and jackdaw (Corvus monedula), nesting in either single- or mixed-species colonies. By recording reproductive outcome as a measure of relative fitness, our results reveal an asymmetric system wherein only jackdaw breeding performance was affected by the activity phenotypes of both conspecific and heterospecific neighbors. Our model incorporating heterospecific social phenotypes is applicable to animal communities where interacting species share a common social trait, thus allowing an assessment of the selection pressure imposed by interspecific interactions in nature. Finally, we discuss the potential role of ecological limitations accounting for random or preferential assortments among interspecific social phenotypes, and the implications of such processes to community evolution. © 2015 The Author(s).

  6. Ensuring standards for the extended role of optometry.

    Science.gov (United States)

    Jarvis, Ian; Ker, Jean

    2014-06-01

    In optometry, as in other health professions, such as pharmacy and nursing, there has been an expansion in the role of the optometrist in primary eye care, taking on aspects of care such as prescribing, which was traditionally undertaken by general practitioners. In optometry, this extended role involves monitoring eye conditions that do not require treatment or monitoring in secondary care, roles that were traditionally referred on. However, any extended role requires educational support. The development of a national educational programme can prevent any duplication of effort and provide a system of disseminating support to practitioners, who often work in isolation. The National Education Programme was developed and supported by the National Health Service (NHS) Education for Scotland (NES). This a national body responsible for supporting NHS services in Scotland, by developing and delivering education and training for those who work in NHS Scotland. Optometrists were given educational support in three main stages: simulation of component skills; safe learning in a simulated health care context; and support for the transfer of those skills to practice, including the use of mentoring via e-mail. The evaluation of this three-stage process has been positive. To further develop any expanded role in health care practice, safe guidelines for practice are required. Simulation can provide support at the start of this process in setting standards. However, mentorship in the practice area, as reported in this study, was required to embed newly acquired skills. © 2014 John Wiley & Sons Ltd.

  7. The Extended Role of the Communication Partner in AAC interaction

    DEFF Research Database (Denmark)

    Pilesjö, Maja Sigurd

    in the field.FindingsThe findings demonstrate that the speaking co-participant is sensitive to the actions of the person with impairments’ display of attention and actions within the local ‘contextual configuration’ (Goodwin, 2000). Due to differing resources, the relevant options for the next move......The Extended Role of the Communication Partner in AAC interactionIntroductionThe speaking communication partner in AAC interaction has a unique role (Blackstone et al., 2007). Interactional research in the field of AAC has, for instance, found that the interaction is characterized by a great deal......-analyses on naturally occurring social interaction, this session will demonstrate tasks that the speaking communication partner can undertake in AAC- interaction.Method and dataThe method of Conversation analysis (CA) is used in the current study (Higginbotham & Engelke, 2013). The general aim of CA is at getting...

  8. Longitudinal changes in extended roles in radiography: A new perspective

    Energy Technology Data Exchange (ETDEWEB)

    Price, R.C. [School of Paramedic Sciences, Physiotherapy and Radiography, Faculty of Health and Human Sciences, University of Hertfordshire, College Lane, Hatfield, Hertfordshire AL10 9AB (United Kingdom)]. E-mail: r.c.price@herts.ac.uk; Le Masurier, S.B. [University of Hertfordshire, College Lane, Hatfield, Hertfordshire AL10 9AB (United Kingdom)

    2007-02-15

    Purpose: The purpose of this research was to update data on the scope of changes to radiographic practice since the principal author's last survey in 2000. The study also sought to identify any regional patterns in the adoption and diffusion of extended roles and to map the implementation of the 4-tier structure. Method: Structured questionnaires were sent to radiology managers at acute National Health Service (NHS) trusts across the United Kingdom. Information sought included region, teaching/non-teaching status, the nature of extended role tasks undertaken and the year in which these tasks were adopted, numbers of radiographers and radiologists in post. Information was also sought on the implementation of the '4-tier structure'. Results: Some 177 questionnaires were returned from a total of 258 dispatched giving a 68.6% response rate. In 166 trusts, radiographers administered intravenous injections; they performed barium enemas in 147 trusts and barium meals in 19 trusts, while a red dot system was in operation in 143 trusts. Each category showed an increase from that reported in 2000. Likewise reporting by radiographers had increased since 2000. Responses indicated that at 146 trusts, radiographers were reporting in ultrasound; reporting of the appendicular skeleton was undertaken at 81 trusts and axial skeletal reporting at 70 trusts. Barium enemas were reported by radiographers in 78 trusts. Reporting was also undertaken by radiographers on barium meals, mammography, nuclear medicine, paediatric and chest radiography; and all showed an increase in frequency since 2000. Regional differences were again apparent in reporting, with a greater prevalence in the English regions, with the exception of London. In respect of the 4-tier structure, 59% of the sample employed assistant practitioners, 47% advanced practitioners and 3% employed consultants. The numbers reported in each category (excluding practitioners) were 158 assistants, 623 advanced

  9. Longitudinal changes in extended roles in radiography: A new perspective

    International Nuclear Information System (INIS)

    Price, R.C.; Le Masurier, S.B.

    2007-01-01

    Purpose: The purpose of this research was to update data on the scope of changes to radiographic practice since the principal author's last survey in 2000. The study also sought to identify any regional patterns in the adoption and diffusion of extended roles and to map the implementation of the 4-tier structure. Method: Structured questionnaires were sent to radiology managers at acute National Health Service (NHS) trusts across the United Kingdom. Information sought included region, teaching/non-teaching status, the nature of extended role tasks undertaken and the year in which these tasks were adopted, numbers of radiographers and radiologists in post. Information was also sought on the implementation of the '4-tier structure'. Results: Some 177 questionnaires were returned from a total of 258 dispatched giving a 68.6% response rate. In 166 trusts, radiographers administered intravenous injections; they performed barium enemas in 147 trusts and barium meals in 19 trusts, while a red dot system was in operation in 143 trusts. Each category showed an increase from that reported in 2000. Likewise reporting by radiographers had increased since 2000. Responses indicated that at 146 trusts, radiographers were reporting in ultrasound; reporting of the appendicular skeleton was undertaken at 81 trusts and axial skeletal reporting at 70 trusts. Barium enemas were reported by radiographers in 78 trusts. Reporting was also undertaken by radiographers on barium meals, mammography, nuclear medicine, paediatric and chest radiography; and all showed an increase in frequency since 2000. Regional differences were again apparent in reporting, with a greater prevalence in the English regions, with the exception of London. In respect of the 4-tier structure, 59% of the sample employed assistant practitioners, 47% advanced practitioners and 3% employed consultants. The numbers reported in each category (excluding practitioners) were 158 assistants, 623 advanced practitioners and six

  10. Limited role for extended maintenance temozolomide for newly diagnosed glioblastoma.

    Science.gov (United States)

    Gramatzki, Dorothee; Kickingereder, Philipp; Hentschel, Bettina; Felsberg, Jörg; Herrlinger, Ulrich; Schackert, Gabriele; Tonn, Jörg-Christian; Westphal, Manfred; Sabel, Michael; Schlegel, Uwe; Wick, Wolfgang; Pietsch, Torsten; Reifenberger, Guido; Loeffler, Markus; Bendszus, Martin; Weller, Michael

    2017-04-11

    To explore an association with survival of modifying the current standard of care for patients with newly diagnosed glioblastoma of surgery followed by radiotherapy plus concurrent and 6 cycles of maintenance temozolomide chemotherapy (TMZ/RT → TMZ) by extending TMZ beyond 6 cycles. The German Glioma Network cohort was screened for patients with newly diagnosed glioblastoma who received TMZ/RT → TMZ and completed ≥6 cycles of maintenance chemotherapy without progression. Associations of clinical patient characteristics, molecular markers, and residual tumor determined by magnetic resonance imaging after 6 cycles of TMZ with progression-free survival (PFS) and overall survival (OS) were analyzed with the log-rank test. Multivariate analyses using the Cox proportional hazards model were performed to assess associations of prolonged TMZ use with outcome. Sixty-one of 142 identified patients received at least 7 maintenance TMZ cycles (median 11, range 7-20). Patients with extended maintenance TMZ treatment had better PFS (20.5 months, 95% confidence interval [CI] 17.7-23.3, vs 17.2 months, 95% CI 10.2-24.2, p = 0.035) but not OS (32.6 months, 95% CI 28.9-36.4, vs 33.2 months, 95% CI 25.3-41.0, p = 0.126). However, there was no significant association of prolonged TMZ chemotherapy with PFS (hazard ratio [HR] = 0.8, 95% CI 0.4-1.6, p = 0.559) or OS (HR = 1.6, 95% CI 0.8-3.3, p = 0.218) adjusted for age, extent of resection, Karnofsky performance score, presence of residual tumor, O 6 -methylguanine DNA methyltransferase (MGMT) promoter methylation status, or isocitrate dehydrogenase ( IDH ) mutation status. These data may not support the practice of prolonging maintenance TMZ chemotherapy beyond 6 cycles. This study provides Class III evidence that in patients with newly diagnosed glioblastoma, prolonged TMZ chemotherapy does not significantly increase PFS or OS. © 2017 American Academy of Neurology.

  11. An Extended Role Based Access Control Method for XML Documents

    Institute of Scientific and Technical Information of China (English)

    MENG Xiao-feng; LUO Dao-feng; OU Jian-bo

    2004-01-01

    As XML has been increasingly important as the Data-change format of Internet and Intranet, access-control-on-XML-properties rises as a new issue.Role-based access control (RBAC) is an access control method that has been widely used in Internet, Operation System and Relation Data Base these 10 years.Though RBAC is already relatively mature in the above fields, new problems occur when it is used in XML properties.This paper proposes an integrated model to resolve these problems, after the fully analysis on the features of XML and RBAC.

  12. Approaching an Extended Role for Meeting Special Educational Needs in High Schools.

    Science.gov (United States)

    Mills, Keith

    1988-01-01

    The paper looks at staffing issues in meeting special educational needs in high schools in Manchester, United Kingdom. The concerns of Heads of Departments (N=26) included improved training and inservice, their extended roles within their schools, the support role of special needs staff, and the need for more planning and evaluation. (Author/DB)

  13. Copy number variants in extended autism spectrum disorder families reveal candidates potentially involved in autism risk.

    Directory of Open Access Journals (Sweden)

    Daria Salyakina

    Full Text Available Copy number variations (CNVs are a major cause of genetic disruption in the human genome with far more nucleotides being altered by duplications and deletions than by single nucleotide polymorphisms (SNPs. In the multifaceted etiology of autism spectrum disorders (ASDs, CNVs appear to contribute significantly to our understanding of the pathogenesis of this complex disease. A unique resource of 42 extended ASD families was genotyped for over 1 million SNPs to detect CNVs that may contribute to ASD susceptibility. Each family has at least one avuncular or cousin pair with ASD. Families were then evaluated for co-segregation of CNVs in ASD patients. We identified a total of five deletions and seven duplications in eleven families that co-segregated with ASD. Two of the CNVs overlap with regions on 7p21.3 and 15q24.1 that have been previously reported in ASD individuals and two additional CNVs on 3p26.3 and 12q24.32 occur near regions associated with schizophrenia. These findings provide further evidence for the involvement of ICA1 and NXPH1 on 7p21.3 in ASD susceptibility and highlight novel ASD candidates, including CHL1, FGFBP3 and POUF41. These studies highlight the power of using extended families for gene discovery in traits with a complex etiology.

  14. Extended Impact of Pin1 Catalytic Loop Phosphorylation Revealed by S71E Phosphomimetic.

    Science.gov (United States)

    Mahoney, Brendan J; Zhang, Meiling; Zintsmaster, John S; Peng, Jeffrey W

    2018-03-02

    Pin1 is a two-domain human protein that catalyzes the cis-trans isomerization of phospho-Ser/Thr-Pro (pS/T-P) motifs in numerous cell-cycle regulatory proteins. These pS/T-P motifs bind to Pin1's peptidyl-prolyl isomerase (PPIase) domain in a catalytic pocket, between an extended catalytic loop and the PPIase domain core. Previous studies showed that post-translational phosphorylation of S71 in the catalytic loop decreases substrate binding affinity and isomerase activity. To define the origins for these effects, we investigated a phosphomimetic Pin1 mutant, S71E-Pin1, using solution NMR. We find that S71E perturbs not only its host loop but also the nearby PPIase core. The perturbations identify a local network of hydrogen bonds and salt bridges that is more extended than previously thought, and includes interactions between the catalytic loop and the α2/α3 turn in the PPIase core. Explicit-solvent molecular dynamics simulations and phylogenetic analysis suggest that these interactions act as conserved "latches" between the loop and PPIase core that enhance binding of phosphorylated substrates, as they are absent in PPIases lacking pS/T-P specificity. Our results suggest that S71 is a hub residue within an electrostatic network primed for phosphorylation, and may illustrate a common mechanism of phosphorylation-mediated allostery. Copyright © 2018 Elsevier Ltd. All rights reserved.

  15. Developmental Reorganization of the Core and Extended Face Networks Revealed by Global Functional Connectivity.

    Science.gov (United States)

    Wang, Xu; Zhu, Qi; Song, Yiying; Liu, Jia

    2017-08-28

    Prior studies on development of functional specialization in human brain mainly focus on age-related increases in regional activation and connectivity among regions. However, a few recent studies on the face network demonstrate age-related decrease in face-specialized activation in the extended face network (EFN), in addition to increase in activation in the core face network (CFN). Here we used a voxel-based global brain connectivity approach to investigate whether development of the face network exhibited both increase and decrease in network connectivity. We found the voxel-wise resting-state functional connectivity (FC) within the CFN increased with age in bilateral posterior superior temporal sulcus, suggesting the integration of the CFN during development. Interestingly, the FC of the voxels in the EFN to the right fusiform face area and occipital face area decreased with age, suggesting that the CFN segregated from the EFN during development. Moreover, the age-related connectivity in the CFN was related to behavioral performance in face processing. Overall, our study demonstrated developmental reorganization of the face network achieved by both integration within the CFN and segregation of the CFN from the EFN, which may account for the simultaneous increases and decreases in neural activation during the development of the face network. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  16. Extended Schools: The School's Role in Encouraging Behaviour for Learning Outside the Classroom that Supports Learning within. A Response to the "Every Child Matters" and Extended Schools Initiatives

    Science.gov (United States)

    Lewis, Jeff

    2006-01-01

    Extended schools have an important role to play in a society that is undergoing significant change. This article moves beyond the normal "extended schools are a good thing" rhetoric. It points out the risks of "bolt on" policy initiatives, especially with regard to "pupils at risk" and argues against the superficial adoption of such policy. In a…

  17. What Can Students Learn in an Extended Role-Play Simulation on Technology and Society?

    Science.gov (United States)

    Loui, Michael C.

    2009-01-01

    In a small course on technology and society, students participated in an extended role-play simulation for two weeks. Each student played a different adult character in a fictional community, which faces technological decisions in three scenarios set in the near future. The three scenarios involved stem cell research, nanotechnology, and privacy.…

  18. Canadian physiotherapists' views on certification, specialisation, extended role practice, and entry-level training in rheumatology

    Directory of Open Access Journals (Sweden)

    Sutton Evelyn

    2009-06-01

    Full Text Available Abstract Background Since the last decade there has been a gradual change of boundaries of health professions in providing arthritis care. In Canada, some facilities have begun to adopt new arthritis care models, some of which involve physiotherapists (PT working in extended roles. However, little is known about PTs' interests in these new roles. The primary objective of this survey was to determine the interests among orthopaedic physiotherapists (PTs in being a certified arthritis therapist, a PT specialized in arthritis, or an extended scope practitioner in rheumatology, and to explore the associated factors, including the coverage of arthritis content in the entry-level physiotherapy training. Methods Six hundred PTs practicing in orthopaedics in Canada were randomly selected to receive a postal survey. The questionnaire covered areas related to clinical practice, perceptions of rheumatology training received, and attitudes toward PT roles in arthritis care. Logistic regression models were developed to explore the associations between PTs' interests in pursuing each of the three extended scope practice designations and the personal/professional/attitudinal variables. Results We received 286 questionnaires (response rate = 47.7%; 258 contained usable data. The average length of time in practice was 15.4 years (SD = 10.4. About 1 in 4 PTs agreed that they were interested in assuming advanced practice roles (being a certified arthritis therapist = 28.9%, being a PT specialized in rheumatology = 23.3%, being a PT practitioner = 20.9%. Having a caseload of ≥ 40% in arthritis, having a positive attitude toward advanced practice roles in arthritis care and toward the formal credentialing process, and recognizing the difference between certification and specialisation were associated with an interest in pursing advanced practice roles. Conclusion Orthopaedic PTs in Canada indicated a fair level of interest in pursuing certification, specialisation

  19. The role of extended scope physiotherapists in managing patients with inflammatory arthropathies: a systematic review

    Directory of Open Access Journals (Sweden)

    Stanhope J

    2012-05-01

    Full Text Available Jessica Stanhope,1 Kate Beaton,1 Karen Grimmer-Somers,1 Joanne Morris21International Centre for Allied Health Evidence (iCAHE, University of South Australia, Adelaide, South Australia; 2ACT Government Health Directorate, Canberra, Australian Capital Territory, AustraliaObjectives: To review the literature to identify whether, and how, physiotherapists working in extended scope of practice (ESP engage with patients with inflammatory arthropathies. Measures of effectiveness of ESP were particularly sought.Methods: A comprehensive library database search was conducted to identify English language studies published in full text in peer-reviewed journals during the years 2002–2012. Studies were allocated into the National Health and Medical Research Council hierarchy of evidence, but were not critically appraised. Data was extracted on conditions treated, ESP roles and responsibilities, and effectiveness. Data was analyzed and reported descriptively.Results: We identified 123 studies, and included four. All were low hierarchy (highest being one level III_2 study. Commonly reported conditions were rheumatoid arthritis and ankylosing spondylitis. Information was provided on activities of role extension, such as triaging patients, monitoring and recommending changes to medications, referring to other health and medical professionals, and ordering and interpreting imaging. There was blurring between ESP and non-ESP roles. No study reported measures of effectiveness.Conclusion: There are descriptors of ESP physiotherapy activities, but no evidence of effectiveness of ESP physiotherapy in managing patients with inflammatory arthropathies.Keywords: ESP, extended scope, rheumatoid arthritis, ankylosing spondylitis, inflammatory arthropathy, physiotherapy

  20. Pivotal role of extended linker 2 in the activation of Gα by G protein-coupled receptor.

    Science.gov (United States)

    Huang, Jianyun; Sun, Yutong; Zhang, J Jillian; Huang, Xin-Yun

    2015-01-02

    G protein-coupled receptors (GPCRs) relay extracellular signals mainly to heterotrimeric G-proteins (Gαβγ) and they are the most successful drug targets. The mechanisms of G-protein activation by GPCRs are not well understood. Previous studies have revealed a signal relay route from a GPCR via the C-terminal α5-helix of Gα to the guanine nucleotide-binding pocket. Recent structural and biophysical studies uncover a role for the opening or rotating of the α-helical domain of Gα during the activation of Gα by a GPCR. Here we show that β-adrenergic receptors activate eight Gαs mutant proteins (from a screen of 66 Gαs mutants) that are unable to bind Gβγ subunits in cells. Five of these eight mutants are in the αF/Linker 2/β2 hinge region (extended Linker 2) that connects the Ras-like GTPase domain and the α-helical domain of Gαs. This extended Linker 2 is the target site of a natural product inhibitor of Gq. Our data show that the extended Linker 2 is critical for Gα activation by GPCRs. We propose that a GPCR via its intracellular loop 2 directly interacts with the β2/β3 loop of Gα to communicate to Linker 2, resulting in the opening and closing of the α-helical domain and the release of GDP during G-protein activation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Physiological roles revealed by ghrelin and ghrelin receptor deficient mice

    Science.gov (United States)

    Ghrelin is a hormone made in the stomach and known primarily for its growth hormone releasing and orexigenic properties. Nevertheless, ghrelin through its receptor, the GHS-R1a, has been shown to exert many roles including regulation of glucose homeostasis, memory & learning, food addiction and neur...

  2. Lipidomics profiling reveals the role of glycerophospholipid metabolism in psoriasis.

    Science.gov (United States)

    Zeng, Chunwei; Wen, Bo; Hou, Guixue; Lei, Li; Mei, Zhanlong; Jia, Xuekun; Chen, Xiaomin; Zhu, Wu; Li, Jie; Kuang, Yehong; Zeng, Weiqi; Su, Juan; Liu, Siqi; Peng, Cong; Chen, Xiang

    2017-10-01

    Psoriasis is a common and chronic inflammatory skin disease that is complicated by gene-environment interactions. Although genomic, transcriptomic, and proteomic analyses have been performed to investigate the pathogenesis of psoriasis, the role of metabolites in psoriasis, particularly of lipids, remains unclear. Lipids not only comprise the bulk of the cellular membrane bilayers but also regulate a variety of biological processes such as cell proliferation, apoptosis, immunity, angiogenesis, and inflammation. In this study, an untargeted lipidomics approach was used to study the lipid profiles in psoriasis and to identify lipid metabolite signatures for psoriasis through ultra-performance liquid chromatography-tandem quadrupole mass spectrometry. Plasma samples from 90 participants (45 healthy and 45 psoriasis patients) were collected and analyzed. Statistical analysis was applied to find different metabolites between the disease and healthy groups. In addition, enzyme-linked immunosorbent assay was performed to validate differentially expressed lipids in psoriatic patient plasma. Finally, we identified differential expression of several lipids including lysophosphatidic acid (LPA), lysophosphatidylcholine (LysoPC), phosphatidylinositol (PI), phosphatidylcholine (PC), and phosphatidic acid (PA); among these metabolites, LPA, LysoPC, and PA were significantly increased, while PC and PI were down-regulated in psoriasis patients. We found that elements of glycerophospholipid metabolism such as LPA, LysoPC, PA, PI, and PC were significantly altered in the plasma of psoriatic patients; this study characterizes the circulating lipids in psoriatic patients and provides novel insight into the role of lipids in psoriasis. © The Author 2017. Published by Oxford University Press.

  3. When proteomics reveals unsuspected roles: the plastoglobule example

    Directory of Open Access Journals (Sweden)

    Claire eBréhélin

    2013-04-01

    Full Text Available Plastoglobules are globular compartments found in plastids. Before initial proteomic studies were published, these particles were often viewed as passive lipid droplets whose unique role was to store lipids coming from the thylakoid turn-over, or to accumulate carotenoids in the chromoplasts. Yet, two proteomic studies, published concomitantly, suggested for the first time that plastoglobules are more than "junk cupboards" for lipids. Indeed, both studies demonstrated that plastoglobules do not only include structural proteins belonging to the plastoglobulin / fibrillin family, but also contain active enzymes. The specific plastoglobule localization of these enzymes has been confirmed by different approaches such as immunogold localization and GFP protein fusions, thus providing evidence that plastoglobules actively participate in diverse pathways of plastid metabolism. These proteomic studies have been the basis for numerous recent works investigating plastoglobule function. However, a lot still needs to be discovered about the molecular composition and the role of plastoglobules. In this chapter, we will describe how the proteomic approaches have launched new perspectives on plastoglobule functions.

  4. Jazz musicians reveal role of expectancy in human creativity.

    Science.gov (United States)

    Przysinda, Emily; Zeng, Tima; Maves, Kellyn; Arkin, Cameron; Loui, Psyche

    2017-12-01

    Creativity has been defined as the ability to produce work that is novel, high in quality, and appropriate to an audience. While the nature of the creative process is under debate, many believe that creativity relies on real-time combinations of known neural and cognitive processes. One useful model of creativity comes from musical improvisation, such as in jazz, in which musicians spontaneously create novel sound sequences. Here we use jazz musicians to test the hypothesis that individuals with training in musical improvisation, which entails creative generation of musical ideas, might process expectancy differently. We compare jazz improvisers, non-improvising musicians, and non-musicians in the domain-general task of divergent thinking, as well as the musical task of preference ratings for chord progressions that vary in expectation while EEGs were recorded. Behavioral results showed for the first time that jazz musicians preferred unexpected chord progressions. ERP results showed that unexpected stimuli elicited larger early and mid-latency ERP responses (ERAN and P3b), followed by smaller long-latency responses (Late Positivity Potential) in jazz musicians. The amplitudes of these ERP components were significantly correlated with behavioral measures of fluency and originality on the divergent thinking task. Together, results highlight the role of expectancy in creativity. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. The beneficial role of simultaneous splenectomy after extended hepatectomy: experimental study in pigs.

    Science.gov (United States)

    Athanasiou, Antonios; Papalois, Apostolos; Kontos, Michael; Griniatsos, John; Liakopoulos, Dimitrios; Spartalis, Eleftherios; Agrogiannis, George; Liakakos, Theodoros; Pikoulis, Emmanouil

    2017-02-01

    The role of hepatic hemodynamic modulation in the development of "small-for-size" syndrome (SFSS) after extended hepatectomy (EH) or living-donor liver transplantation is still controversial. We have designed an experimental study to investigate the effect of hemodynamic parameters of the liver circulation on the development of SFSS after EH in a porcine model. Eighteen pigs were randomly divided into two groups: group A has received EH (75%-80%) without splenectomy, and group B with EH and simultaneous splenectomy was carried out. Portal hemodynamics, liver function tests, histologic findings, injury and survival rates were compared between groups A and B. The 7-d survival rate in the splenectomy group was significantly improved compared with group A (88.9% versus 44.4%, P splenectomy group were significantly lower than in group A immediately after splenectomy and postoperatively until the day of sacrifice. Histologic findings in group A clearly illustrate severe inflammation, bridging necrosis, ischemic cholangitis, and severe congestion, while in group B there were less serious histologic changes. Our experimental study indicates that perioperative portal modulation can successfully prevent the manifestation of SFSS after EH. Therefore, by focusing on "flow" rather than on "size," researchers may understand better the pathophysiology of this syndrome. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. An Extended Functional Analysis Protocol Assesses the Role of Stereotypy in Aggression in Two Young Children with Autism Spectrum Disorder

    Science.gov (United States)

    White, Pamela; O'Reilly, Mark; Fragale, Christina; Kang, Soyeon; Muhich, Kimberly; Falcomata, Terry; Lang, Russell; Sigafoos, Jeff; Lancioni, Giulio

    2011-01-01

    Two children with autism who engaged in aggression and stereotypy were assessed using common analogue functional analysis procedures. Aggression was maintained by access to specific preferred items. Data on the rates of stereotypy and appropriate play were collected during an extended functional analysis tangible condition. These data reveal that…

  7. Extending role by Japanese pharmacists after training for performing vital signs monitoring

    Directory of Open Access Journals (Sweden)

    Hasegawa F

    2014-09-01

    Full Text Available Background: In Japan, the circumstances in which pharmacists work are changing. Pharmacists are expected to assess conditions of patients subject to medication to ensure proper use of pharmaceutical products. To ensure fulfilment of these roles, there have already been pharmacists’ efforts in performing vital signs monitoring. Objective: To clarify the necessity and related issues, by investigating the state of vital sign monitoring in clinical field by pharmacists who have been trained in vital sign monitoring. Method: A web survey was conducted from 4th October to 3rd December 2012, subjecting 1,026 pharmacists who completed the vital signs training hosted by The Japanese Association of Home Care Pharmacies (JAHCP. Survey items were 1 basic information of a respondent, 2 situation of homecare conducted by pharmacists, 3 seminar attendance status, and 4 vital signs monitoring status after the seminar. Results: The number of valid respondents was 430 and the response rate was 41.9%. As a result of the present research, it was revealed that 168 pharmacists (41.4%, had the opportunity to perform vital signs monitoring. By conducting vital sign monitoring, effects such as 1 improved motivation of pharmacists and better communication with patients, 2 proper use of medication, and 3 cost reduction were confirmed. Conclusion: Judging from the results of the survey, pharmacists can improve medication therapy for patients by attaining vital sign skills and conduct vital sign monitoring. Pharmacists who perform vital sign monitoring should share cases where they experienced positive patient outcomes.

  8. Competing through operations and supply: The role of classic and extended resource-based advantage

    OpenAIRE

    Lewis, Michael; Brandon-Jones, Alistair; Slack, Nigel; Howard, Mickey

    2010-01-01

    Purpose: The paper seeks to analyze the evolution of competitive advantage using both "classic" and "extended" resource-based theory (RBT). The aim is to examine the different ways in which "classic" and "extended" resource-based advantage develops and how they might combine to create long-term advantage. Design/methodology/approach: A single case study method is used to examine the process by which competitive advantage has accumulated over a 50-year period at Food Services Group Inc., a hig...

  9. Extending the role of a healthcare digital library environment to support orthopaedic research.

    Science.gov (United States)

    Miles-Board, Timothy; Carr, Leslie; Wills, Gary; Power, Guillermo; Bailey, Christopher; Hall, Wendy; Stenning, Matthew; Grange, Simon

    2006-06-01

    A digital archive, together with its users and its contents, does not exist in isolation; there is a cycle of activities which provides the context for the archive's existence. In arguing for the broadening of the traditional view of digital libraries as merely collections towards the processes of collecting and deploying, we have developed an extend ed digital library environment for orthopaedic surgeons which bridges the gap between the undertaking of experimental work and the dissemination of its results through electronic publication.

  10. Contemporary internal auditing practices : (new) roles and influencing variables. Evidence from extended case studies

    OpenAIRE

    G. SARENS; I. DE BEELDE

    2004-01-01

    Following the recommendation made by Rittenberg (1999), this study encompasses the broader nature of internal auditing that is evolving in practice and confirmed by the revised definition of internal auditing and the new Professional Practices Framework, both issued by the IIA in 1999. This study, based on six extended case studies, attempts to contribute to the literature by studying contemporary internal auditing practices in Belgium (assurance and / or consulting oriented), refining the in...

  11. Examining the Roles of Reasoning and Working Memory in Predicting Casual Game Performance across Extended Gameplay.

    Science.gov (United States)

    Kranz, Michael B; Baniqued, Pauline L; Voss, Michelle W; Lee, Hyunkyu; Kramer, Arthur F

    2017-01-01

    The variety and availability of casual video games presents an exciting opportunity for applications such as cognitive training. Casual games have been associated with fluid abilities such as working memory (WM) and reasoning, but the importance of these cognitive constructs in predicting performance may change across extended gameplay and vary with game structure. The current investigation examined the relationship between cognitive abilities and casual game performance over time by analyzing first and final session performance over 4-5 weeks of game play. We focused on two groups of subjects who played different types of casual games previously shown to relate to WM and reasoning when played for a single session: (1) puzzle-based games played adaptively across sessions and (2) speeded switching games played non-adaptively across sessions. Reasoning uniquely predicted first session casual game scores for both groups and accounted for much of the relationship with WM. Furthermore, over time, WM became uniquely important for predicting casual game performance for the puzzle-based adaptive games but not for the speeded switching non-adaptive games. These results extend the burgeoning literature on cognitive abilities involved in video games by showing differential relationships of fluid abilities across different game types and extended play. More broadly, the current study illustrates the usefulness of using multiple cognitive measures in predicting performance, and provides potential directions for game-based cognitive training research.

  12. Examining the Roles of Reasoning and Working Memory in Predicting Casual Game Performance across Extended Gameplay

    Science.gov (United States)

    Kranz, Michael B.; Baniqued, Pauline L.; Voss, Michelle W.; Lee, Hyunkyu; Kramer, Arthur F.

    2017-01-01

    The variety and availability of casual video games presents an exciting opportunity for applications such as cognitive training. Casual games have been associated with fluid abilities such as working memory (WM) and reasoning, but the importance of these cognitive constructs in predicting performance may change across extended gameplay and vary with game structure. The current investigation examined the relationship between cognitive abilities and casual game performance over time by analyzing first and final session performance over 4–5 weeks of game play. We focused on two groups of subjects who played different types of casual games previously shown to relate to WM and reasoning when played for a single session: (1) puzzle-based games played adaptively across sessions and (2) speeded switching games played non-adaptively across sessions. Reasoning uniquely predicted first session casual game scores for both groups and accounted for much of the relationship with WM. Furthermore, over time, WM became uniquely important for predicting casual game performance for the puzzle-based adaptive games but not for the speeded switching non-adaptive games. These results extend the burgeoning literature on cognitive abilities involved in video games by showing differential relationships of fluid abilities across different game types and extended play. More broadly, the current study illustrates the usefulness of using multiple cognitive measures in predicting performance, and provides potential directions for game-based cognitive training research. PMID:28326042

  13. Spontaneous Extensor Tendon Rupture in the Rheumatoid Wrist: Risk Factors and Preventive Role of Extended Tenosynovectomy.

    Science.gov (United States)

    Hsueh, Jung-Hua; Liu, Wen-Chung; Yang, Kuo-Chung; Hsu, Kuei-Chang; Lin, Cheng-Ta; Chen, Lee-Wei

    2016-03-01

    Spontaneous extensor tendon rupture is often seen in rheumatoid arthritis (RA) patients, but the risk factors are not clearly defined. We therefore collected the data of RA patients with previous extensor tendon rupture and those with tenosynovitis and analyzed the relationship between extended tenosynovectomy and spontaneous extensor tendon rupture. We retrospectively reviewed 17 spontaneous extensor tendon rupture episodes in 15 RA patients and 14 tenosynovitis episodes that required tenosynovectomy in 12 RA patients from 1997 to 2013. Correlations between the incidence of tendon rupture, X-ray findings, and clinical findings in the affected wrists before tendon rupture were analyzed statistically using the test for proportion. The following parameters were significantly correlated with spontaneous extensor tendon rupture: disease duration longer than 8 years, persistent tenosynovitis longer than 1 year duration, and Larsen grade greater than 4 (P = 0.02, 0.03, and 0.01, respectively). Dislocation of the distal end of the ulna, carpal collapse, and the scallop sign on X-ray contributed to a higher spontaneous extensor tendon rupture rate among RA patients (P = 0.01, 0.05, and 0.03, respectively). Extended tenosynovectomy was performed on 14 wrists in 12 RA patients with persistent tenosynovitis longer than 6 months, and Larsen grade did not deteriorate in this group compared with those who did not undergo the surgery. No spontaneous extensor tendon rupture occurred following the surgery. Risk factors of spontaneous extensor tendon rupture included disease duration longer than 8 years, persistent tenosynovitis longer than 1 year, and wrist Larsen grade greater than 4. Dislocation of the distal end of the ulna, carpal collapse, and the scallop sign on X-ray indicated a higher probability of extensor tendon rupture. Rheumatologists should consult with hand surgeons promptly to preserve hand function before tendon rupture. Prophylactic extended tenosynovectomy

  14. Management of obesity and cardiometabolic risk – role of phentermine/extended release topiramate

    Directory of Open Access Journals (Sweden)

    Sweeting AN

    2014-02-01

    Full Text Available Arianne N Sweeting,1 Eddy Tabet,1 Ian D Caterson,1,2 Tania P Markovic1,2 1Department of Endocrinology, Royal Prince Alfred Hospital, Sydney, NSW, Australia; 2Boden Institute of Obesity, Nutrition, Exercise & Eating Disorders, University of Sydney, NSW, Australia Abstract: The US Food and Drug Administration (FDA recently approved lorcaserin and the combination of phentermine and extended release topiramate (phentermine/topiramate ER for the treatment of obesity in conjunction with a lifestyle intervention, expanding the therapeutic options for long-term obesity pharmacotherapy, which was previously limited to orlistat. Combination phentermine/topiramate ER is associated with greater weight loss compared to its constituent monotherapy, with a more favorable adverse effect profile. Phentermine/topiramate ER also appears to have beneficial effects on cardiometabolic risk, although longer-term cardiovascular safety data are required. While there are no head-to-head studies among the currently available obesity pharmacotherapy agents, phentermine/topiramate ER appears to have a superior weight loss profile. This review will discuss the epidemiology, natural history, and cardiometabolic risk associated with obesity, provide an overview on current obesity pharmacotherapy, and summarize the recent clinical efficacy and safety data underpinning the FDA's approval of both phentermine/topiramate ER and lorcaserin as pharmacotherapy for a long-term obesity intervention. Keywords: obesity, phentermine/topiramate extended release, safety and efficacy, review

  15. Extended Pancreatectomy: Does It Have a Role in the Contemporary Management of Pancreatic Adenocarcinoma?

    Science.gov (United States)

    Kaiser, Joerg; Hackert, Thilo; Büchler, Markus W

    2017-01-01

    Pancreatic cancer is a low-incident but highly mortal disease. Surgery is still the preferred treatment option for resectable pancreatic cancer as it offers the only realistic chance for cure. As many patients present with locally advanced disease, which is generally considered as not amenable to surgical treatment, it is important to know the limits of surgical therapy in this disease. In this review, the indication and outcomes of extended pancreatectomies as well as the alternative treatment options for locally advanced pancreatic cancer are described. Furthermore, controversies as well as ongoing and future directions for the treatment options of locally advanced pancreatic cancer are discussed. Extended pancreatectomy can be performed with higher morbidity and mortality rates in patients with locally advanced pancreatic cancer compared to patients undergoing formal pancreatic resections. These procedures offer significant advantages with respect to both perioperative results and to long-term outcome when compared to chemotherapy. Due to the higher morbidity and mortality rates, these operations should be limited to specialist units with great experience in pancreatic surgery as well as experience in peri- and post-operative management of patients with pancreatic diseases. © 2017 S. Karger AG, Basel.

  16. The imidazopyridine derivative JK184 reveals dual roles for microtubules in Hedgehog signaling.

    Science.gov (United States)

    Cupido, Tommaso; Rack, Paul G; Firestone, Ari J; Hyman, Joel M; Han, Kyuho; Sinha, Surajit; Ocasio, Cory A; Chen, James K

    2009-01-01

    Eradicating hedgehogs: The title molecule has been previously identified as a potent inhibitor of the Hedgehog signaling pathway, which gives embryonic cells information needed to develop properly. This molecule is shown to modulate Hedgehog target gene expression by depolymerizing microtubules, thus revealing dual roles of the cytoskeleton in pathway regulation (see figure).

  17. Role of superconducting energy gap in extended BCS-Bose crossover theory

    Science.gov (United States)

    Chávez, I.; García, L. A.; de Llano, M.; Grether, M.

    2017-10-01

    The generalized Bose-Einstein condensation (GBEC) theory of superconductivity (SC) is briefly surveyed. It hinges on three distinct new ingredients: (i) Treatment of Cooper pairs (CPs) as actual bosons since they obey Bose statistics, in contrast to BCS pairs which do not obey Bose commutation relations; (ii) inclusion of two-hole Cooper pairs (2hCPs) on an equal footing with two-electron Cooper pairs (2eCPs), thus making this a complete boson-fermion (BF) model; and (iii) inclusion in the resulting ternary ideal BF gas with particular BF vertex interactions that drive boson formation/disintegration processes. GBEC subsumes as special cases both BCS (having its 50-50 symmetry of both kinds of CPs) and ordinary BEC theories (having no 2hCPs), as well as the now familiar BCS-Bose crossover theory. We extended the crossover theory with the explicit inclusion of 2hCPs and construct a phase diagram of Tc/TF versus n/nf, where Tc and TF are the critical and Fermi temperatures, n is the total number density and nf that of unbound electrons at T = 0. Also, with this extended crossover one can construct the energy gap Δ(T)/Δ(0) versus T/Tc for some elemental SCs by solving at least two equations numerically: a gap-like and a number equation. In 50-50 symmetry, the energy gap curve agrees quite well with experimental data. But ignoring 2hCPs altogether leads to the gap curve falling substantially below that with 50-50 symmetry which already fits the data quite well, showing that 2hCPs are indispensable to describe SCs.

  18. Role of paliperidone extended-release in treatment of schizoaffective disorder.

    Science.gov (United States)

    Canuso, Carla M; Turkoz, Ibrahim; Fu, Dong Jing; Bossie, Cynthia A

    2010-10-05

    Schizoaffective disorder is characterized by the presence of symptoms of both schizophrenia and a major mood disorder. The coexistence of these symptoms can be difficult to manage, and these patients are generally treated with antipsychotics as well as mood stabilizers and/or antidepressants. Additionally, no established treatment guidelines exist for this disorder. This review describes the combined results of two international, double-blind, placebo-controlled clinical studies of paliperidone extended-release (ER), an atypical antipsychotic recently approved in the US for the treatment of schizoaffective disorder. Subjects in these six-week trials were aged 18-65 years, had a diagnosis of schizoaffective disorder based on the Structural Clinical Interview for DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, 4th Edition) Disorders, and were experiencing an acute exacerbation. The subjects from these studies had significant symptomatology as evidenced by a mean (standard deviation) baseline Positive and Negative Syndrome Scale total score of 92.8 (13.0). Based on Young Mania Rating Scale and/or a 21-item Hamilton Rating Scale for Depression score of ≥16 at baseline, 79.5% and 66.9% of subjects presented with prominent manic and depressive symptoms, respectively, and 46.4% presented with mixed symptoms. Approximately half (45%) of subjects were taking adjunctive mood stabilizers and/or antidepressants. Paliperidone ER was found to be effective in improving psychotic and mood symptoms in these subjects. Paliperidone ER was also effective as monotherapy or adjunctive to mood stabilizers and/or antidepressants for subjects with prominent manic, depressive, or mixed symptoms at baseline. No new tolerability signals were observed in this population. To the best of our awareness, these pooled data provide the largest data set of patients with schizoaffective disorder, and extend our knowledge of disease characteristics and treatment response.

  19. The role of family configuration in early childhood intellectual development in the context of an extended family system in Pakistan

    Directory of Open Access Journals (Sweden)

    Avan B

    2007-01-01

    Full Text Available Context: The confluence theory of intelligence by Zajonc and Markus emphasizes that individual intellectual difference of children manifests itself in the context of family configuration. Instead of assuming its generalizability, careful scientific work is required before applying the model to South Asian cultures where, predominantly, an extended family type exists. Aims: To assess the role of extended family configuration on the child′s intellectual development in a South Asian setting. Settings and Design: A cross-sectional study was conducted on 4-5-year-old preschool children residing in Karachi, Pakistan. Materials and Methods: Three hundred and forty-two child and mother dyads were assessed through a validated cognitive psychometric tool and through a structured questionnaire. Children who were registered at the main Mother and Child Health Centres (MCH of the Aga Khan Health Services, Pakistan (AKHSP Karachi and who were born between July 1st 1993-June 30th 1994 with traceable birth records at the maternity homes, were considered for this study. Statistical Analysis: Multivariate linear regression models were used to identify the individual effect of family configuration on the intellectual scores. Results: Family configuration variables such as number of co-residents ( P < 0.05 and the number of siblings ( P < 0.01 in the house were significantly correlated with the psychometric score. Even after controlling for gender, socio-economic status, birth order and birth intervals, significant differentials were observed in favor of an extended family system on a child′s intellectual development. Conclusion: The findings suggest the positive role of co-residents of an extended family environment on the intellectual development in early childhood.

  20. Extending hierarchical achievement motivation models: the role of motivational needs for achievement goals and academic performance

    NARCIS (Netherlands)

    Bipp, T.; Dam, van K.

    2014-01-01

    In the current study, we investigated the role of three basic motivational needs (need for power, affiliation, achievement) as antecedents of goals within the 2 × 2 achievement goal framework, and examined their combined predictive validity with regard to academic performance in a sample of 120

  1. The role of religious values in extending social protection: A South ...

    African Journals Online (AJOL)

    This article reflects critically on the role played by religious networks – in particular the African Independent Churches or African Initiated Churches (AICs) – in enhancing social protection by means of informal coping mechanisms in the South African context. It also examines various factors that contribute towards informal ...

  2. Extending the Challenge-Hindrance Model of Occupational Stress: The Role of Appraisal

    Science.gov (United States)

    Webster, Jennica R.; Beehr, Terry A.; Love, Kevin

    2011-01-01

    Interest regarding the challenge-hindrance occupational stress model has increased in recent years, however its theoretical foundation has not been tested. Drawing from the transactional theory of stress, this study tests the assumptions made in past research (1) that workload and responsibility are appraised as challenges and role ambiguity and…

  3. Extending and expanding the Darwinian synthesis: the role of complex systems dynamics.

    Science.gov (United States)

    Weber, Bruce H

    2011-03-01

    Darwinism is defined here as an evolving research tradition based upon the concepts of natural selection acting upon heritable variation articulated via background assumptions about systems dynamics. Darwin's theory of evolution was developed within a context of the background assumptions of Newtonian systems dynamics. The Modern Evolutionary Synthesis, or neo-Darwinism, successfully joined Darwinian selection and Mendelian genetics by developing population genetics informed by background assumptions of Boltzmannian systems dynamics. Currently the Darwinian Research Tradition is changing as it incorporates new information and ideas from molecular biology, paleontology, developmental biology, and systems ecology. This putative expanded and extended synthesis is most perspicuously deployed using background assumptions from complex systems dynamics. Such attempts seek to not only broaden the range of phenomena encompassed by the Darwinian Research Tradition, such as neutral molecular evolution, punctuated equilibrium, as well as developmental biology, and systems ecology more generally, but to also address issues of the emergence of evolutionary novelties as well as of life itself. Copyright © 2010 Elsevier Ltd. All rights reserved.

  4. Extending the role of peritoneal dialysis: can we win hearts and minds?

    Science.gov (United States)

    Davies, Simon; Lally, Frank; Satchithananda, Duwarakan; Kadam, Umesh; Roffe, Christine

    2014-09-01

    The ability of peritoneal dialysis (PD) to achieve low-molecular weight solute clearance and ultrafiltration at low haemodynamic cost makes it an attractive therapy in situations where more aggressive therapy may be undesirable due to sudden reductions in cerebral, coronary or renal blood flow. We undertook a review of the literature to examine the recent evidence for this in two specific examples: the removal of glutamate following acute stroke and ultrafiltration for the treatment of diuretic resistant heart failure. In acute stroke, glutamate, when released into the extracellular tissues, causes neuronal cell death due to its excitotoxic properties. Experimental evidence from animal models indicates that its removal, including via PD, can reduce infarct size and restore functional brain tissue. PD is effective in removing glutamate in patients treated for renal failure. In heart failure, PD has a number of both theoretical and practical advantages for extending treatment, especially as an established home therapy. Several recent cohort studies describing its use in approaching 300 patients with diuretic resistance show consistent benefits in hospitalization and severity. Both these applications require substantial further clinical evaluation before they can justify wider adoption but their potential to alleviate morbidity on a large and potentially highly cost-effective scale demands further study. © The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

  5. Potential role of gabapentin and extended- release gabapentin in the management of menopausal hot flashes

    Directory of Open Access Journals (Sweden)

    Yadav M

    2013-08-01

    Full Text Available Manisha Yadav, Judith Volkar Center for Specialized Women’s Health, Cleveland Clinic Foundation, Cleveland, Ohio, USA Abstract: About 80% of postmenopausal women experience vasomotor symptoms, such as hot flashes and night sweats – symptoms that are associated with sleep disruption and can lead to fatigue and mood changes. Moreover, hot flashes can be embarrassing for women, causing difficulties at work and in their social lives. Many therapies have been advocated for relief of vasomotor symptoms, but only hormone therapy has been US Food and Drug Administration approved. However, after the Women's Health Initiative Study suggested that there was a correlation between hormone therapy and increased risk for breast cancer and cardiovascular events, many women stopped taking hormone therapy, and many do not want to initiate it. Hormone therapy is also contraindicated in certain women, such as those with a history of hormone-stimulated cancer like breast and uterine cancer. Gabapentin (Neurontin has shown efficacy in relieving vasomotor symptoms and is used as off-label for this indication. A new extended-release formulation of gabapentin has also shown efficacy in treating hot flashes and improving sleep quality with possibly fewer side effects than regular gabapentin. Keywords: Hot flushes, vasomotor symptoms, postmenopausal, hormone-sensitive cancer, non-hormonal therapy, gastric-retentive, Breeze

  6. Mitochondrial-Targeted Catalase: Extended Longevity and the Roles in Various Disease Models.

    Science.gov (United States)

    Dai, D-F; Chiao, Y-A; Martin, G M; Marcinek, D J; Basisty, N; Quarles, E K; Rabinovitch, P S

    2017-01-01

    The free-radical theory of aging was proposed more than 50 years ago. As one of the most popular mechanisms explaining the aging process, it has been extensively studied in several model organisms. However, the results remain controversial. The mitochondrial version of free-radical theory of aging proposes that mitochondria are both the primary sources of reactive oxygen species (ROS) and the primary targets of ROS-induced damage. One critical ROS is hydrogen peroxide, which is naturally degraded by catalase in peroxisomes or glutathione peroxidase within mitochondria. Our laboratory developed mice-overexpressing catalase targeted to mitochondria (mCAT), peroxisomes (pCAT), or the nucleus (nCAT) in order to investigate the role of hydrogen peroxide in different subcellular compartments in aging and age-related diseases. The mCAT mice have demonstrated the largest effects on life span and healthspan extension. This chapter will discuss the mCAT phenotype and review studies using mCAT to investigate the roles of mitochondrial oxidative stresses in various disease models, including metabolic syndrome and atherosclerosis, cardiac aging, heart failure, skeletal muscle pathology, sensory defect, neurodegenerative diseases, and cancer. As ROS has been increasingly recognized as essential signaling molecules that may be beneficial in hormesis, stress response and immunity, the potential pleiotropic, or adverse effects of mCAT are also discussed. Finally, the development of small-molecule mitochondrial-targeted therapeutic approaches is reviewed. © 2017 Elsevier Inc. All rights reserved.

  7. Dissecting a role for melanopsin in behavioural light aversion reveals a response independent of conventional photoreception.

    Directory of Open Access Journals (Sweden)

    Ma'ayan Semo

    Full Text Available Melanopsin photoreception plays a vital role in irradiance detection for non-image forming responses to light. However, little is known about the involvement of melanopsin in emotional processing of luminance. When confronted with a gradient in light, organisms exhibit spatial movements relative to this stimulus. In rodents, behavioural light aversion (BLA is a well-documented but poorly understood phenomenon during which animals attribute salience to light and remove themselves from it. Here, using genetically modified mice and an open field behavioural paradigm, we investigate the role of melanopsin in BLA. While wildtype (WT, melanopsin knockout (Opn4(-/- and rd/rd cl (melanopsin only (MO mice all exhibit BLA, our novel methodology reveals that isolated melanopsin photoreception produces a slow, potentiating response to light. In order to control for the involvement of pupillary constriction in BLA we eliminated this variable with topical atropine application. This manipulation enhanced BLA in WT and MO mice, but most remarkably, revealed light aversion in triple knockout (TKO mice, lacking three elements deemed essential for conventional photoreception (Opn4(-/- Gnat1(-/- Cnga3(-/-. Using a number of complementary strategies, we determined this response to be generated at the level of the retina. Our findings have significant implications for the understanding of how melanopsin signalling may modulate aversive responses to light in mice and humans. In addition, we also reveal a clear potential for light perception in TKO mice.

  8. X-ray fluorescence microscopy reveals the role of selenium in spermatogenesis

    Science.gov (United States)

    Kehr, Sebastian; Malinouski, Mikalai; Finney, Lydia; Vogt, Stefan; Labunskyy, Vyacheslav M.; Kasaikina, Marina V.; Carlson, Bradley A.; Zhou, You; Hatfield, Dolph L.; Gladyshev, Vadim N.

    2009-01-01

    Selenium (Se) is a trace element with important roles in human health. Several selenoproteins have essential functions in development. However, the cellular and tissue distribution of Se remains largely unknown because of the lack of analytical techniques that image this element with sufficient sensitivity and resolution. Herein, we report that X-ray fluorescence microscopy (XFM) can be used to visualize and quantify the tissue, cellular and subcellular topography of Se. We applied this technique to characterize the role of Se in spermatogenesis and identified a dramatic Se enrichment specifically in late spermatids, a pattern that was not seen in any other elemental maps. This enrichment was due to elevated levels of the mitochondrial form of glutathione peroxidase 4 and was fully dependent on the supplies of Se by Selenoprotein P. High-resolution scans revealed that Se concentrated near the lumen side of elongating spermatids, where structural components of sperm are formed. During spermatogenesis, maximal Se associated with decreased phosphorus, whereas Zn did not change. In sperm, Se was primarily in the midpiece and co-localized with Cu and Fe. XFM allowed quantification of Se in the midpiece (0.8 fg) and head (0.14 fg) of individual sperm cells, revealing the ability of sperm cells to handle the amounts of this element well above its toxic levels. Overall, the use of XFM allowed visualization of tissue and cellular Se and provided important insights in the role of this and other trace elements in spermatogenesis. PMID:19379757

  9. Gene disruption reveals a dispensable role for plasmepsin VII in the Plasmodium berghei life cycle.

    Science.gov (United States)

    Mastan, Babu S; Kumari, Anchala; Gupta, Dinesh; Mishra, Satish; Kumar, Kota Arun

    2014-06-01

    Plasmepsins (PM), aspartic proteases of Plasmodium, comprises a family of ten proteins that perform critical functions in Plasmodium life cycle. Except VII and VIII, functions of the remaining plasmepsin members have been well characterized. Here, we have generated a mutant parasite lacking PM VII in Plasmodium berghei using reverse genetics approach. Systematic comparison of growth kinetics and infection in both mosquito and vertebrate host revealed that PM VII depleted mutants exhibited no defects in development and progressed normally throughout the parasite life cycle. These studies suggest a dispensable role for PM VII in Plasmodium berghei life cycle. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Extended producer responsibility for packaging wastes and WEEE - a comparison of implementation and the role of local authorities across Europe.

    Science.gov (United States)

    Cahill, Rachel; Grimes, Sue M; Wilson, David C

    2011-05-01

    A comparison of the implementation of extended producer responsibility (EPR) to packaging waste and waste electrical and electronic equipment (WEEE) is presented for a representative sample of eleven European Union countries based on five indicators: stakeholders and responsibilities; compliance mechanisms; role of local authorities; financing mechanisms and merits and limitations, with four countries selected for more detailed case study analysis. Similarities, trends and differences in national systems are highlighted with particular focus on the role of local authorities and their relationship with obligated producers and the effect on the operation and success of each system. The national systems vary considerably in design, in terms of influence of pre-existing policy and systems, methods of achieving producer compliance (multiple or single collective schemes), fee structures, targets, waste stream prioritization and local authority involvement. Differing approaches are evident across all member states with respect to the role played by local authorities, responsibility apportioned to them, and the evolution of working relationships between obligated producers and municipalities. On the whole, EPR for packaging and WEEE has been successfully implemented throughout Europe in terms of Directive targets. It is, however, clear that the EPR systems currently in application across Europe differ primarily due to contrasting opinion on the legitimacy of local authorities as stakeholders and, in some cases, a fear on the part of industry of associated costs. Where local authorities have been engaged in the design and implementation of national systems, existing infrastructure used and defined roles established for producers and local authorities, results have been significantly more positive than in the cases where local authorities have had limited engagement.

  11. Targeted deletion of fibrinogen like protein 1 reveals a novel role in energy substrate utilization.

    Directory of Open Access Journals (Sweden)

    Valeriy Demchev

    Full Text Available Fibrinogen like protein 1(Fgl1 is a secreted protein with mitogenic activity on primary hepatocytes. Fgl1 is expressed in the liver and its expression is enhanced following acute liver injury. In animals with acute liver failure, administration of recombinant Fgl1 results in decreased mortality supporting the notion that Fgl1 stimulates hepatocyte proliferation and/or protects hepatocytes from injury. However, because Fgl1 is secreted and detected in the plasma, it is possible that the role of Fgl1 extends far beyond its effect on hepatocytes. In this study, we show that Fgl1 is additionally expressed in brown adipose tissue. We find that signals elaborated following liver injury also enhance the expression of Fgl1 in brown adipose tissue suggesting that there is a cross talk between the injured liver and adipose tissues. To identify extra hepatic effects, we generated Fgl1 deficient mice. These mice exhibit a phenotype suggestive of a global metabolic defect: Fgl1 null mice are heavier than wild type mates, have abnormal plasma lipid profiles, fasting hyperglycemia with enhanced gluconeogenesis and exhibit differences in white and brown adipose tissue morphology when compared to wild types. Because Fgl1 shares structural similarity to Angiopoietin like factors 2, 3, 4 and 6 which regulate lipid metabolism and energy utilization, we postulate that Fgl1 is a member of an emerging group of proteins with key roles in metabolism and liver regeneration.

  12. Extending the mirror neuron system model, II: what did I just do? A new role for mirror neurons.

    Science.gov (United States)

    Bonaiuto, James; Arbib, Michael A

    2010-04-01

    A mirror system is active both when an animal executes a class of actions (self-actions) and when it sees another execute an action of that class. Much attention has been given to the possible roles of mirror systems in responding to the actions of others but there has been little attention paid to their role in self-actions. In the companion article (Bonaiuto et al. Biol Cybern 96:9-38, 2007) we presented MNS2, an extension of the Mirror Neuron System model of the monkey mirror system trained to recognize the external appearance of its own actions as a basis for recognizing the actions of other animals when they perform similar actions. Here we further extend the study of the mirror system by introducing the novel hypotheses that a mirror system may additionally help in monitoring the success of a self-action and may also be activated by recognition of one's own apparent actions as well as efference copy from one's intended actions. The framework for this computational demonstration is a model of action sequencing, called augmented competitive queuing, in which action choice is based on the desirability of executable actions. We show how this "what did I just do?" function of mirror neurons can contribute to the learning of both executability and desirability which in certain cases supports rapid reorganization of motor programs in the face of disruptions.

  13. Quantitative proteomics of the tonoplast reveals a role for glycolytic enzymes in salt tolerance.

    Science.gov (United States)

    Barkla, Bronwyn J; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

    2009-12-01

    To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na(+) sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H(+)-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H(+)-pump activity.

  14. Genome-wide analyses reveal a role for peptide hormones in planarian germline development.

    Directory of Open Access Journals (Sweden)

    James J Collins

    Full Text Available Bioactive peptides (i.e., neuropeptides or peptide hormones represent the largest class of cell-cell signaling molecules in metazoans and are potent regulators of neural and physiological function. In vertebrates, peptide hormones play an integral role in endocrine signaling between the brain and the gonads that controls reproductive development, yet few of these molecules have been shown to influence reproductive development in invertebrates. Here, we define a role for peptide hormones in controlling reproductive physiology of the model flatworm, the planarian Schmidtea mediterranea. Based on our observation that defective neuropeptide processing results in defects in reproductive system development, we employed peptidomic and functional genomic approaches to characterize the planarian peptide hormone complement, identifying 51 prohormone genes and validating 142 peptides biochemically. Comprehensive in situ hybridization analyses of prohormone gene expression revealed the unanticipated complexity of the flatworm nervous system and identified a prohormone specifically expressed in the nervous system of sexually reproducing planarians. We show that this member of the neuropeptide Y superfamily is required for the maintenance of mature reproductive organs and differentiated germ cells in the testes. Additionally, comparative analyses of our biochemically validated prohormones with the genomes of the parasitic flatworms Schistosoma mansoni and Schistosoma japonicum identified new schistosome prohormones and validated half of all predicted peptide-encoding genes in these parasites. These studies describe the peptide hormone complement of a flatworm on a genome-wide scale and reveal a previously uncharacterized role for peptide hormones in flatworm reproduction. Furthermore, they suggest new opportunities for using planarians as free-living models for understanding the reproductive biology of flatworm parasites.

  15. Hypomorphic conditional deletion of E11/Podoplanin reveals a role in osteocyte dendrite elongation.

    Science.gov (United States)

    Staines, Katherine A; Javaheri, Behzad; Hohenstein, Peter; Fleming, Robert; Ikpegbu, Ekele; Unger, Erin; Hopkinson, Mark; Buttle, David J; Pitsillides, Andrew A; Farquharson, Colin

    2017-11-01

    The transmembrane glycoprotein E11/Podoplanin (Pdpn) has been implicated in the initial stages of osteocyte differentiation. However, its precise function and regulatory mechanisms are still unknown. Due to the known embryonic lethality induced by global Pdpn deletion, we have herein explored the effect of bone-specific Pdpn knockdown on osteocyte form and function in the post-natal mouse. Extensive skeletal phenotyping of male and female 6-week-old Oc-cre;Pdpn flox/flox (cKO) mice and their Pdpn flox/flox controls (fl/fl) has revealed that Pdpn deletion significantly compromises tibial cortical bone microarchitecture in both sexes, albeit to different extents (p < 0.05). Consistent with this, we observed an increase in stiffness in female cKO mice in comparison to fl/fl mice (p < 0.01). Moreover, analysis of the osteocyte phenotype by phalloidin staining revealed a significant decrease in the dendrite volume (p < 0.001) and length (p < 0.001) in cKO mice in which deletion of Pdpn also modifies the bone anabolic loading response (p < 0.05) in comparison to age-matched fl/fl mice. Together, these data confirm a regulatory role for Pdpn in osteocyte dendrite formation and as such, in the control of osteocyte function. As the osteocyte dendritic network is known to play vital roles in regulating bone modeling/remodeling, this highlights an essential role for Pdpn in bone homeostasis. © 2017 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

  16. Metabolomics analysis reveals the metabolic and functional roles of flavonoids in light-sensitive tea leaves.

    Science.gov (United States)

    Zhang, Qunfeng; Liu, Meiya; Ruan, Jianyun

    2017-03-20

    As the predominant secondary metabolic pathway in tea plants, flavonoid biosynthesis increases with increasing temperature and illumination. However, the concentration of most flavonoids decreases greatly in light-sensitive tea leaves when they are exposed to light, which further improves tea quality. To reveal the metabolism and potential functions of flavonoids in tea leaves, a natural light-sensitive tea mutant (Huangjinya) cultivated under different light conditions was subjected to metabolomics analysis. The results showed that chlorotic tea leaves accumulated large amounts of flavonoids with ortho-dihydroxylated B-rings (e.g., catechin gallate, quercetin and its glycosides etc.), whereas total flavonoids (e.g., myricetrin glycoside, epigallocatechin gallate etc.) were considerably reduced, suggesting that the flavonoid components generated from different metabolic branches played different roles in tea leaves. Furthermore, the intracellular localization of flavonoids and the expression pattern of genes involved in secondary metabolic pathways indicate a potential photoprotective function of dihydroxylated flavonoids in light-sensitive tea leaves. Our results suggest that reactive oxygen species (ROS) scavenging and the antioxidation effects of flavonoids help chlorotic tea plants survive under high light stress, providing new evidence to clarify the functional roles of flavonoids, which accumulate to high levels in tea plants. Moreover, flavonoids with ortho-dihydroxylated B-rings played a greater role in photo-protection to improve the acclimatization of tea plants.

  17. Ecological roles of dominant and rare prokaryotes in acid mine drainage revealed by metagenomics and metatranscriptomics.

    Science.gov (United States)

    Hua, Zheng-Shuang; Han, Yu-Jiao; Chen, Lin-Xing; Liu, Jun; Hu, Min; Li, Sheng-Jin; Kuang, Jia-Liang; Chain, Patrick S G; Huang, Li-Nan; Shu, Wen-Sheng

    2015-06-01

    High-throughput sequencing is expanding our knowledge of microbial diversity in the environment. Still, understanding the metabolic potentials and ecological roles of rare and uncultured microbes in natural communities remains a major challenge. To this end, we applied a 'divide and conquer' strategy that partitioned a massive metagenomic data set (>100 Gbp) into subsets based on K-mer frequency in sequence assembly to a low-diversity acid mine drainage (AMD) microbial community and, by integrating with an additional metatranscriptomic assembly, successfully obtained 11 draft genomes most of which represent yet uncultured and/or rare taxa (relative abundance 90%) and its metabolic potentials and gene expression profile, providing initial molecular insights into the ecological role of these lesser known, but potentially important, microorganisms in the AMD environment. Gene transcriptional analysis of the active taxa revealed major metabolic capabilities executed in situ, including carbon- and nitrogen-related metabolisms associated with syntrophic interactions, iron and sulfur oxidation, which are key in energy conservation and AMD generation, and the mechanisms of adaptation and response to the environmental stresses (heavy metals, low pH and oxidative stress). Remarkably, nitrogen fixation and sulfur oxidation were performed by the rare taxa, indicating their critical roles in the overall functioning and assembly of the AMD community. Our study demonstrates the potential of the 'divide and conquer' strategy in high-throughput sequencing data assembly for genome reconstruction and functional partitioning analysis of both dominant and rare species in natural microbial assemblages.

  18. The Structure of Neurexin 1[alpha] Reveals Features Promoting a Role as Synaptic Organizer

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Fang; Venugopal, Vandavasi; Murray, Beverly; Rudenko, Gabby (Michigan)

    2014-10-02

    {alpha}-Neurexins are essential synaptic adhesion molecules implicated in autism spectrum disorder and schizophrenia. The {alpha}-neurexin extracellular domain consists of six LNS domains interspersed by three EGF-like repeats and interacts with many different proteins in the synaptic cleft. To understand how {alpha}-neurexins might function as synaptic organizers, we solved the structure of the neurexin 1{alpha} extracellular domain (n1{alpha}) to 2.65 {angstrom}. The L-shaped molecule can be divided into a flexible repeat I (LNS1-EGF-A-LNS2), a rigid horseshoe-shaped repeat II (LNS3-EGF-B-LNS4) with structural similarity to so-called reelin repeats, and an extended repeat III (LNS5-EGF-B-LNS6) with controlled flexibility. A 2.95 {angstrom} structure of n1{alpha} carrying splice insert SS3 in LNS4 reveals that SS3 protrudes as a loop and does not alter the rigid arrangement of repeat II. The global architecture imposed by conserved structural features enables {alpha}-neurexins to recruit and organize proteins in distinct and variable ways, influenced by splicing, thereby promoting synaptic function.

  19. Myc inhibition is effective against glioma and reveals a role for Myc in proficient mitosis.

    Science.gov (United States)

    Annibali, Daniela; Whitfield, Jonathan R; Favuzzi, Emilia; Jauset, Toni; Serrano, Erika; Cuartas, Isabel; Redondo-Campos, Sara; Folch, Gerard; Gonzàlez-Juncà, Alba; Sodir, Nicole M; Massó-Vallés, Daniel; Beaulieu, Marie-Eve; Swigart, Lamorna B; Mc Gee, Margaret M; Somma, Maria Patrizia; Nasi, Sergio; Seoane, Joan; Evan, Gerard I; Soucek, Laura

    2014-08-18

    Gliomas are the most common primary tumours affecting the adult central nervous system and respond poorly to standard therapy. Myc is causally implicated in most human tumours and the majority of glioblastomas have elevated Myc levels. Using the Myc dominant negative Omomyc, we previously showed that Myc inhibition is a promising strategy for cancer therapy. Here, we preclinically validate Myc inhibition as a therapeutic strategy in mouse and human glioma, using a mouse model of spontaneous multifocal invasive astrocytoma and its derived neuroprogenitors, human glioblastoma cell lines, and patient-derived tumours both in vitro and in orthotopic xenografts. Across all these experimental models we find that Myc inhibition reduces proliferation, increases apoptosis and remarkably, elicits the formation of multinucleated cells that then arrest or die by mitotic catastrophe, revealing a new role for Myc in the proficient division of glioma cells.

  20. A genomewide screen for suppressors of Alu-mediated rearrangements reveals a role for PIF1.

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    Karen M Chisholm

    Full Text Available Alu-mediated rearrangement of tumor suppressor genes occurs frequently during carcinogenesis. In breast cancer, this mechanism contributes to loss of the wild-type BRCA1 allele in inherited disease and to loss of heterozygosity in sporadic cancer. To identify genes required for suppression of Alu-mediated recombination we performed a genomewide screen of a collection of 4672 yeast gene deletion mutants using a direct repeat recombination assay. The primary screen and subsequent analysis identified 12 candidate genes including TSA, ELG1, and RRM3, which are known to play a significant role in maintaining genomic stability. Genetic analysis of the corresponding human homologs was performed in sporadic breast tumors and in inherited BRCA1-associated carcinomas. Sequencing of these genes in high risk breast cancer families revealed a potential role for the helicase PIF1 in cancer predisposition. PIF1 variant L319P was identified in three breast cancer families; importantly, this variant, which is predicted to be functionally damaging, was not identified in a large series of controls nor has it been reported in either dbSNP or the 1000 Genomes Project. In Schizosaccharomyces pombe, Pfh1 is required to maintain both mitochondrial and nuclear genomic integrity. Functional studies in yeast of human PIF1 L319P revealed that this variant cannot complement the essential functions of Pfh1 in either the nucleus or mitochondria. Our results provide a global view of nonessential genes involved in suppressing Alu-mediated recombination and implicate variation in PIF1 in breast cancer predisposition.

  1. Spatiotemporal mechanical variation reveals critical role for rho kinase during primitive streak morphogenesis.

    Science.gov (United States)

    Henkels, Julia; Oh, Jaeho; Xu, Wenwei; Owen, Drew; Sulchek, Todd; Zamir, Evan

    2013-02-01

    Large-scale morphogenetic movements during early embryo development are driven by complex changes in biochemical and biophysical factors. Current models for amniote primitive streak morphogenesis and gastrulation take into account numerous genetic pathways but largely ignore the role of mechanical forces. Here, we used atomic force microscopy (AFM) to obtain for the first time precise biomechanical properties of the early avian embryo. Our data reveal that the primitive streak is significantly stiffer than neighboring regions of the epiblast, and that it is stiffer than the pre-primitive streak epiblast. To test our hypothesis that these changes in mechanical properties are due to a localized increase of actomyosin contractility, we inhibited actomyosin contractility via the Rho kinase (ROCK) pathway using the small-molecule inhibitor Y-27632. Our results using several different assays show the following: (1) primitive streak formation was blocked; (2) the time-dependent increase in primitive streak stiffness was abolished; and (3) convergence of epiblast cells to the midline was inhibited. Taken together, our data suggest that actomyosin contractility is necessary for primitive streak morphogenesis, and specifically, ROCK plays a critical role. To better understand the underlying mechanisms of this fundamental process, future models should account for the findings presented in this study.

  2. Targeted Disruption of ALK Reveals a Potential Role in Hypogonadotropic Hypogonadism.

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    Barbara Witek

    Full Text Available Mice lacking ALK activity have previously been reported to exhibit subtle behavioral phenotypes. In this study of ALK of loss of function mice we present data supporting a role for ALK in hypogonadotropic hypogonadism in male mice. We observed lower level of serum testosterone at P40 in ALK knock-out males, accompanied by mild disorganization of seminiferous tubules exhibiting decreased numbers of GATA4 expressing cells. These observations highlight a role for ALK in testis function and are further supported by experiments in which chemical inhibition of ALK activity with the ALK TKI crizotinib was employed. Oral administration of crizotinib resulted in a decrease of serum testosterone levels in adult wild type male mice, which reverted to normal levels after cessation of treatment. Analysis of GnRH expression in neurons of the hypothalamus revealed a significant decrease in the number of GnRH positive neurons in ALK knock-out mice at P40 when compared with control littermates. Thus, ALK appears to be involved in hypogonadotropic hypogonadism by regulating the timing of pubertal onset and testis function at the upper levels of the hypothalamic-pituitary gonadal axis.

  3. Separable roles of UFO during floral development revealed by conditional restoration of gene function.

    Science.gov (United States)

    Laufs, Patrick; Coen, Enrico; Kronenberger, Jocelyne; Traas, Jan; Doonan, John

    2003-02-01

    The UNUSUAL FLORAL ORGANS (UFO) gene is required for several aspects of floral development in Arabidopsis including specification of organ identity in the second and third whorls and the proper pattern of primordium initiation in the inner three whorls. UFO is expressed in a dynamic pattern during the early phases of flower development. Here we dissect the role of UFO by ubiquitously expressing it in ufo loss-of-function flowers at different developmental stages and for various durations using an ethanol-inducible expression system. The previously known functions of UFO could be separated and related to its expression at specific stages of development. We show that a 24- to 48-hour period of UFO expression from floral stage 2, before any floral organs are visible, is sufficient to restore normal petal and stamen development. The earliest requirement for UFO is during stage 2, when the endogenous UFO gene is transiently expressed in the centre of the wild-type flower and is required to specify the initiation patterns of petal, stamen and carpel primordia. Petal and stamen identity is determined during stages 2 or 3, when UFO is normally expressed in the presumptive second and third whorl. Although endogenous UFO expression is absent from the stamen whorl from stage 4 onwards, stamen identity can be restored by UFO activation up to stage 6. We also observed floral phenotypes not observed in loss-of-function or constitutive gain-of-function backgrounds, revealing additional roles of UFO in outgrowth of petal primordia.

  4. A novel immunotoxin reveals a new role for CD321 in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Takeshi Fukuhara

    Full Text Available There are currently several antibody therapies that directly target tumors, and antibody-drug conjugates represent a novel moiety as next generation therapeutics. Here, we used a unique screening probe, DT3C, to identify functional antibodies that recognized surface molecules and functional epitopes, and which provided toxin delivery capability. Accordingly, we generated the 90G4 antibody, which induced DT3C-dependent cytotoxicity in endothelial cells. Molecular analysis revealed that 90G4 recognized CD321, a protein localized at tight junctions. Although CD321 plays a pivotal role in inflammation and lymphocyte trans-endothelial migration, little is known about its mechanism of action in endothelial cells. Targeting of CD321 by the 90G4 immunotoxin induced cell death. Moreover, 90G4 immunotoxin caused cytotoxicity primarily in migratory endothelial cells, but not in those forming sheets, suggesting a critical role for CD321 in tumor angiogenesis. We also found that hypoxia triggered redistribution of CD321 to a punctate localization on the basal side of cells, resulting in functional impairment of tight junctions and increased motility. Thus, our findings raise the intriguing possibility that endothelial CD321 presented cellular localization in tight junction as well as multifunctional dynamics in several conditions, leading to illuminate the importance of widely-expressed CD321 as a potential target for antitumor therapy.

  5. Targeted Disruption of ALK Reveals a Potential Role in Hypogonadotropic Hypogonadism

    Science.gov (United States)

    Nord, Christoffer; Ahlgren, Ulf; Eriksson, Maria; Vernersson-Lindahl, Emma; Helland, Åslaug; Alexeyev, Oleg A.; Hallberg, Bengt; Palmer, Ruth H.

    2015-01-01

    Mice lacking ALK activity have previously been reported to exhibit subtle behavioral phenotypes. In this study of ALK of loss of function mice we present data supporting a role for ALK in hypogonadotropic hypogonadism in male mice. We observed lower level of serum testosterone at P40 in ALK knock-out males, accompanied by mild disorganization of seminiferous tubules exhibiting decreased numbers of GATA4 expressing cells. These observations highlight a role for ALK in testis function and are further supported by experiments in which chemical inhibition of ALK activity with the ALK TKI crizotinib was employed. Oral administration of crizotinib resulted in a decrease of serum testosterone levels in adult wild type male mice, which reverted to normal levels after cessation of treatment. Analysis of GnRH expression in neurons of the hypothalamus revealed a significant decrease in the number of GnRH positive neurons in ALK knock-out mice at P40 when compared with control littermates. Thus, ALK appears to be involved in hypogonadotropic hypogonadism by regulating the timing of pubertal onset and testis function at the upper levels of the hypothalamic-pituitary gonadal axis. PMID:25955180

  6. Molecular Typing of Enterobacteriaceae from Pig Holdings in North-Western Germany Reveals Extended- Spectrum and AmpC β-Lactamases Producing but no Carbapenem Resistant Ones

    NARCIS (Netherlands)

    Garcia-Cobos, Silvia; Koeck, Robin; Mellmann, Alexander; Frenzel, Julia; Friedrich, Alexander W.; Rossen, John W. A.

    2015-01-01

    The increase of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) in humans and in food-producing animals is of public health concern. The latter could contribute to spreading of these bacteria or their resistance genes to humans. Several studies have reported the isolation of

  7. Comprehensive RNA Polymerase II Interactomes Reveal Distinct and Varied Roles for Each Phospho-CTD Residue

    Directory of Open Access Journals (Sweden)

    Kevin M. Harlen

    2016-06-01

    Full Text Available Transcription controls splicing and other gene regulatory processes, yet mechanisms remain obscure due to our fragmented knowledge of the molecular connections between the dynamically phosphorylated RNA polymerase II (Pol II C-terminal domain (CTD and regulatory factors. By systematically isolating phosphorylation states of the CTD heptapeptide repeat (Y1S2P3T4S5P6S7, we identify hundreds of protein factors that are differentially enriched, revealing unappreciated connections between the Pol II CTD and co-transcriptional processes. These data uncover a role for threonine-4 in 3′ end processing through control of the transition between cleavage and termination. Furthermore, serine-5 phosphorylation seeds spliceosomal assembly immediately downstream of 3′ splice sites through a direct interaction with spliceosomal subcomplex U1. Strikingly, threonine-4 phosphorylation also impacts splicing by serving as a mark of co-transcriptional spliceosome release and ensuring efficient post-transcriptional splicing genome-wide. Thus, comprehensive Pol II interactomes identify the complex and functional connections between transcription machinery and other gene regulatory complexes.

  8. IL-15 deficient tax mice reveal a role for IL-1α in tumor immunity.

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    Daniel A Rauch

    Full Text Available IL-15 is recognized as a promising candidate for tumor immunotherapy and has been described as both a promoter of cancer and a promoter of anti-cancer immunity. IL-15 was discovered in cells transformed by HTLV-1, the etiologic agent of adult T cell leukemia/lymphoma (ATL and the human retrovirus that carries the Tax oncogene. We have developed the TAX-LUC mouse model of ATL in which Tax expression drives both malignant transformation and luciferase expression, enabling non-invasive imaging of tumorigenesis in real time. To identify the role of IL-15 in spontaneous development of lymphoma in vivo, an IL-15(-/- TAX-LUC strain was developed and examined. The absence of IL-15 resulted in aggressive tumor growth and accelerated mortality and demonstrated that IL-15 was not required for Tax-mediated lymphoma but was essential for anti-tumor immunity. Further analysis revealed a unique transcriptional profile in tumor cells that arise in the absence of IL-15 that included a significant increase in the expression of IL-1α and IL-1α-regulated cytokines. Moreover, anti-IL-1α antibodies and an IL-1 receptor antagonist (Anakinra were used to interrogate the potential of IL-1α targeted therapies in this model. Taken together, these findings identify IL-15 and IL-1α as therapeutic targets in lymphoma.

  9. Automatic morphological subtyping reveals new roles of caspases in mitochondrial dynamics.

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    Jyh-Ying Peng

    2011-10-01

    Full Text Available Morphological dynamics of mitochondria is associated with key cellular processes related to aging and neuronal degenerative diseases, but the lack of standard quantification of mitochondrial morphology impedes systematic investigation. This paper presents an automated system for the quantification and classification of mitochondrial morphology. We discovered six morphological subtypes of mitochondria for objective quantification of mitochondrial morphology. These six subtypes are small globules, swollen globules, straight tubules, twisted tubules, branched tubules and loops. The subtyping was derived by applying consensus clustering to a huge collection of more than 200 thousand mitochondrial images extracted from 1422 micrographs of Chinese hamster ovary (CHO cells treated with different drugs, and was validated by evidence of functional similarity reported in the literature. Quantitative statistics of subtype compositions in cells is useful for correlating drug response and mitochondrial dynamics. Combining the quantitative results with our biochemical studies about the effects of squamocin on CHO cells reveals new roles of Caspases in the regulatory mechanisms of mitochondrial dynamics. This system is not only of value to the mitochondrial field, but also applicable to the investigation of other subcellular organelle morphology.

  10. Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells.

    Science.gov (United States)

    Rusin, Scott F; Schlosser, Kate A; Adamo, Mark E; Kettenbach, Arminja N

    2015-10-13

    Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry-based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c-dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2-dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. Copyright © 2015, American Association for the Advancement of Science.

  11. Quantitative proteomics reveals that peroxidases play key roles in post-flooding recovery in soybean roots.

    Science.gov (United States)

    Khan, Mudassar Nawaz; Sakata, Katsumi; Hiraga, Susumu; Komatsu, Setsuko

    2014-12-05

    Soybean is an important legume crop that exhibits markedly reduced growth and yields under flooding conditions. To unravel the mechanisms involved in recovery after flooding in soybean root, gel-free proteomic analysis was performed. Morphological analysis revealed that growth suppression was more severe with increased flooding duration. Out of a total of 1645 and 1707 identified proteins, 73 and 21 proteins were changed significantly during the recovery stage following 2 and 4 days flooding, respectively. Based on the proteomic, clustering, and in silico protein-protein interaction analyses, six key enzymes were analyzed at the mRNA level. Lipoxygenase 1, which was increased at the protein level during the recovery period, was steadily down-regulated at the mRNA level. The peroxidase superfamily protein continuously increased in abundance during the course of recovery and was up-regulated at the mRNA level. HAD acid phosphatase was decreased at the protein level and down-regulated at the transcript level, while isoflavone reductase and an unknown protein were increased at both the protein and mRNA levels. Consistent with these findings, the enzymatic activity of peroxidase was decreased under flooding stress but increased significantly during the recovery sage. These results suggest that peroxidases might play key roles in post-flooding recovery in soybean roots through the scavenging of toxic radicals.

  12. Genetic Screen Reveals the Role of Purine Metabolism in Staphylococcus aureus Persistence to Rifampicin

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    Rebecca Yee

    2015-12-01

    Full Text Available Chronic infections with Staphylococcus aureus such as septicemia, osteomyelitis, endocarditis, and biofilm infections are difficult to treat because of persisters. Despite many efforts in understanding bacterial persistence, the mechanisms of persister formation in S. aureus remain elusive. Here, we performed a genome-wide screen of a transposon mutant library to study the molecular mechanisms involved in persistence of community-acquired S. aureus. Screening of the library for mutants defective in persistence or tolerance to rifampicin revealed many genes involved in metabolic pathways that are important for antibiotic persistence. In particular, the identified mutants belonged to metabolic pathways involved in carbohydrate, amino acid, lipid, vitamin and purine biosynthesis. Five mutants played a role in purine biosynthesis and two mutants, purB, an adenylosuccinate lyase, and purM, a phosphoribosylaminoimidazole synthetase, were selected for further confirmation. Mutants purB and purM showed defective persistence compared to the parental strain USA300 in multiple stress conditions including various antibiotics, low pH, and heat stress. The defect in persistence was restored by complementation with the wildtype purB and purM gene in the respective mutants. These findings provide new insights into the mechanisms of persistence in S. aureus and provide novel therapeutic targets for developing more effective treatment for persistent infections due to S. aureus.

  13. Potential Role of the Last Half Repeat in TAL Effectors Revealed by a Molecular Simulation Study

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    Hua Wan

    2016-01-01

    Full Text Available TAL effectors (TALEs contain a modular DNA-binding domain that is composed of tandem repeats. In all naturally occurring TALEs, the end of tandem repeats is invariantly a truncated half repeat. To investigate the potential role of the last half repeat in TALEs, we performed comparative molecular dynamics simulations for the crystal structure of DNA-bound TALE AvrBs3 lacking the last half repeat and its modeled structure having the last half repeat. The structural stability analysis indicates that the modeled system is more stable than the nonmodeled system. Based on the principle component analysis, it is found that the AvrBs3 increases its structural compactness in the presence of the last half repeat. The comparison of DNA groove parameters of the two systems implies that the last half repeat also causes the change of DNA major groove binding efficiency. The following calculation of hydrogen bond reveals that, by stabilizing the phosphate binding with DNA at the C-terminus, the last half repeat helps to adopt a compact conformation at the protein-DNA interface. It further mediates more contacts between TAL repeats and DNA nucleotide bases. Finally, we suggest that the last half repeat is required for the high-efficient recognition of DNA by TALE.

  14. Extending the baseline of tropical dry forest loss in Ghana (1984–2015) reveals drivers of major deforestation inside a protected area

    OpenAIRE

    Janssen, T; Ametsisi, G; Collins, M; Adu-Bredu, S; Oliveras-Menor, I; Mitchard, ETA; Veenendaal, EM

    2017-01-01

    Abstract Tropical dry forests experience the highest deforestation rates on Earth, with major implications for the biodiversity of these ecosystems, as well as for its human occupants. Global remote sensing based forest cover data (2000 − 2012) point to the rapid loss of tropical dry forest in South America and Africa, also, if not foremost, inside formally protected areas. Here, we significantly extend the baseline of tropical dry forest loss inside a protected area in Ghana using a generali...

  15. Bcl2-associated Athanogene 3 Interactome Analysis Reveals a New Role in Modulating Proteasome Activity*

    Science.gov (United States)

    Chen, Ying; Yang, Li-Na; Cheng, Li; Tu, Shun; Guo, Shu-Juan; Le, Huang-Ying; Xiong, Qian; Mo, Ran; Li, Chong-Yang; Jeong, Jun-Seop; Jiang, Lizhi; Blackshaw, Seth; Bi, Li-Jun; Zhu, Heng; Tao, Sheng-Ce; Ge, Feng

    2013-01-01

    Bcl2-associated athanogene 3 (BAG3), a member of the BAG family of co-chaperones, plays a critical role in regulating apoptosis, development, cell motility, autophagy, and tumor metastasis and in mediating cell adaptive responses to stressful stimuli. BAG3 carries a BAG domain, a WW domain, and a proline-rich repeat (PXXP), all of which mediate binding to different partners. To elucidate BAG3's interaction network at the molecular level, we employed quantitative immunoprecipitation combined with knockdown and human proteome microarrays to comprehensively profile the BAG3 interactome in humans. We identified a total of 382 BAG3-interacting proteins with diverse functions, including transferase activity, nucleic acid binding, transcription factors, proteases, and chaperones, suggesting that BAG3 is a critical regulator of diverse cellular functions. In addition, we characterized interactions between BAG3 and some of its newly identified partners in greater detail. In particular, bioinformatic analysis revealed that the BAG3 interactome is strongly enriched in proteins functioning within the proteasome-ubiquitination process and that compose the proteasome complex itself, suggesting that a critical biological function of BAG3 is associated with the proteasome. Functional studies demonstrated that BAG3 indeed interacts with the proteasome and modulates its activity, sustaining cell survival and underlying resistance to therapy through the down-modulation of apoptosis. Taken as a whole, this study expands our knowledge of the BAG3 interactome, provides a valuable resource for understanding how BAG3 affects different cellular functions, and demonstrates that biologically relevant data can be harvested using this kind of integrated approach. PMID:23824909

  16. Quantitative membrane proteomics reveals a role for tetraspanin enriched microdomains during entry of human cytomegalovirus.

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    Kasinath Viswanathan

    Full Text Available Human cytomegalovirus (HCMV depends on and modulates multiple host cell membrane proteins during each stage of the viral life cycle. To gain a global view of the impact of HCMV-infection on membrane proteins, we analyzed HCMV-induced changes in the abundance of membrane proteins in fibroblasts using stable isotope labeling with amino acids (SILAC, membrane fractionation and protein identification by two-dimensional liquid chromatography and tandem mass spectrometry. This systematic approach revealed that CD81, CD44, CD98, caveolin-1 and catenin delta-1 were down-regulated during infection whereas GRP-78 was up-regulated. Since CD81 downregulation was also observed during infection with UV-inactivated virus we hypothesized that this tetraspanin is part of the viral entry process. Interestingly, additional members of the tetraspanin family, CD9 and CD151, were also downregulated during HCMV-entry. Since tetraspanin-enriched microdomains (TEM cluster host cell membrane proteins including known CMV receptors such as integrins, we studied whether TEMs are required for viral entry. When TEMs were disrupted with the cholesterol chelator methyl-β-cylcodextrin, viral entry was inhibited and this inhibition correlated with reduced surface levels of CD81, CD9 and CD151, whereas integrin levels remained unchanged. Furthermore, simultaneous siRNA-mediated knockdown of multiple tetraspanins inhibited viral entry whereas individual knockdown had little effect suggesting essential, but redundant roles for individual tetraspanins during entry. Taken together, our data suggest that TEM act as platforms for receptors utilized by HCMV for entry into cells.

  17. Epistaxis revealing the rupture of a carotid aneurysm of the cavernous sinus extending into the sphenoid: treatment using an uncovered stent and coils. Review of literature.

    Science.gov (United States)

    Lehmann, Pierre; Saliou, Guillaume; Page, Cyril; Balut, Antonia; Le Gars, Daniel; Vallée, Jean Noel

    2009-05-01

    True carotid aneurysms with sphenoid extension and revealed by epistaxis are rare. A review of the literature shows the mortality risk of this pathology and the different therapeutic options. A 41-year-old female presented with a cavernous carotid aneurysm with sphenoid extension revealed by massive epistaxis. We propose a combined treatment of the affected vessel using coils and an uncovered stent. The first stage to stop the hemorrhages and occlude the aneurysm using the coil and the second stage several days later after anticoagulation using the stent to prevent revascularization. This treatment has been shown to be effective in producing immediate hemostasis and stable long-term occlusion.

  18. A national survey of pain clinics within the United Kingdom and Ireland focusing on the multidisciplinary team and the incorporation of the extended nursing role.

    Science.gov (United States)

    Kailainathan, Pungavi; Humble, Stephen; Dawson, Helen; Cameron, Fiona; Gokani, Shyam; Lidder, Gursimren

    2018-02-01

    Inconsistencies in the availability and quality of pain service provision have been noted nationally, as have lengthy waiting times for appointments and lack of awareness of the Pain Clinic role. The 2013 NHS England report stated that specialist pain services must offer multispecialty and multidisciplinary pain clinics. This national survey of multidisciplinary pain service provision in the United Kingdom and Ireland provides a snapshot of pain service provision in order to review and highlight what variations exist in multidisciplinary team (MDT) provision and working patterns. A common perception among clinicians is that financial pressures have led to alternate ways of staff utilisation with variable degrees of success. The survey included 143 pain clinics, focusing principally on MDT working patterns, MDT composition and adoption of the extended role. The results identified that the majority of Pain Clinics utilise the MDT approach. However, provision of critical components such as regular MDT meetings is highly variable as is the composition of the MDT itself and also working patterns of the individual clinicians. The survey reports the successful use of the extended roles for specialist nurses in follow up clinics. In contrast, the survey highlights that a large proportion of clinicians surveyed have reservations about both the effectiveness and the safety of utilising specialist nurses in the extended role to see new referrals of complex pain patients to pain clinics. This survey underlines the essential requirement for incorporation of greater MDT working locally and nationally and allocation of appropriate resources to facilitate this.

  19. Extended objects

    International Nuclear Information System (INIS)

    Creutz, M.

    1976-01-01

    After some disconnected comments on the MIT bag and string models for extended hadrons, I review current understanding of extended objects in classical conventional relativistic field theories and their quantum mechanical interpretation

  20. Modelling Facebook Usage among University Students in Thailand: The Role of Emotional Attachment in an Extended Technology Acceptance Model

    Science.gov (United States)

    Teo, Timothy

    2016-01-01

    The aim of this study is to examine the factors that influenced the use of Facebook among university students. Using an extended technology acceptance model (TAM) with emotional attachment (EA) as an external variable, a sample of 498 students from a public-funded Thailand university were surveyed on their responses to five variables hypothesized…

  1. Reconciling the Knowledge of Scholars & Practitioners: An Extended Case Analysis of the Role of Theory in Student Affairs

    Science.gov (United States)

    Kimball, Ezekiel

    2016-01-01

    This paper utilizes a critical post-pragmatist epistemological lens in tandem with an extended case analysis to explore how student affairs professionals process truth claims related to student experience. Findings from the study, which include the limited usage of formal theory and the iterative reconstruction of informal theory, are used to…

  2. Comparing Sexuality Communication Among Offspring of Teen Parents and Adult Parents: a Different Role for Extended Family

    OpenAIRE

    Grossman, Jennifer M.; Tracy, Allison J.; Richer, Amanda M.; Erkut, Sumru

    2015-01-01

    This brief report examined teenagers’ sexuality communication with their parents and extended families. It compared who teens of early parents (those who had children when they were adolescents) and teens of later parents (those who were adults when they had children) talk to about sex. Eighth grade students (N=1281) in 24 schools completed survey items about their communication about sex. Structural equation modeling was used to predict communication profiles, while adjusting for the nesting...

  3. How Far Can Extended Knowledge Be Extended?

    DEFF Research Database (Denmark)

    Wray, K. Brad

    2018-01-01

    by an artifact, like a notebook or telescope. The chapter illustrates this by applying Pritchard’s account of extended knowledge to collaborating scientists. The beliefs acquired through collaborative research cannot satisfy both of Pritchard’s conditions of creditability. Further, there is evidence......Duncan Pritchard (2010) has developed a theory of extended knowledge based on the notion of extended cognition initially developed by Clark and Chalmers (1998). Pritchard’s account gives a central role to the notion of creditability, which requires the following two conditions to be met: (i...... that scientists are not prepared to take responsibility for the actions of the scientists with whom they collaborate....

  4. Extended local similarity analysis (eLSA) reveals unique associations between bacterial community structure and odor emission during pig carcasses decomposition.

    Science.gov (United States)

    Ki, Bo-Min; Ryu, Hee Wook; Cho, Kyung-Suk

    2018-02-22

    Soil burial and composting methods have been widely used for the disposal of pig carcasses. The relationship between bacterial community structure and odor emission was examined using extended local similarity analysis (eLSA) during the degradation of pig carcasses in soil and compost. In soil, Hyphomicrobium, Niastella, Rhodanobacter, Polaromonas, Dokdonella and Mesorhizobium were associated with the emission of sulfur-containing odors such as hydrogen sulfide, methyl mercaptan and dimethyl disulfide. Sphingomonas, Rhodanobacter, Mesorhizobium, Dokdonella, Leucobacter and Truepera were associated with the emission of nitrogen-containing odors including ammonia and trimetylamine. In compost, however, Carnobacteriaceae, Lachnospiaceae and Clostridiales were highly correlated with the emission of sulfur-containing odors, while Rumincoccaceae was associated with the emission of nitrogen-containing odors. The emission of organic acids was closely related to Massilia, Sphaerobacter and Bradyrhizobiaceae in soil, but to Actinobacteria, Sporacetigenium, Micromonosporaceae and Solirubrobacteriales in compost. This study suggests that network analysis using eLSA is a useful strategy for exploring the mechanisms of odor emission during biodegradation of pig carcasses.

  5. The Military and Domestic Disaster Response: Lead Role Revealed Through the Eye of Hurricane Katrina?

    National Research Council Canada - National Science Library

    Walker, Juliana M

    2006-01-01

    .... During and in the aftermath of Hurricane Katrina however the slow and perceived inept response to the massive disaster prompted a national debate on the appropriate role of the military in major domestic disasters...

  6. The transcriptional response to the olive fruit fly (Bactrocera oleae) reveals extended differences between tolerant and susceptible olive (Olea europaea L.) varieties.

    Science.gov (United States)

    Grasso, Filomena; Coppola, Mariangela; Carbone, Fabrizio; Baldoni, Luciana; Alagna, Fiammetta; Perrotta, Gaetano; Pérez-Pulido, Antonio J; Garonna, Antonio; Facella, Paolo; Daddiego, Loretta; Lopez, Loredana; Vitiello, Alessia; Rao, Rosa; Corrado, Giandomenico

    2017-01-01

    The olive fruit fly Bactrocera oleae (Diptera: Tephritidae) is the most devastating pest of cultivated olive (Olea europaea L.). Intraspecific variation in plant resistance to B. oleae has been described only at phenotypic level. In this work, we used a transcriptomic approach to study the molecular response to the olive fruit fly in two olive cultivars with contrasting level of susceptibility. Using next-generation pyrosequencing, we first generated a catalogue of more than 80,000 sequences expressed in drupes from approximately 700k reads. The assembled sequences were used to develop a microarray layout with over 60,000 olive-specific probes. The differential gene expression analysis between infested (i.e. with II or III instar larvae) and control drupes indicated a significant intraspecific variation between the more tolerant and susceptible cultivar. Around 2500 genes were differentially regulated in infested drupes of the tolerant variety. The GO annotation of the differentially expressed genes implies that the inducible resistance to the olive fruit fly involves a number of biological functions, cellular processes and metabolic pathways, including those with a known role in defence, oxidative stress responses, cellular structure, hormone signalling, and primary and secondary metabolism. The difference in the induced transcriptional changes between the cultivars suggests a strong genetic role in the olive inducible defence, which can ultimately lead to the discovery of factors associated with a higher level of tolerance to B. oleae.

  7. Third Year Medical School Students' Experiences of Revealing Patients' Stories through Role Playing.

    Science.gov (United States)

    Cansever, Zeliha; Avsar, Zeynep; Tastan, Kenan

    2015-02-01

    Studying medicine is hard and it takes longer time compared to other majors. In addition, medical students find medical education boring. It is now necessary to turn medical education into an enjoyable and interesting way. The aim of this study is to evaluate the impact of an educational program related to how to learn taking medical history and how an effective patient-doctor interview should be. The program is structured in various scenarios, on the students learning skills, by the "role playing" method. A scenario prepared by the lecturer was employed in this study. While one of the students acted in a doctor role, the other one played in the role of patient's relative. The lecturer always played in the role of patient. After performing the role playing, students' written and oral feedbacks were gathered. Data were analysed by using SPSS 20.0 program. A total of 470 feedbacks (51.3% were given by the female students) were taken from the students. Thirty-three volunteer students, nineteen of them were male, took part in the role playing. In the patient-doctor interview, the field that students were best were greeting the patients and dealing only with patients during the examination. The mean scores were 3.81±0.95 and 3.79±0.94 respectively. The ability to "summarize" and to "address the patient with his/her name" had the lowest scores; the mean scores of the students in these areas were 2.94±1.11 and 2.70±1.31, respectively. Medical education is a long and tough process. Therefore, it should be interesting, attention getting and cheerful. Role playing can be effective in meeting that need.

  8. An extended role for thermoluminescent phosphors in personnel, environmental and accident dosimetry using sensitisation, re-estimation and fast fading

    International Nuclear Information System (INIS)

    Charles, M.W.

    1983-01-01

    This paper summarises some techniques for extending the usefulness of conventional phosphors in personnel, environmental and accident dosimetry. An optimised procedure for utilising radiation sensitisation and UV re-estimation in thermoluminescent LiF is presented. In particular it is shown that optimum performance is achieved by using a UV wavelength of 250 +- 10 nm for both the UV/thermal anneal following sensitisation, and for the UV re-estimation procedure. In the case of Harshaw Lif Chips (3x3x0.9 mm 3 ) the sensitivity is increased by a factor of 4-5 to achieve a minimum detectable dose of approx.=10 μGy (2sigma) and a minimum re-estimable dose of 50-100 mGy (2sigma), dependent on batch. Sensitized LiF also exhibits improved tissue equivalence, extended linearity and improved precision at low doses. The information from fast-fading glow peaks, which is normally rejected, is shown to have a useful application to the evaluation of short-term increases in environmental dose rates such as may occur following accidental releases of radioactivity. (orig.)

  9. Role of the extended MAPT haplotype in the worsening of psychotic symptoms and treatment response in Alzheimer disease.

    Science.gov (United States)

    Creese, Byron; Corbett, Anne; Jones, Emma; Fox, Chris; Ballard, Clive

    2014-12-01

    There is evidence that neurofibrillary tangle (NFT) burden is associated with psychotic symptoms in Alzheimer disease (AD). However, it is not clear whether this association is direct or mediated through the increased cognitive impairment associated with NFTs. We sought to determine whether the extended MAPT haplotype was associated with the worsening of delusions and hallucinations in a combined cohort of 95 patients who participated in 2 clinical trials of treatment with memantine. After controlling for baseline dementia severity, exposure to memantine, and antipsychotics, analysis shows that carriers of at least one H2 allele had a 5.4-fold (P = .03) increased risk of worsening hallucinations. There was some evidence of association with worsening delusions but only in analysis by allele. These results are the first to indicate that the H2 allele of the extended MAPT haplotype negatively affects the course of psychotic symptoms in AD independently of disease severity. It will be important for future research to examine MAPT transcription in people with AD with and without psychotic symptoms to understand the exact mechanisms underlying these findings. Copyright © 2014 AMDA – The Society for Post-Acute and Long-Term Care Medicine. Published by Elsevier Inc. All rights reserved.

  10. Lateralized odor preference training in rat pups reveals an enhanced network response in anterior piriform cortex to olfactory input that parallels extended memory.

    Science.gov (United States)

    Fontaine, Christine J; Harley, Carolyn W; Yuan, Qi

    2013-09-18

    The present study examines synaptic plasticity in the anterior piriform cortex (aPC) using ex vivo slices from rat pups given lateralized odor preference training. In the early odor preference learning model, a brief 10 min training session yields 24 h memory, while four daily sessions yield 48 h memory. Odor preference memory can be lateralized through naris occlusion as the anterior commissure is not yet functional. AMPA receptor-mediated postsynaptic responses in the aPC to lateral olfactory tract input, shown to be enhanced at 24 h, are no longer enhanced 48 h after a single training session. Following four spaced lateralized trials, the AMPA receptor-mediated fEPSP is enhanced in the trained aPC at 48 h. Calcium imaging of aPC pyramidal cells within 48 h revealed decreased firing thresholds in the pyramidal cell network. Thus multiday odor preference training induced increased odor input responsiveness in previously weakly activated aPC cells. These results support the hypothesis that increased synaptic strength in olfactory input networks mediates odor preference memory. The increase in aPC network activation parallels behavioral memory.

  11. Extended Emotions

    DEFF Research Database (Denmark)

    Krueger, Joel; Szanto, Thomas

    2016-01-01

    beyond the neurophysiological confines of organisms; some even argue that emotions can be socially extended and shared by multiple agents. Call this the extended emotions thesis (ExE). In this article, we consider different ways of understanding ExE in philosophy, psychology, and the cognitive sciences...

  12. Proximal humeral fractures: the role of calcium sulphate augmentation and extended deltoid splitting approach in internal fixation using locking plates.

    Science.gov (United States)

    Somasundaram, K; Huber, C P; Babu, V; Zadeh, H

    2013-04-01

    The aim of our study is to analyse the results of our surgical technique for the treatment of proximal humeral fractures and fracture dislocations using locking plates in conjunction with calcium sulphate bone-substitute augmentation and tuberosity repair using high-strength sutures. We used the extended deltoid-splitting approach for fracture patterns involving displacement of both lesser and greater tuberosities and for fracture-dislocations. Optimal surgical management of proximal humeral fractures remains controversial. Locking plates have become a popular method of fixation. However, failure of fixation may occur if they are used as the sole method of fixation in comminuted fractures, especially in osteopenic bone. We retrospectively analysed 22 proximal humeral fractures in 21 patients; 10 were male and 11 female with an average age of 64.6 years (range 37-77). Average follow-up was 24 months. Eleven of these fractures were exposed by the extended deltoid-splitting approach. Fractures were classified according to Neer and Hertel systems. Preoperative radiographs and computed tomography (CT) scans in three- and four-part fractures were done to assess the displacement and medial calcar length for predicting the humeral head vascularity. According to the Neer classification, there were five two-part, six three-part, five four-part fractures and six fracture-dislocations (two anterior and four posterior). Results were assessed clinically with disabilities of the arm, shoulder and hand (DASH) scores, modified Constant and Murley scores and serial postoperative radiographs. The mean DASH score was 16.18 and the modified Constant and Murley score was 64.04 at the last follow-up. Eighteen out of twenty-two cases achieved good clinical outcome. All the fractures united with no evidence of infection, failure of fixation, malunion, tuberosity failure, avascular necrosis or adverse reaction to calcium sulphate bone substitute. There was no evidence of axillary nerve

  13. Proteomic investigations reveal a role for RNA processing factor THRAP3 in the DNA damage response

    DEFF Research Database (Denmark)

    Beli, Petra; Lukashchuk, Natalia; Wagner, Sebastian A

    2012-01-01

    /ATR/DNA-PK target consensus motif, suggesting an important role of downstream kinases in amplifying DDR signals. We show that the splicing-regulator phosphatase PPM1G is recruited to sites of DNA damage, while the splicing-associated protein THRAP3 is excluded from these regions. Moreover, THRAP3 depletion causes...

  14. Metabolomic profiling reveals a role for CPT1c in neuronal oxidative metabolism.

    Science.gov (United States)

    Lee, Jieun; Wolfgang, Michael J

    2012-10-25

    Carnitine Palmitoyltransferase-1c (CPT1c) is a neuron specific homologue of the carnitine acyltransferase family of enzymes. CPT1 isoenzymes transfer long chain acyl groups to carnitine. This constitutes a rate setting step for mitochondrial fatty acid beta-oxidation by facilitating the initial step in acyl transfer to the mitochondrial matrix. In general, neurons do not heavily utilize fatty acids for bioenergetic needs and definitive enzymatic activity has been unable to be demonstrated for CPT1c. Although there are studies suggesting an enzymatic role of CPT1c, its role in neurochemistry remains elusive. In order to better understand how CPT1c functions in neural metabolism, we performed unbiased metabolomic profiling on wild-type (WT) and CPT1c knockout (KO) mouse brains. Consistent with the notion that CPT1c is not involved in fatty acid beta-oxidation, there were no changes in metabolites associated with fatty acid oxidation. Endocannabinoids were suppressed in the CPT1c KO, which may explain the suppression of food intake seen in CPT1c KO mice. Although products of beta-oxidation were unchanged, small changes in carnitine and carnitine metabolites were observed. Finally, we observed changes in redox homeostasis including a greater than 2-fold increase in oxidized glutathione. This indicates that CPT1c may play a role in neural oxidative metabolism. Steady-state metabolomic analysis of CPT1c WT and KO mouse brains identified a small number of metabolites that differed between CPT1c WT and KO mice. The subtle changes in a broad range of metabolites in vivo indicate that CPT1c does not play a significant or required role in fatty acid oxidation; however, it could play an alternative role in neuronal oxidative metabolism.

  15. Metabolomic profiling reveals a role for CPT1c in neuronal oxidative metabolism

    Directory of Open Access Journals (Sweden)

    Lee Jieun

    2012-10-01

    Full Text Available Abstract Background Carnitine Palmitoyltransferase-1c (CPT1c is a neuron specific homologue of the carnitine acyltransferase family of enzymes. CPT1 isoenzymes transfer long chain acyl groups to carnitine. This constitutes a rate setting step for mitochondrial fatty acid beta-oxidation by facilitating the initial step in acyl transfer to the mitochondrial matrix. In general, neurons do not heavily utilize fatty acids for bioenergetic needs and definitive enzymatic activity has been unable to be demonstrated for CPT1c. Although there are studies suggesting an enzymatic role of CPT1c, its role in neurochemistry remains elusive. Results In order to better understand how CPT1c functions in neural metabolism, we performed unbiased metabolomic profiling on wild-type (WT and CPT1c knockout (KO mouse brains. Consistent with the notion that CPT1c is not involved in fatty acid beta-oxidation, there were no changes in metabolites associated with fatty acid oxidation. Endocannabinoids were suppressed in the CPT1c KO, which may explain the suppression of food intake seen in CPT1c KO mice. Although products of beta-oxidation were unchanged, small changes in carnitine and carnitine metabolites were observed. Finally, we observed changes in redox homeostasis including a greater than 2-fold increase in oxidized glutathione. This indicates that CPT1c may play a role in neural oxidative metabolism. Conclusions Steady-state metabolomic analysis of CPT1c WT and KO mouse brains identified a small number of metabolites that differed between CPT1c WT and KO mice. The subtle changes in a broad range of metabolites in vivo indicate that CPT1c does not play a significant or required role in fatty acid oxidation; however, it could play an alternative role in neuronal oxidative metabolism.

  16. 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.

  17. A multigene mutation classification of 468 colorectal cancers reveals a prognostic role for APC

    Science.gov (United States)

    Schell, Michael J.; Yang, Mingli; Teer, Jamie K.; Lo, Fang Yin; Madan, Anup; Coppola, Domenico; Monteiro, Alvaro N. A.; Nebozhyn, Michael V.; Yue, Binglin; Loboda, Andrey; Bien-Willner, Gabriel A.; Greenawalt, Danielle M.; Yeatman, Timothy J.

    2016-01-01

    Colorectal cancer (CRC) is a highly heterogeneous disease, for which prognosis has been relegated to clinicopathologic staging for decades. There is a need to stratify subpopulations of CRC on a molecular basis to better predict outcome and assign therapies. Here we report targeted exome-sequencing of 1,321 cancer-related genes on 468 tumour specimens, which identified a subset of 17 genes that best classify CRC, with APC playing a central role in predicting overall survival. APC may assume 0, 1 or 2 truncating mutations, each with a striking differential impact on survival. Tumours lacking any APC mutation carry a worse prognosis than single APC mutation tumours; however, two APC mutation tumours with mutant KRAS and TP53 confer the poorest survival among all the subgroups examined. Our study demonstrates a prognostic role for APC and suggests that sequencing of APC may have clinical utility in the routine staging and potential therapeutic assignment for CRC. PMID:27302369

  18. Selective spider toxins reveal a role for Nav1.1 channel in mechanical pain

    OpenAIRE

    Osteen, Jeremiah D.; Herzig, Volker; Gilchrist, John; Emrick, Joshua J.; Zhang, Chuchu; Wang, Xidao; Castro, Joel; Garcia-Caraballo, Sonia; Grundy, Luke; Rychkov, Grigori Y.; Weyer, Andy D.; Dekan, Zoltan; Undheim, Eivind A. B.; Alewood, Paul; Stucky, Cheryl L.

    2016-01-01

    Voltage-gated sodium (Nav) channels initiate action potentials in most neurons, including primary afferent nerve fibers of the pain pathway. Local anesthetics block pain through non-specific actions at all Nav channels, but the discovery of selective modulators would facilitate the analysis of individual subtypes and their contributions to chemical, mechanical, or thermal pain. Here, we identify and characterize spider toxins that selectively activate the Nav1.1 subtype, whose role in nocicep...

  19. X-ray fluorescence microscopy reveals the role of selenium in spermatogenesis

    OpenAIRE

    Kehr, Sebastian; Malinouski, Mikalai; Finney, Lydia; Vogt, Stefan; Labunskyy, Vyacheslav M.; Kasaikina, Marina V.; Carlson, Bradley A.; Zhou, You; Hatfield, Dolph L.; Gladyshev, Vadim N.

    2009-01-01

    Selenium (Se) is a trace element with important roles in human health. Several selenoproteins have essential functions in development. However, the cellular and tissue distribution of Se remains largely unknown because of the lack of analytical techniques that image this element with sufficient sensitivity and resolution. Herein, we report that X-ray fluorescence microscopy (XFM) can be used to visualize and quantify the tissue, cellular and subcellular topography of Se. We applied this techn...

  20. Pam heterozygous mice reveal essential role for Cu in amygdalar behavioral and synaptic function.

    Science.gov (United States)

    Gaier, Eric D; Eipper, Betty A; Mains, Richard E

    2014-05-01

    Copper (Cu) is an essential element with many biological roles, but its roles in the mammalian nervous system are poorly understood. Mice deficient in the cuproenzyme peptidylglycine α-amidating monooxygenase (Pam(+/-) mice) were initially generated to study neuropeptide amidation. Pam(+/-) mice exhibit profound deficits in a few behavioral tasks, including enhancements in innate fear along with deficits in acquired fear. Interestingly, several Pam(+/-) phenotypes were recapitulated in Cu-restricted wild-type mice and rescued in Cu-supplemented Pam(+/-) mice. These behaviors correspond to enhanced excitability and deficient synaptic plasticity in the amygdala of Pam(+/-) mice, which are also rescued by Cu supplementation. Cu and ATP7A are present at synapses, in key positions to respond to and influence synaptic activity. Further study demonstrated that extracellular Cu is necessary for wild-type synaptic plasticity and sufficient to induce long-term potentiation. These experiments support roles for PAM in Cu homeostasis and for synaptic Cu in amygdalar function. © 2014 New York Academy of Sciences.

  1. Don’t let up: implementing and sustaining change in a new post-licensure education model for developing extended role practitioners involved in arthritis care

    Directory of Open Access Journals (Sweden)

    Lundon K

    2015-08-01

    Full Text Available Katie Lundon,1,3 Rachel Shupak,1–3 Sonya Canzian,4 Ed Ziesmann,5 Rayfel Schneider,6,71Office of Continuing Professional Development, Faculty of Medicine, University of Toronto, 2Division of Rheumatology, St Michael's Hospital, 3Department of Medicine, University of Toronto, 4Trauma/Neurosurgery and Mobility Programs, St Michael's Hospital, 5Programs and Services, The Arthritis Society, 6Division of Rheumatology, The Hospital for Sick Children, 7Department of Paediatrics, University of Toronto, Toronto, ON, CanadaKey message: Across a 9-year period, the Advanced Clinician Practitioner in Arthritis Care program has achieved a set of short-term “wins” giving direction and momentum to the development of new roles for health care practitioners providing arthritis care.Implications: This is a viable model for post-licensure training offered to multiple allied health professionals to support the development of competent extended role practitioners (extended scope practice. Challenges at this critical juncture include: retain focus, drive, and commitment; develop academic and financial partnerships transferring short-term success to long-term sustainability; advanced, context-driven, system-level evaluation including fiscal outcome; health care policy adaptation to new human health resource development.Supporting evidence: Success includes: completed 2-year health services research evaluating 37 graduates; leadership, innovation, educational excellence, and human health resource benefit awards; influential publications/presentations addressing post-licensure education/outcome, interprofessional collaboration, and improved patient care. Keywords: human health resource development, post-licensure education, arthritis, extended role practitioners, allied health professionals

  2. Taking a fresh look at optical crowding of TiO2: the role of nanocarbonate as synergistic optical extender

    Science.gov (United States)

    Ridgway, Cathy J.; Gane, Patrick A. C.

    2013-10-01

    The light scattering potential of titanium dioxide (TiO2) is maximized when the TiO2 particles are correctly spaced in relation to the size of the particles, their spacing distance and the refractive index contrast with the surrounding medium. Nanoparticle extenders are claimed to improve TiO2 spacing due to the small particles acting to separate larger TiO2 particles (void size in respect to the separation of the surfaces ˜0.2 µm) and so preventing the TiO2 particles from reaching optical crowding. This concept has been challenged by previous work (Diebold 2011 J. Coat. Technol. Res. 8 541-52) using a combination of Monte Carlo simulations and image analysis techniques. Surprisingly in the light of traditional interpretations, the results showed that nanoparticles did not, in fact, space TiO2 particles under any conditions examined. Instead, TiO2 distributions and spacings were completely indifferent to the presence of smaller particles. This current paper undertakes to study why in certain cases extenders do in fact deliver significant synergy allowing TiO2 levels to be reduced in practice. The use of nano-calcium carbonate is demonstrated to provide a wide range of synergistic extension of TiO2 when in combination with synthetic latex binder, but, as found in the previous theoretical work, only a limited synergistic range in direct pigment packing studies using slurry pigment mixes only. A schematic model of the action of calcium carbonate containing a high nanoparticle fraction in the presence of binder is proposed, and is related to a combination of the potential reduction of contact between latex and TiO2 related to an increase in effective pigment volume fraction and surface area as well as the potential for differential flocculation. This model is supported by electron microscopy images and a measure of the ‘hardness’ of the skeletal elements making up the coating structure, determined via the bulk modulus using mercury intrusion porosimetry, enabling

  3. Taking a fresh look at optical crowding of TiO2: the role of nanocarbonate as synergistic optical extender

    International Nuclear Information System (INIS)

    Ridgway, Cathy J; Gane, Patrick A C

    2013-01-01

    The light scattering potential of titanium dioxide (TiO 2 ) is maximized when the TiO 2 particles are correctly spaced in relation to the size of the particles, their spacing distance and the refractive index contrast with the surrounding medium. Nanoparticle extenders are claimed to improve TiO 2 spacing due to the small particles acting to separate larger TiO 2 particles (void size in respect to the separation of the surfaces ∼0.2 µm) and so preventing the TiO 2 particles from reaching optical crowding. This concept has been challenged by previous work (Diebold 2011 J. Coat. Technol. Res. 8 541–52) using a combination of Monte Carlo simulations and image analysis techniques. Surprisingly in the light of traditional interpretations, the results showed that nanoparticles did not, in fact, space TiO 2 particles under any conditions examined. Instead, TiO 2 distributions and spacings were completely indifferent to the presence of smaller particles. This current paper undertakes to study why in certain cases extenders do in fact deliver significant synergy allowing TiO 2 levels to be reduced in practice. The use of nano-calcium carbonate is demonstrated to provide a wide range of synergistic extension of TiO 2 when in combination with synthetic latex binder, but, as found in the previous theoretical work, only a limited synergistic range in direct pigment packing studies using slurry pigment mixes only. A schematic model of the action of calcium carbonate containing a high nanoparticle fraction in the presence of binder is proposed, and is related to a combination of the potential reduction of contact between latex and TiO 2 related to an increase in effective pigment volume fraction and surface area as well as the potential for differential flocculation. This model is supported by electron microscopy images and a measure of the ‘hardness’ of the skeletal elements making up the coating structure, determined via the bulk modulus using mercury intrusion

  4. Evaluation of bone marrow examinations performed by an advanced nurse practitioner: an extended role within a haematology service.

    LENUS (Irish Health Repository)

    Kelly, Mary

    2012-01-31

    PURPOSE: Traditionally, medical personnel have undertaken bone marrow (BM) examination. However, specially trained nurses in advanced practice roles are increasingly undertaking this role. This paper presents the findings from an audit of BM examinations undertaken by an advanced nurse practitioner (ANP) at a regional haematology specialist centre. METHODS: The audit evaluated the quality of BM examinations performed by the ANP over the past two years (September 2007-September 2009). Over the two year period, 324 BM examinations were performed at the centre of which 156 (48.1%) were performed by the ANP. A random sample of 30 BM examinations undertaken by the ANP were analysed by the consultant haematologist. RESULTS: All 30 BM examinations undertaken by the ANP were sufficient for diagnosis. CONCLUSIONS: The ANP is capable and competent to obtain BM samples which are of a sufficient quality to permit diagnosis.

  5. Extended education and the re-definition of the role of educational psychology for science and mathematics teachers

    OpenAIRE

    Maria Marques Zanforlin Pires de Almeida, Inês; Francesca Conte de Almeida, Sandra

    2012-01-01

    This article aims to address the topic of continued teacher education, one of the main themes of the PhD thesis in psychology Re-definition of the Role cif Educational Psychology in Continued Education of Science and Mathetnatics Teachers. By tracing the historical path of the different trends, concepts and movements regarding continued education and the relationship between psychology and education, it has been possible to develop severa! guidelines and reflections related to pedagogical...

  6. Gender roles, physical and sexual violence prevention in primary extend to secondary school in Samutsakorn Province, Thailand.

    Science.gov (United States)

    Chamroonsawasdi, Kanittha; Suparp, Jarueyporn; Kittipichai, Wirin; Khajornchaikul, Piyathida

    2010-03-01

    To enhance positive attitude and life skills on gender roles to prevent physical and sexual violence. A whole school-based participatory learning program using a quasi-experimental study with pre and post test design was conducted among 2 schools during June-September, 2005. The experimental group, were 134 students in a primary school and 179 students in a secondary school. While the control group, were 122 students in a primary school and 95 students in a secondary school. Means score of attitude toward gender roles before implementation in the experimental group was significantly lower than the control group (p 0.05). Means paired different score (after-before) between the two groups was significantly different (p = 0.002). A whole school-based program on gender roles and violence prevention is suitable for youths and should be merged as school curricula and expanded as a nationwide program at all level of education. Gender equity should be taught at an early childhood. Parental involvement in school-based activities should be negotiated.

  7. Mcph1-deficient mice reveal a role for MCPH1 in otitis media.

    Directory of Open Access Journals (Sweden)

    Jing Chen

    Full Text Available Otitis media is a common reason for hearing loss, especially in children. Otitis media is a multifactorial disease and environmental factors, anatomic dysmorphology and genetic predisposition can all contribute to its pathogenesis. However, the reasons for the variable susceptibility to otitis media are elusive. MCPH1 mutations cause primary microcephaly in humans. So far, no hearing impairment has been reported either in the MCPH1 patients or mouse models with Mcph1 deficiency. In this study, Mcph1-deficient (Mcph1(tm1a (/tm1a mice were produced using embryonic stem cells with a targeted mutation by the Sanger Institute's Mouse Genetics Project. Auditory brainstem response measurements revealed that Mcph1(tm1a (/tm1a mice had mild to moderate hearing impairment with around 70% penetrance. We found otitis media with effusion in the hearing-impaired Mcph1(tm1a (/tm1a mice by anatomic and histological examinations. Expression of Mcph1 in the epithelial cells of middle ear cavities supported its involvement in the development of otitis media. Other defects of Mcph1(tm1a (/tm1a mice included small skull sizes, increased micronuclei in red blood cells, increased B cells and ocular abnormalities. These findings not only recapitulated the defects found in other Mcph1-deficient mice or MCPH1 patients, but also revealed an unexpected phenotype, otitis media with hearing impairment, which suggests Mcph1 is a new gene underlying genetic predisposition to otitis media.

  8. Extended spawning in brown trout (Salmo trutta populations from the Southern Iberian Peninsula: the role of climate variability

    Directory of Open Access Journals (Sweden)

    José E. Larios-López

    2014-02-01

    Full Text Available The reproductive periods of brown trout (Salmo trutta populations in 12 rivers in the Baetic Mountains in southern Spain were studied from 2008 to 2013. This area is an ecological and geographical limit for the distribution of this species in Europe. We found that the spawning period has been markedly extended in these fish. The mean spawning dates in the studied populations are consistent with the European trend at this latitude, but our data suggest that females from most of the populations that we studied are able to produce eggs from early October through late April or early May, yielding a reproductive period of between 150 and 170 days, the longest and most delayed brown trout reproduction periods that have been reported in the literature. We believe that such expanded spawning periods result primarily from the unpredictability of the Mediterranean climate, although it is possible that other factors may have contributed to the development of this reproductive behaviour. This hypothesis is discussed in the context of a comparison of our results with those found for other European S. trutta populations.

  9. Simple analytical model reveals the functional role of embodied sensorimotor interaction in hexapod gaits

    Science.gov (United States)

    Aoi, Shinya; Nachstedt, Timo; Manoonpong, Poramate; Wörgötter, Florentin; Matsuno, Fumitoshi

    2018-01-01

    Insects have various gaits with specific characteristics and can change their gaits smoothly in accordance with their speed. These gaits emerge from the embodied sensorimotor interactions that occur between the insect’s neural control and body dynamic systems through sensory feedback. Sensory feedback plays a critical role in coordinated movements such as locomotion, particularly in stick insects. While many previously developed insect models can generate different insect gaits, the functional role of embodied sensorimotor interactions in the interlimb coordination of insects remains unclear because of their complexity. In this study, we propose a simple physical model that is amenable to mathematical analysis to explain the functional role of these interactions clearly. We focus on a foot contact sensory feedback called phase resetting, which regulates leg retraction timing based on touchdown information. First, we used a hexapod robot to determine whether the distributed decoupled oscillators used for legs with the sensory feedback generate insect-like gaits through embodied sensorimotor interactions. The robot generated two different gaits and one had similar characteristics to insect gaits. Next, we proposed the simple model as a minimal model that allowed us to analyze and explain the gait mechanism through the embodied sensorimotor interactions. The simple model consists of a rigid body with massless springs acting as legs, where the legs are controlled using oscillator phases with phase resetting, and the governed equations are reduced such that they can be explained using only the oscillator phases with some approximations. This simplicity leads to analytical solutions for the hexapod gaits via perturbation analysis, despite the complexity of the embodied sensorimotor interactions. This is the first study to provide an analytical model for insect gaits under these interaction conditions. Our results clarified how this specific foot contact sensory

  10. Application of a hierarchical enzyme classification method reveals the role of gut microbiome in human metabolism.

    Science.gov (United States)

    Mohammed, Akram; Guda, Chittibabu

    2015-01-01

    Enzymes are known as the molecular machines that drive the metabolism of an organism; hence identification of the full enzyme complement of an organism is essential to build the metabolic blueprint of that species as well as to understand the interplay of multiple species in an ecosystem. Experimental characterization of the enzymatic reactions of all enzymes in a genome is a tedious and expensive task. The problem is more pronounced in the metagenomic samples where even the species are not adequately cultured or characterized. Enzymes encoded by the gut microbiota play an essential role in the host metabolism; thus, warranting the need to accurately identify and annotate the full enzyme complements of species in the genomic and metagenomic projects. To fulfill this need, we develop and apply a method called ECemble, an ensemble approach to identify enzymes and enzyme classes and study the human gut metabolic pathways. ECemble method uses an ensemble of machine-learning methods to accurately model and predict enzymes from protein sequences and also identifies the enzyme classes and subclasses at the finest resolution. A tenfold cross-validation result shows accuracy between 97 and 99% at different levels in the hierarchy of enzyme classification, which is superior to comparable methods. We applied ECemble to predict the entire complements of enzymes from ten sequenced proteomes including the human proteome. We also applied this method to predict enzymes encoded by the human gut microbiome from gut metagenomic samples, and to study the role played by the microbe-derived enzymes in the human metabolism. After mapping the known and predicted enzymes to canonical human pathways, we identified 48 pathways that have at least one bacteria-encoded enzyme, which demonstrates the complementary role of gut microbiome in human gut metabolism. These pathways are primarily involved in metabolizing dietary nutrients such as carbohydrates, amino acids, lipids, cofactors and

  11. Application of a hierarchical enzyme classification method reveals the role of gut microbiome in human metabolism

    Science.gov (United States)

    2015-01-01

    Background Enzymes are known as the molecular machines that drive the metabolism of an organism; hence identification of the full enzyme complement of an organism is essential to build the metabolic blueprint of that species as well as to understand the interplay of multiple species in an ecosystem. Experimental characterization of the enzymatic reactions of all enzymes in a genome is a tedious and expensive task. The problem is more pronounced in the metagenomic samples where even the species are not adequately cultured or characterized. Enzymes encoded by the gut microbiota play an essential role in the host metabolism; thus, warranting the need to accurately identify and annotate the full enzyme complements of species in the genomic and metagenomic projects. To fulfill this need, we develop and apply a method called ECemble, an ensemble approach to identify enzymes and enzyme classes and study the human gut metabolic pathways. Results ECemble method uses an ensemble of machine-learning methods to accurately model and predict enzymes from protein sequences and also identifies the enzyme classes and subclasses at the finest resolution. A tenfold cross-validation result shows accuracy between 97 and 99% at different levels in the hierarchy of enzyme classification, which is superior to comparable methods. We applied ECemble to predict the entire complements of enzymes from ten sequenced proteomes including the human proteome. We also applied this method to predict enzymes encoded by the human gut microbiome from gut metagenomic samples, and to study the role played by the microbe-derived enzymes in the human metabolism. After mapping the known and predicted enzymes to canonical human pathways, we identified 48 pathways that have at least one bacteria-encoded enzyme, which demonstrates the complementary role of gut microbiome in human gut metabolism. These pathways are primarily involved in metabolizing dietary nutrients such as carbohydrates, amino acids, lipids

  12. Genotypic Resistance Tests Sequences Reveal the Role of Marginalized Populations in HIV-1 Transmission in Switzerland.

    Science.gov (United States)

    Shilaih, Mohaned; Marzel, Alex; Yang, Wan Lin; Scherrer, Alexandra U; Schüpbach, Jörg; Böni, Jürg; Yerly, Sabine; Hirsch, Hans H; Aubert, Vincent; Cavassini, Matthias; Klimkait, Thomas; Vernazza, Pietro L; Bernasconi, Enos; Furrer, Hansjakob; Günthard, Huldrych F; Kouyos, Roger

    2016-06-14

    Targeting hard-to-reach/marginalized populations is essential for preventing HIV-transmission. A unique opportunity to identify such populations in Switzerland is provided by a database of all genotypic-resistance-tests from Switzerland, including both sequences from the Swiss HIV Cohort Study (SHCS) and non-cohort sequences. A phylogenetic tree was built using 11,127 SHCS and 2,875 Swiss non-SHCS sequences. Demographics were imputed for non-SHCS patients using a phylogenetic proximity approach. Factors associated with non-cohort outbreaks were determined using logistic regression. Non-B subtype (univariable odds-ratio (OR): 1.9; 95% confidence interval (CI): 1.8-2.1), female gender (OR: 1.6; 95% CI: 1.4-1.7), black ethnicity (OR: 1.9; 95% CI: 1.7-2.1) and heterosexual transmission group (OR:1.8; 95% CI: 1.6-2.0), were all associated with underrepresentation in the SHCS. We found 344 purely non-SHCS transmission clusters, however, these outbreaks were small (median 2, maximum 7 patients) with a strong overlap with the SHCS'. 65% of non-SHCS sequences were part of clusters composed of >= 50% SHCS sequences. Our data suggests that marginalized-populations are underrepresented in the SHCS. However, the limited size of outbreaks among non-SHCS patients in-care implies that no major HIV outbreak in Switzerland was missed by the SHCS surveillance. This study demonstrates the potential of sequence data to assess and extend the scope of infectious-disease surveillance.

  13. Selective spider toxins reveal a role for Nav1.1 channel in mechanical pain

    Science.gov (United States)

    Osteen, Jeremiah D.; Herzig, Volker; Gilchrist, John; Emrick, Joshua J.; Zhang, Chuchu; Wang, Xidao; Castro, Joel; Garcia-Caraballo, Sonia; Grundy, Luke; Rychkov, Grigori Y.; Weyer, Andy D.; Dekan, Zoltan; Undheim, Eivind A. B.; Alewood, Paul; Stucky, Cheryl L.; Brierley, Stuart M.; Basbaum, Allan I.; Bosmans, Frank; King, Glenn F.; Julius, David

    2016-01-01

    Voltage-gated sodium (Nav) channels initiate action potentials in most neurons, including primary afferent nerve fibers of the pain pathway. Local anesthetics block pain through non-specific actions at all Nav channels, but the discovery of selective modulators would facilitate the analysis of individual subtypes and their contributions to chemical, mechanical, or thermal pain. Here, we identify and characterize spider toxins that selectively activate the Nav1.1 subtype, whose role in nociception and pain has not been explored. We exploit these probes to demonstrate that Nav1.1-expressing fibers are modality-specific nociceptors: their activation elicits robust pain behaviors without neurogenic inflammation and produces profound hypersensitivity to mechanical, but not thermal, stimuli. In the gut, high-threshold mechanosensitive fibers also express Nav1.1 and show enhanced toxin sensitivity in a model of irritable bowel syndrome. Altogether, these findings establish an unexpected role for Nav1.1 in regulating the excitability of sensory nerve fibers that underlie mechanical pain. PMID:27281198

  14. Genetic Disruption of 2-Arachidonoylglycerol Synthesis Reveals a Key Role for Endocannabinoid Signaling in Anxiety Modulation

    Directory of Open Access Journals (Sweden)

    Brian C. Shonesy

    2014-12-01

    Full Text Available Summary: Endocannabinoid (eCB signaling has been heavily implicated in the modulation of anxiety and depressive behaviors and emotional learning. However, the role of the most-abundant endocannabinoid 2-arachidonoylglycerol (2-AG in the physiological regulation of affective behaviors is not well understood. Here, we show that genetic deletion of the 2-AG synthetic enzyme diacylglycerol lipase α (DAGLα in mice reduces brain, but not circulating, 2-AG levels. DAGLα deletion also results in anxiety-like and sex-specific anhedonic phenotypes associated with impaired activity-dependent eCB retrograde signaling at amygdala glutamatergic synapses. Importantly, acute pharmacological normalization of 2-AG levels reverses both phenotypes of DAGLα-deficient mice. These data suggest 2-AG deficiency could contribute to the pathogenesis of affective disorders and that pharmacological normalization of 2-AG signaling could represent an approach for the treatment of mood and anxiety disorders. : The role of the primary endogenous cannabinoid 2-AG in mood and anxiety regulation is not well understood. Shonesy et al. show that deletion of a primary 2-AG synthetic enzyme, DAGLα, results in anxiety and sex-specific depressive phenotypes, which can be rapidly reversed by pharmacological normalization of endocannabinoid levels.

  15. Comparative Phosphoproteomics Reveals an Important Role of MKK2 in Banana (Musa spp.) Cold Signal Network

    Science.gov (United States)

    Gao, Jie; Zhang, Sheng; He, Wei-Di; Shao, Xiu-Hong; Li, Chun-Yu; Wei, Yue-Rong; Deng, Gui-Ming; Kuang, Rui-Bin; Hu, Chun-Hua; Yi, Gan-Jun; Yang, Qiao-Song

    2017-01-01

    Low temperature is one of the key environmental stresses, which greatly affects global banana production. However, little is known about the global phosphoproteomes in Musa spp. and their regulatory roles in response to cold stress. In this study, we conducted a comparative phosphoproteomic profiling of cold-sensitive Cavendish Banana and relatively cold tolerant Dajiao under cold stress. Phosphopeptide abundances of five phosphoproteins involved in MKK2 interaction network, including MKK2, HY5, CaSR, STN7 and kinesin-like protein, show a remarkable difference between Cavendish Banana and Dajiao in response to cold stress. Western blotting of MKK2 protein and its T31 phosphorylated peptide verified the phosphoproteomic results of increased T31 phosphopeptide abundance with decreased MKK2 abundance in Daojiao for a time course of cold stress. Meanwhile increased expression of MKK2 with no detectable T31 phosphorylation was found in Cavendish Banana. These results suggest that the MKK2 pathway in Dajiao, along with other cold-specific phosphoproteins, appears to be associated with the molecular mechanisms of high tolerance to cold stress in Dajiao. The results also provide new evidence that the signaling pathway of cellular MKK2 phosphorylation plays an important role in abiotic stress tolerance that likely serves as a universal plant cold tolerance mechanism. PMID:28106078

  16. Spatially resolved metabolic analysis reveals a central role for transcriptional control in carbon allocation to wood.

    Science.gov (United States)

    Roach, Melissa; Arrivault, Stéphanie; Mahboubi, Amir; Krohn, Nicole; Sulpice, Ronan; Stitt, Mark; Niittylä, Totte

    2017-06-15

    The contribution of transcriptional and post-transcriptional regulation to modifying carbon allocation to developing wood of trees is not well defined. To clarify the role of transcriptional regulation, the enzyme activity patterns of eight central primary metabolism enzymes across phloem, cambium, and developing wood of aspen (Populus tremula L.) were compared with transcript levels obtained by RNA sequencing of sequential stem sections from the same trees. Enzymes were selected on the basis of their importance in sugar metabolism and in linking primary metabolism to lignin biosynthesis. Existing enzyme assays were adapted to allow measurements from ~1 mm3 sections of dissected stem tissue. These experiments provided high spatial resolution of enzyme activity changes across different stages of wood development, and identified the gene transcripts probably responsible for these changes. In most cases, there was a clear positive relationship between transcripts and enzyme activity. During secondary cell wall formation, the increases in transcript levels and enzyme activities also matched with increased levels of glucose, fructose, hexose phosphates, and UDP-glucose, emphasizing an important role for transcriptional regulation in carbon allocation to developing aspen wood. These observations corroborate the efforts to increase carbon allocation to wood by engineering gene regulatory networks. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  17. New roles for Nanos in neural cell fate determination revealed by studies in a cnidarian.

    Science.gov (United States)

    Kanska, Justyna; Frank, Uri

    2013-07-15

    Nanos is a pan-metazoan germline marker, important for germ cell development and maintenance. In flies, Nanos also acts in posterior and neural development, but these functions have not been demonstrated experimentally in other animals. Using the cnidarian Hydractinia we have uncovered novel roles for Nanos in neural cell fate determination. Ectopic expression of Nanos2 increased the numbers of embryonic stinging cell progenitors, but decreased the numbers of neurons. Downregulation of Nanos2 had the opposite effect. Furthermore, Nanos2 blocked maturation of committed, post-mitotic nematoblasts. Hence, Nanos2 acts as a switch between two differentiation pathways, increasing the numbers of nematoblasts at the expense of neuroblasts, but preventing nematocyte maturation. Nanos2 ectopic expression also caused patterning defects, but these were not associated with deregulation of Wnt signaling, showing that the basic anterior-posterior polarity remained intact, and suggesting that numerical imbalance between nematocytes and neurons might have caused these defects, affecting axial patterning only indirectly. We propose that the functions of Nanos in germ cells and in neural development are evolutionarily conserved, but its role in posterior patterning is an insect or arthropod innovation.

  18. Discovery of methylfarnesoate as the annelid brain hormone reveals an ancient role of sesquiterpenoids in reproduction.

    Science.gov (United States)

    Schenk, Sven; Krauditsch, Christian; Frühauf, Peter; Gerner, Christopher; Raible, Florian

    2016-11-29

    Animals require molecular signals to determine when to divert resources from somatic functions to reproduction. This decision is vital in animals that reproduce in an all-or-nothing mode, such as bristle worms: females committed to reproduction spend roughly half their body mass for yolk and egg production; following mass spawning, the parents die. An enigmatic brain hormone activity suppresses reproduction. We now identify this hormone as the sesquiterpenoid methylfarnesoate. Methylfarnesoate suppresses transcript levels of the yolk precursor Vitellogenin both in cell culture and in vivo , directly inhibiting a central energy-costly step of reproductive maturation. We reveal that contrary to common assumptions, sesquiterpenoids are ancient animal hormones present in marine and terrestrial lophotrochozoans. In turn, insecticides targeting this pathway suppress vitellogenesis in cultured worm cells. These findings challenge current views of animal hormone evolution, and indicate that non-target species and marine ecosystems are susceptible to commonly used insect larvicides.

  19. Spaceflight Alters Bacterial Gene Expression and Virulence and Reveals Role for Global Regulator Hfq

    Science.gov (United States)

    Wilson, J. W.; Ott, C. M.; zuBentrup, K. Honer; Ramamurthy R.; Quick, L.; Porwollik, S.; Cheng, P.; McClellan, M.; Tsaprailis, G.; Radabaugh, T.; hide

    2007-01-01

    A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the spaceflight environment has never been accomplished due to significant technological and logistical hurdles. Moreover, the effects of spaceflight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared to identical ground control cultures. Global microarray and proteomic analyses revealed 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground based microgravity culture model. Spaceflight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during spaceflight missions and provide novel therapeutic options on Earth.

  20. Rapid centriole assembly in Naegleria reveals conserved roles for both de novo and mentored assembly.

    Science.gov (United States)

    Fritz-Laylin, Lillian K; Levy, Yaron Y; Levitan, Edward; Chen, Sean; Cande, W Zacheus; Lai, Elaine Y; Fulton, Chandler

    2016-03-01

    Centrioles are eukaryotic organelles whose number and position are critical for cilia formation and mitosis. Many cell types assemble new centrioles next to existing ones ("templated" or mentored assembly). Under certain conditions, centrioles also form without pre-existing centrioles (de novo). The synchronous differentiation of Naegleria amoebae to flagellates represents a unique opportunity to study centriole assembly, as nearly 100% of the population transitions from having no centrioles to having two within minutes. Here, we find that Naegleria forms its first centriole de novo, immediately followed by mentored assembly of the second. We also find both de novo and mentored assembly distributed among all major eukaryote lineages. We therefore propose that both modes are ancestral and have been conserved because they serve complementary roles, with de novo assembly as the default when no pre-existing centriole is available, and mentored assembly allowing precise regulation of number, timing, and location of centriole assembly. © 2016 Wiley Periodicals, Inc.

  1. The Role of polarized positrons and electrons in revealing fundamental interactions at the linear collider

    CERN Document Server

    Moortgat-Pick, G.; Alexander, G.; Ananthanarayan, B.; Babich, A.A.; Bharadwaj, V.; Barber, D.; Bartl, A.; Brachmann, A.; Chen, S.; Clarke, J.; Clendenin, J.E.; Dainton, J.; Desch, K.; Diehl, M.; Dobos, B.; Dorland, Tyler McMillan; Dreiner, H.K.; Eberl, H.; Ellis, John R.; Flottmann, K.; Fraas, H.; Franco-Sollova, F.; Franke, F.; Freitas, A.; Goodson, J.; Gray, J.; Han, A.; Heinemeyer, S.; Hesselbach, S.; Hirose, T.; Hohenwarter-Sodek, K.; Juste, A.; Kalinowski, J.; Kernreiter, T.; Kittel, O.; Kraml, S.; Langenfeld, U.; Majerotto, W.; Martinez, A.; Martyn, H.U.; Mikhailichenko, A.; Milstene, C.; Menges, W.; Meyners, N.; Monig, K.; Moffeit, K.; Moretti, S.; Nachtmann, O.; Nagel, F.; Nakanishi, T.; Nauenberg, U.; Nowak, H.; Omori, T.; Osland, P.; Pankov, A.A.; Paver, N.; Pitthan, R.; Poschl, R.; Porod, W.; Proulx, J.; Richardson, P.; Riemann, S.; Rindani, S.D.; Rizzo, T.G.; Schalicke, A.; Schuler, P.; Schwanenberger, C.; Scott, D.; Sheppard, J.; Singh, R.K.; Sopczak, A.; Spiesberger, H.; Stahl, A.; Steiner, H.; Wagner, A.; Weber, A.M.; Weiglein, G.; Wilson, G.W.; Woods, M.; Zerwas, P.; Zhang, J.; Zomer, F.

    2008-01-01

    The proposed International Linear Collider (ILC) is well-suited for discovering physics beyond the Standard Model and for precisely unraveling the structure of the underlying physics. The physics return can be maximized by the use of polarized beams. This report shows the paramount role of polarized beams and summarizes the benefits obtained from polarizing the positron beam, as well as the electron beam. The physics case for this option is illustrated explicitly by analyzing reference reactions in different physics scenarios. The results show that positron polarization, combined with the clean experimental environment provided by the linear collider, allows to improve strongly the potential of searches for new particles and the identification of their dynamics, which opens the road to resolve shortcomings of the Standard Model. The report also presents an overview of possible designs for polarizing both beams at the ILC, as well as for measuring their polarization.

  2. The Role of polarized positrons and electrons in revealing fundamental interactions at the linear collider

    Energy Technology Data Exchange (ETDEWEB)

    Moortgat-Pick, G.; /CERN /Durham U., IPPP; Abe, T.; Alexander, G.; Ananthanarayan, B.; Babich, A.A.; Bharadwaj, V.; Barber, D.; Bartl, A.; Brachmann, A.; Chen, S.; Clarke,; Clendenin, J.E.; Dainton, J.; Desch, K.; Diehl, M.; Dobos, B.; Dorland, T.; Eberl, H.; Ellis, John R.; Flottman, K.; Frass, H.; /CERN /Durham U., IPPP /Colorado U. /Tel-Aviv

    2005-07-01

    The proposed International Linear Collider (ILC) is well-suited for discovering physics beyond the Standard Model and for precisely unraveling the structure of the underlying physics. The physics return can be maximized by the use of polarized beams. This report shows the paramount role of polarized beams and summarizes the benefits obtained from polarizing the positron beam, as well as the electron beam. The physics case for this option is illustrated explicitly by analyzing reference reactions in different physics scenarios. The results show that positron polarization, combined with the clean experimental environment provided by the linear collider, allows to improve strongly the potential of searches for new particles and the identification of their dynamics, which opens the road to resolve shortcomings of the Standard Model. The report also presents an overview of possible designs for polarizing both beams at the ILC, as well as for measuring their polarization.

  3. Revealing Fundamental Interactions: the Role of Polarized Positrons and Electrons at the Linear Collider

    Energy Technology Data Exchange (ETDEWEB)

    Moortgat-Pick, G.; /CERN /Durham U., IPPP; Abe, T.; Alexander, G.; Ananthanarayan, B.; Babich, A.A.; Bharadwaj, V.; Barber, D.; Bartl, A.; Brachmann, A.; Chen, S.; Clarke,; Clendenin, J.E.; Dainton, J.; Desch, K.; Diehl, M.; Dobos, B.; Dorland, T.; Eberl, H.; Ellis, John R.; Flottman, K.; Frass, H.; /CERN /Durham U., IPPP /Colorado U. /Tel-Aviv

    2005-07-06

    The proposed International Linear Collider (ILC) is well-suited for discovering physics beyond the Standard Model and for precisely unraveling the structure of the underlying physics. The physics return can be maximized by the use of polarized beams. This report shows the paramount role of polarized beams and summarizes the benefits obtained from polarizing the positron beam, as well as the electron beam. The physics case for this option is illustrated explicitly by analyzing reference reactions in different physics scenarios. The results show that positron polarization, combined with the clean experimental environment provided by the linear collider, allows to improve strongly the potential of searches for new particles and the identification of their dynamics, which opens the road to resolve shortcomings of the Standard Model. The report also presents an overview of possible designs for polarizing both beams at the ILC, as well as for measuring their polarization.

  4. Revealing Fundamental Interactions: the Role of Polarized Positrons and Electrons at the Linear Collider

    International Nuclear Information System (INIS)

    Moortgat-Pick, G.; CERN, Durham U. IPPP; Abe, T.; Alexander, G.; Ananthanarayan, B.; Babich, A.A.; Bharadwaj, V.; Barber, D.; Bartl, A.; Brachmann, A.; Chen, S.; Clarke, J.; Clendenin, J.E.; Dainton, J.; Desch, K.; Diehl, M.; Dobos, B.; Dorland, T.; Eberl, H.; Ellis, John R.; Flottman, K.; Frass, H.

    2005-01-01

    The proposed International Linear Collider (ILC) is well-suited for discovering physics beyond the Standard Model and for precisely unraveling the structure of the underlying physics. The physics return can be maximized by the use of polarized beams. This report shows the paramount role of polarized beams and summarizes the benefits obtained from polarizing the positron beam, as well as the electron beam. The physics case for this option is illustrated explicitly by analyzing reference reactions in different physics scenarios. The results show that positron polarization, combined with the clean experimental environment provided by the linear collider, allows to improve strongly the potential of searches for new particles and the identification of their dynamics, which opens the road to resolve shortcomings of the Standard Model. The report also presents an overview of possible designs for polarizing both beams at the ILC, as well as for measuring their polarization

  5. Virus evolution reveals an exclusive role for LEDGF/p75 in chromosomal tethering of HIV.

    Directory of Open Access Journals (Sweden)

    Anneleen Hombrouck

    2007-03-01

    Full Text Available Retroviruses by definition insert their viral genome into the host cell chromosome. Although the key player of retroviral integration is viral integrase, a role for cellular cofactors has been proposed. Lentiviral integrases use the cellular protein LEDGF/p75 to tether the preintegration complex to the chromosome, although the existence of alternative host proteins substituting for the function of LEDGF/p75 in integration has been proposed. Truncation mutants of LEDGF/p75 lacking the chromosome attachment site strongly inhibit HIV replication by competition for the interaction with integrase. In an attempt to select HIV strains that can overcome the inhibition, we now have used T-cell lines that stably express a C-terminal fragment of LEDGF/p75. Despite resistance development, the affinity of integrase for LEDGF/p75 is reduced and replication kinetics in human primary T cells is impaired. Detection of the integrase mutations A128T and E170G at key positions in the LEDGF/p75-integrase interface provides in vivo evidence for previously reported crystallographic data. Moreover, the complementary inhibition by LEDGF/p75 knockdown and mutagenesis at the integrase-LEDGF/p75 interface points to the incapability of HIV to circumvent LEDGF/p75 function during proviral integration. Altogether, the data provide a striking example of the power of viral molecular evolution. The results underline the importance of the LEDGF/p75 HIV-1 interplay as target for innovative antiviral therapy. Moreover, the role of LEDGF/p75 in targeting integration will stimulate research on strategies to direct gene therapy vectors into safe landing sites.

  6. Zebrafish eda and edar mutants reveal conserved and ancestral roles of ectodysplasin signaling in vertebrates.

    Directory of Open Access Journals (Sweden)

    Matthew P Harris

    2008-10-01

    Full Text Available The genetic basis of the development and variation of adult form of vertebrates is not well understood. To address this problem, we performed a mutant screen to identify genes essential for the formation of adult skeletal structures of the zebrafish. Here, we describe the phenotypic and molecular characterization of a set of mutants showing loss of adult structures of the dermal skeleton, such as the rays of the fins and the scales, as well as the pharyngeal teeth. The mutations represent adult-viable, loss of function alleles in the ectodysplasin (eda and ectodysplasin receptor (edar genes. These genes are frequently mutated in the human hereditary disease hypohidrotic ectodermal dysplasia (HED; OMIM 224900, 305100 that affects the development of integumentary appendages such as hair and teeth. We find mutations in zebrafish edar that affect similar residues as mutated in human cases of HED and show similar phenotypic consequences. eda and edar are not required for early zebrafish development, but are rather specific for the development of adult skeletal and dental structures. We find that the defects of the fins and scales are due to the role of Eda signaling in organizing epidermal cells into discrete signaling centers of the scale epidermal placode and fin fold. Our genetic analysis demonstrates dose-sensitive and organ-specific response to alteration in levels of Eda signaling. In addition, we show substantial buffering of the effect of loss of edar function in different genetic backgrounds, suggesting canalization of this developmental system. We uncover a previously unknown role of Eda signaling in teleosts and show conservation of the developmental mechanisms involved in the formation and variation of both integumentary appendages and limbs. Lastly, our findings point to the utility of adult genetic screens in the zebrafish in identifying essential developmental processes involved in human disease and in morphological evolution.

  7. Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium

    Energy Technology Data Exchange (ETDEWEB)

    Liberton, Michelle; Saha, Rajib; Jacobs, Jon M.; Nguyen, Amelia Y.; Gritsenko, Marina A.; Smith, Richard D.; Koppenaal, David W.; Pakrasi, Himadri B.

    2016-04-07

    Cyanobacteria are photosynthetic microbes with highly differentiated membrane systems. These organisms contain an outer membrane, plasma membrane, and an internal system of thylakoid membranes where the photosynthetic and respiratory machinery are found. This existence of compartmentalization and differentiation of membrane systems poses a number of challenges for cyanobacterial cells in terms of organization and distribution of proteins to the correct membrane system. Proteomics studies have long sought to identify the components of the different membrane systems, and to date about 450 different proteins have been attributed to either the plasma membrane or thylakoid membrane. Given the complexity of these membranes, many more proteins remain to be identified in these membrane systems, and a comprehensive catalog of plasma membrane and thylakoid membrane proteins is needed. Here we describe the identification of 635 proteins in Synechocystis sp. PCC 6803 by quantitative iTRAQ isobaric labeling; of these, 459 proteins were localized to the plasma membrane and 176 were localized to the thylakoid membrane. Surprisingly, we found over 2.5 times the number of unique proteins identified in the plasma membrane compared to the thylakoid membrane. This suggests that the protein composition of the thylakoid membrane is more homogeneous than the plasma membrane, consistent with the role of the plasma membrane in diverse cellular processes including protein trafficking and nutrient import, compared to a more specialized role for the thylakoid membrane in cellular energetics. Overall, the protein composition of the Synechocystis 6803 plasma membrane and thylakoid membrane is quite similar to the E.coli plasma membrane and Arabidopsis thylakoid membrane, respectively. Synechocystis 6803 can therefore be described as a gram-negative bacterium that has an additional internal membrane system that fulfils the energetic requirements of the cell.

  8. Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium

    Energy Technology Data Exchange (ETDEWEB)

    Liberton, Michelle; Saha, Rajib; Jacobs, Jon M.; Nguyen, Amelia Y.; Gritsenko, Marina A.; Smith, Richard D.; Koppenaal, David W.; Pakrasi, Himadri B.

    2016-04-07

    Cyanobacteria are photosynthetic microbes with highlydifferentiated membrane systems. These organisms contain an outer membrane, plasma membrane, and an internal system of thylakoid membranes where the photosynthetic and respiratory machinery are found. This existence of compartmentalization and differentiation of membrane systems poses a number of challenges for cyanobacterial cells in terms of organization and distribution of proteins to the correct membrane system. Proteomics studies have long sought to identify the components of the different membrane systems in cyanobacteria, and to date about 450 different proteins have been attributed to either the plasma membrane or thylakoid membrane. Given the complexity of these membranes, many more proteins remain to be identified, and a comprehensive catalogue of plasma membrane and thylakoid membrane proteins is needed. Here we describe the identification of 635 differentially localized proteins in Synechocystis sp. PCC 6803 by quantitative iTRAQ isobaric labeling; of these, 459 proteins were localized to the plasma membrane and 176 were localized to the thylakoid membrane. Surprisingly, we found over 2.5 times the number of unique proteins identified in the plasma membrane compared with the thylakoid membrane. This suggests that the protein composition of the thylakoid membrane is more homogeneous than the plasma membrane, consistent with the role of the plasma membrane in diverse cellular processes including protein trafficking and nutrient import, compared with a more specialized role for the thylakoid membrane in cellular energetics. Thus, our data clearly define the two membrane systems with distinct functions. Overall, the protein compositions of the Synechocystis 6803 plasma membrane and thylakoid membrane are quite similar to that of the plasma membrane of Escherichia coli and thylakoid membrane of Arabidopsis chloroplasts, respectively. Synechocystis 6803 can therefore be described as a Gram

  9. Zebrafish eda and edar Mutants Reveal Conserved and Ancestral Roles of Ectodysplasin Signaling in Vertebrates

    Science.gov (United States)

    Harris, Matthew P.; Rohner, Nicolas; Schwarz, Heinz; Perathoner, Simon; Konstantinidis, Peter; Nüsslein-Volhard, Christiane

    2008-01-01

    The genetic basis of the development and variation of adult form of vertebrates is not well understood. To address this problem, we performed a mutant screen to identify genes essential for the formation of adult skeletal structures of the zebrafish. Here, we describe the phenotypic and molecular characterization of a set of mutants showing loss of adult structures of the dermal skeleton, such as the rays of the fins and the scales, as well as the pharyngeal teeth. The mutations represent adult-viable, loss of function alleles in the ectodysplasin (eda) and ectodysplasin receptor (edar) genes. These genes are frequently mutated in the human hereditary disease hypohidrotic ectodermal dysplasia (HED; OMIM 224900, 305100) that affects the development of integumentary appendages such as hair and teeth. We find mutations in zebrafish edar that affect similar residues as mutated in human cases of HED and show similar phenotypic consequences. eda and edar are not required for early zebrafish development, but are rather specific for the development of adult skeletal and dental structures. We find that the defects of the fins and scales are due to the role of Eda signaling in organizing epidermal cells into discrete signaling centers of the scale epidermal placode and fin fold. Our genetic analysis demonstrates dose-sensitive and organ-specific response to alteration in levels of Eda signaling. In addition, we show substantial buffering of the effect of loss of edar function in different genetic backgrounds, suggesting canalization of this developmental system. We uncover a previously unknown role of Eda signaling in teleosts and show conservation of the developmental mechanisms involved in the formation and variation of both integumentary appendages and limbs. Lastly, our findings point to the utility of adult genetic screens in the zebrafish in identifying essential developmental processes involved in human disease and in morphological evolution. PMID:18833299

  10. The Role of Physiotherapy Extended Scope Practitioners in Musculoskeletal care with Focus on Decision Making and Clinical Outcomes: A Systematic Review of Quantitative and Qualitative Research.

    Science.gov (United States)

    Thompson, Jonathan; Yoward, Samantha; Dawson, Pamela

    2017-06-01

    Physiotherapy extended scope practitioner (ESP) roles are widely utilized in the management of musculoskeletal conditions. The present article reviews the current literature, with particular emphasis on the decision-making process, patient/clinician interaction and clinical outcomes. A systematic review of musculoskeletal extended scope practice was carried out. The review focused on the outcome of interventions, and the interactions and decision-making processes between ESPs and their patients. A wide search strategy was employed, through multiple databases, grey literature and experts in the field. Qualitative and quantitative studies alike were included and a mixed-methods synthesis approach was undertaken in analysing the findings of included studies. A total of 476 articles were identified for inclusion, 25 of which (22 quantitative and three qualitative) meeting the criteria for full quality appraisal and synthesis. It was not possible to conduct a meta-analysis owing to data heterogeneity. The results showed high patient satisfaction with the ESP role, support for ESP staff listing patients for orthopaedic surgery, a high positive correlation of decision making between ESPs and orthopaedic surgeons and evidence of a positive impact on patient outcomes. Qualitative themes reflected the importance of ESP clinical decision making and interpersonal skills and their role in patient education. There is broad support for the physiotherapy ESP role and evidence of favourable outcomes from ESP intervention. Clinical decisions made by ESPs correlate well with those of medical colleagues, although there is a lack of detail explaining the ESP decision-making process itself and the influences and mechanisms by which this occurs. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  11. Single-Cell Analysis of SMN Reveals Its Broader Role in Neuromuscular Disease

    Directory of Open Access Journals (Sweden)

    Natalia Rodriguez-Muela

    2017-02-01

    Full Text Available The mechanism underlying selective motor neuron (MN death remains an essential question in the MN disease field. The MN disease spinal muscular atrophy (SMA is attributable to reduced levels of the ubiquitous protein SMN. Here, we report that SMN levels are widely variable in MNs within a single genetic background and that this heterogeneity is seen not only in SMA MNs but also in MNs derived from controls and amyotrophic lateral sclerosis (ALS patients. Furthermore, cells with low SMN are more susceptible to cell death. These findings raise the important clinical implication that some SMN-elevating therapeutics might be effective in MN diseases besides SMA. Supporting this, we found that increasing SMN across all MN populations using an Nedd8-activating enzyme inhibitor promotes survival in both SMA and ALS-derived MNs. Altogether, our work demonstrates that examination of human neurons at the single-cell level can reveal alternative strategies to be explored in the treatment of degenerative diseases.

  12. Community Lenses Revealing the Role of Sociocultural Environment on Physical Activity

    Science.gov (United States)

    Belon, Ana Paula; Nieuwendyk, Laura M.; Vallianatos, Helen; Nykiforuk, Candace I. J.

    2016-01-01

    Purpose To identify perceptions of how sociocultural environment enabled and hindered physical activity (PA) participation. Design Community-based participatory research. Setting Two semirural and two urban communities located in Alberta, Canada. Participants Thirty-five people (74.3% females, 71.4% aged 25–64 years) across the four communities. Method PhotoVoice activities occurred over 3 months during the spring of 2009. Participants were asked to document perceived environmental attributes that might foster or inhibit PA in their community. Photographs and narratives were shared in one-on-one interviews. Line-by-line coding of the transcripts was independently conducted by two researchers using an inductive approach. Codes were arranged into themes and subthemes, which were then organized into the Analysis Grid for Environments Linked to Obesity (ANGELO) framework. Results Six main themes (accompanied by subthemes) emerged: sociocultural aesthetics, safety, social involvement, PA motivation, cultural ideas of recreation, and car culture. Representative quotes and photographs illustrate enablers and obstacles identified by participants. Conclusion This PhotoVoice study revealed how aspects of participants’ sociocultural environments shaped their decisions to be physically active. Providing more PA resources is only one step in the promotion of supportive environments. Strategies should also account for the beautification and maintenance of communities, increasing feelings of safety, enhancement of social support among community members, popularization of PA, and mitigating car culture, among others. PMID:25973966

  13. Genome editing reveals a role for OCT4 in human embryogenesis.

    Science.gov (United States)

    Fogarty, Norah M E; McCarthy, Afshan; Snijders, Kirsten E; Powell, Benjamin E; Kubikova, Nada; Blakeley, Paul; Lea, Rebecca; Elder, Kay; Wamaitha, Sissy E; Kim, Daesik; Maciulyte, Valdone; Kleinjung, Jens; Kim, Jin-Soo; Wells, Dagan; Vallier, Ludovic; Bertero, Alessandro; Turner, James M A; Niakan, Kathy K

    2017-10-05

    Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.

  14. Community Lenses Revealing the Role of Sociocultural Environment on Physical Activity.

    Science.gov (United States)

    Belon, Ana Paula; Nieuwendyk, Laura M; Vallianatos, Helen; Nykiforuk, Candace I J

    2016-01-01

    To identify perceptions of how sociocultural environment enabled and hindered physical activity (PA) participation. Community-based participatory research. Two semirural and two urban communities located in Alberta, Canada. Thirty-five people (74.3% females, 71.4% aged 25-64 years) across the four communities. PhotoVoice activities occurred over 3 months during the spring of 2009. Participants were asked to document perceived environmental attributes that might foster or inhibit PA in their community. Photographs and narratives were shared in one-on-one interviews. Line-by-line coding of the transcripts was independently conducted by two researchers using an inductive approach. Codes were arranged into themes and subthemes, which were then organized into the Analysis Grid for Environments Linked to Obesity (ANGELO) framework. Six main themes (accompanied by subthemes) emerged: sociocultural aesthetics, safety, social involvement, PA motivation, cultural ideas of recreation, and car culture. Representative quotes and photographs illustrate enablers and obstacles identified by participants. This PhotoVoice study revealed how aspects of participants' sociocultural environments shaped their decisions to be physically active. Providing more PA resources is only one step in the promotion of supportive environments. Strategies should also account for the beautification and maintenance of communities, increasing feelings of safety, enhancement of social support among community members, popularization of PA, and mitigating car culture, among others.

  15. A ChIP-chip approach reveals a novel role for transcription factor IRF1 in the DNA damage response.

    Science.gov (United States)

    Frontini, Mattia; Vijayakumar, Meeraa; Garvin, Alexander; Clarke, Nicole

    2009-03-01

    IRF1 is a transcription factor that regulates key processes in the immune system and in tumour suppression. To gain further insight into IRF1's role in these processes, we searched for new target genes by performing chromatin immunoprecipitation coupled to a CpG island microarray (ChIP-chip). Using this approach we identified 202 new IRF1-binding sites with high confidence. Functional categorization of the target genes revealed a surprising cadre of new roles that can be linked to IRF1. One of the major functional categories was the DNA damage response pathway. In order to further validate our findings, we show that IRF1 can regulate the mRNA expression of a number of the DNA damage response genes in our list. In particular, we demonstrate that the mRNA and protein levels of the DNA repair protein BRIP1 [Fanconi anemia gene J (FANC J)] are upregulated after IRF1 over-expression. We also demonstrate that knockdown of IRF1 by siRNA results in loss of BRIP1 expression, abrogation of BRIP1 foci after DNA interstrand crosslink (ICL) damage and hypersensitivity to the DNA crosslinking agent, melphalan; a characteristic phenotype of FANC J cells. Taken together, our data provides a more complete understanding of the regulatory networks controlled by IRF1 and reveals a novel role for IRF1 in regulating the ICL DNA damage response.

  16. A ChIP–chip approach reveals a novel role for transcription factor IRF1 in the DNA damage response

    Science.gov (United States)

    Frontini, Mattia; Vijayakumar, Meeraa; Garvin, Alexander; Clarke, Nicole

    2009-01-01

    IRF1 is a transcription factor that regulates key processes in the immune system and in tumour suppression. To gain further insight into IRF1's role in these processes, we searched for new target genes by performing chromatin immunoprecipitation coupled to a CpG island microarray (ChIP–chip). Using this approach we identified 202 new IRF1-binding sites with high confidence. Functional categorization of the target genes revealed a surprising cadre of new roles that can be linked to IRF1. One of the major functional categories was the DNA damage response pathway. In order to further validate our findings, we show that IRF1 can regulate the mRNA expression of a number of the DNA damage response genes in our list. In particular, we demonstrate that the mRNA and protein levels of the DNA repair protein BRIP1 [Fanconi anemia gene J (FANC J)] are upregulated after IRF1 over-expression. We also demonstrate that knockdown of IRF1 by siRNA results in loss of BRIP1 expression, abrogation of BRIP1 foci after DNA interstrand crosslink (ICL) damage and hypersensitivity to the DNA crosslinking agent, melphalan; a characteristic phenotype of FANC J cells. Taken together, our data provides a more complete understanding of the regulatory networks controlled by IRF1 and reveals a novel role for IRF1 in regulating the ICL DNA damage response. PMID:19129219

  17. Extended thermodynamics

    CERN Document Server

    Müller, Ingo

    1993-01-01

    Physicists firmly believe that the differential equations of nature should be hyperbolic so as to exclude action at a distance; yet the equations of irreversible thermodynamics - those of Navier-Stokes and Fourier - are parabolic. This incompatibility between the expectation of physicists and the classical laws of thermodynamics has prompted the formulation of extended thermodynamics. After describing the motifs and early evolution of this new branch of irreversible thermodynamics, the authors apply the theory to mon-atomic gases, mixtures of gases, relativistic gases, and "gases" of phonons and photons. The discussion brings into perspective the various phenomena called second sound, such as heat propagation, propagation of shear stress and concentration, and the second sound in liquid helium. The formal mathematical structure of extended thermodynamics is exposed and the theory is shown to be fully compatible with the kinetic theory of gases. The study closes with the testing of extended thermodynamics thro...

  18. Pro-survival role for Parkinson's associated gene DJ-1 revealed in trophically impaired dopaminergic neurons.

    Directory of Open Access Journals (Sweden)

    Liviu Aron

    2010-04-01

    Full Text Available The mechanisms underlying the selective death of substantia nigra (SN neurons in Parkinson disease (PD remain elusive. While inactivation of DJ-1, an oxidative stress suppressor, causes PD, animal models lacking DJ-1 show no overt dopaminergic (DA neuron degeneration in the SN. Here, we show that aging mice lacking DJ-1 and the GDNF-receptor Ret in the DA system display an accelerated loss of SN cell bodies, but not axons, compared to mice that only lack Ret signaling. The survival requirement for DJ-1 is specific for the GIRK2-positive subpopulation in the SN which projects exclusively to the striatum and is more vulnerable in PD. Using Drosophila genetics, we show that constitutively active Ret and associated Ras/ERK, but not PI3K/Akt, signaling components interact genetically with DJ-1. Double loss-of-function experiments indicate that DJ-1 interacts with ERK signaling to control eye and wing development. Our study uncovers a conserved interaction between DJ-1 and Ret-mediated signaling and a novel cell survival role for DJ-1 in the mouse. A better understanding of the molecular connections between trophic signaling, cellular stress and aging could uncover new targets for drug development in PD.

  19. Targeted CRISPR disruption reveals a role for RNase MRP RNA in human preribosomal RNA processing.

    Science.gov (United States)

    Goldfarb, Katherine C; Cech, Thomas R

    2017-01-01

    MRP RNA is an abundant, essential noncoding RNA whose functions have been proposed in yeast but are incompletely understood in humans. Mutations in the genomic locus for MRP RNA cause pleiotropic human diseases, including cartilage hair hypoplasia (CHH). Here we applied CRISPR-Cas9 genome editing to disrupt the endogenous human MRP RNA locus, thereby attaining what has eluded RNAi and RNase H experiments: elimination of MRP RNA in the majority of cells. The resulting accumulation of ribosomal RNA (rRNA) precursor-analyzed by RNA fluorescent in situ hybridization (FISH), Northern blots, and RNA sequencing-implicates MRP RNA in pre-rRNA processing. Amelioration of pre-rRNA imbalance is achieved through rescue of MRP RNA levels by ectopic expression. Furthermore, affinity-purified MRP ribonucleoprotein (RNP) from HeLa cells cleaves the human pre-rRNA in vitro at at least one site used in cells, while RNP isolated from cells with CRISPR-edited MRP loci loses this activity, and ectopic MRP RNA expression restores cleavage activity. Thus, a role for RNase MRP in human pre-rRNA processing is established. As demonstrated here, targeted CRISPR disruption is a valuable tool for functional studies of essential noncoding RNAs that are resistant to RNAi and RNase H-based degradation. © 2017 Goldfarb and Cech; Published by Cold Spring Harbor Laboratory Press.

  20. New inducible genetic method reveals critical roles of GABA in the control of feeding and metabolism.

    Science.gov (United States)

    Meng, Fantao; Han, Yong; Srisai, Dollada; Belakhov, Valery; Farias, Monica; Xu, Yong; Palmiter, Richard D; Baasov, Timor; Wu, Qi

    2016-03-29

    Currently available inducible Cre/loxP systems, despite their considerable utility in gene manipulation, have pitfalls in certain scenarios, such as unsatisfactory recombination rates and deleterious effects on physiology and behavior. To overcome these limitations, we designed a new, inducible gene-targeting system by introducing an in-frame nonsense mutation into the coding sequence of Cre recombinase (nsCre). Mutant mRNAs transcribed from nsCre transgene can be efficiently translated into full-length, functional Cre recombinase in the presence of nonsense suppressors such as aminoglycosides. In a proof-of-concept model, GABA signaling from hypothalamic neurons expressing agouti-related peptide (AgRP) was genetically inactivated within 4 d after treatment with a synthetic aminoglycoside. Disruption of GABA synthesis in AgRP neurons in young adult mice led to a dramatic loss of body weight due to reduced food intake and elevated energy expenditure; they also manifested glucose intolerance. In contrast, older mice with genetic inactivation of GABA signaling by AgRP neurons had only transient reduction of feeding and body weight; their energy expenditure and glucose tolerance were unaffected. These results indicate that GABAergic signaling from AgRP neurons plays a key role in the control of feeding and metabolism through an age-dependent mechanism. This new genetic technique will augment current tools used to elucidate mechanisms underlying many physiological and neurological processes.

  1. CpG dinucleotide frequencies reveal the role of host methylation capabilities in parvovirus evolution.

    Science.gov (United States)

    Upadhyay, Mohita; Samal, Jasmine; Kandpal, Manish; Vasaikar, Suhas; Biswas, Banhi; Gomes, James; Vivekanandan, Perumal

    2013-12-01

    Parvoviruses are rapidly evolving viruses that infect a wide range of hosts, including vertebrates and invertebrates. Extensive methylation of the parvovirus genome has been recently demonstrated. A global pattern of methylation of CpG dinucleotides is seen in vertebrate genomes, compared to "fractional" methylation patterns in invertebrate genomes. It remains unknown if the loss of CpG dinucleotides occurs in all viruses of a given DNA virus family that infect host species spanning across vertebrates and invertebrates. We investigated the link between the extent of CpG dinucleotide depletion among autonomous parvoviruses and the evolutionary lineage of the infected host. We demonstrate major differences in the relative abundance of CpG dinucleotides among autonomous parvoviruses which share similar genome organization and common ancestry, depending on the infected host species. Parvoviruses infecting vertebrate hosts had significantly lower relative abundance of CpG dinucleotides than parvoviruses infecting invertebrate hosts. The strong correlation of CpG dinucleotide depletion with the gain in TpG/CpA dinucleotides and the loss of TpA dinucleotides among parvoviruses suggests a major role for CpG methylation in the evolution of parvoviruses. Our data present evidence that links the relative abundance of CpG dinucleotides in parvoviruses to the methylation capabilities of the infected host. In sum, our findings support a novel perspective of host-driven evolution among autonomous parvoviruses.

  2. Developmental Ethanol Exposure Causes Reduced Feeding and Reveals a Critical Role for Neuropeptide F in Survival

    Science.gov (United States)

    Guevara, Amanda; Gates, Hillary; Urbina, Brianna; French, Rachael

    2018-01-01

    Food intake is necessary for survival, and natural reward circuitry has evolved to help ensure that animals ingest sufficient food to maintain development, growth, and survival. Drugs of abuse, including alcohol, co-opt the natural reward circuitry in the brain, and this is a major factor in the reinforcement of drug behaviors leading to addiction. At the junction of these two aspects of reward are alterations in feeding behavior due to alcohol consumption. In particular, developmental alcohol exposure (DAE) results in a collection of physical and neurobehavioral disorders collectively referred to as Fetal Alcohol Spectrum Disorder (FASD). The deleterious effects of DAE include intellectual disabilities and other neurobehavioral changes, including altered feeding behaviors. Here we use Drosophila melanogaster as a genetic model organism to study the effects of DAE on feeding behavior and the expression and function of Neuropeptide F. We show that addition of a defined concentration of ethanol to food leads to reduced feeding at all stages of development. Further, genetic conditions that reduce or eliminate NPF signaling combine with ethanol exposure to further reduce feeding, and the distribution of NPF is altered in the brains of ethanol-supplemented larvae. Most strikingly, we find that the vast majority of flies with a null mutation in the NPF receptor die early in larval development when reared in ethanol, and provide evidence that this lethality is due to voluntary starvation. Collectively, we find a critical role for NPF signaling in protecting against altered feeding behavior induced by developmental ethanol exposure. PMID:29623043

  3. Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle.

    Science.gov (United States)

    Daimon, Takaaki; Uchibori, Miwa; Nakao, Hajime; Sezutsu, Hideki; Shinoda, Tetsuro

    2015-08-04

    Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow larvae to undergo multiple rounds of status quo molts. However, the roles of JHs during the embryonic and very early larval stages have not been fully understood. We generated and characterized knockout silkworms (Bombyx mori) with null mutations in JH biosynthesis or JH receptor genes using genome-editing tools. We found that embryonic growth and morphogenesis are largely independent of JHs in Bombyx and that, even in the absence of JHs or JH signaling, pupal characters are not formed in first- or second-instar larvae, and precocious metamorphosis is induced after the second instar at the earliest. We also show by mosaic analysis that a pupal specifier gene broad, which is dramatically up-regulated in the late stage of the last larval instar, is essential for pupal commitment in the epidermis. Importantly, the mRNA expression level of broad, which is thought to be repressed by JHs, remained at very low basal levels during the early larval instars of JH-deficient or JH signaling-deficient knockouts. Therefore, our study suggests that the long-accepted paradigm that JHs maintain the juvenile status throughout larval life should be revised because the larval status can be maintained by a JH-independent mechanism in very early larval instars. We propose that the lack of competence for metamorphosis during the early larval stages may result from the absence of an unidentified broad-inducing factor, i.e., a competence factor.

  4. Proteomic analysis reveals heat shock protein 70 has a key role in polycythemia Vera.

    Science.gov (United States)

    Gallardo, Miguel; Barrio, Santiago; Fernandez, Marisol; Paradela, Alberto; Arenas, Alicia; Toldos, Oscar; Ayala, Rosa; Albizua, Enriqueta; Jimenez, Ana; Redondo, Santiago; Garcia-Martin, Rosa Maria; Gilsanz, Florinda; Albar, Juan Pablo; Martinez-Lopez, Joaquin

    2013-11-19

    JAK-STAT signaling through the JAK2V617F mutation is central to the pathogenesis of myeloproliferative neoplasms (MPN). However, other events could precede the JAK2 mutation. The aim of this study is to analyze the phenotypic divergence between polycytemia vera (PV) and essential thrombocytemia (ET) to find novel therapeutics targets by a proteomic and functional approach to identify alternative routes to JAK2 activation. Through 2D-DIGE and mass spectrometry of granulocyte protein from 20 MPN samples, showed differential expression of HSP70 in PV and ET besides other 60 proteins. Immunohistochemistry of 46 MPN bone marrow samples confirmed HSP70 expression. The median of positive granulocytes was 80% in PV (SD 35%) vs. 23% in ET (SD 34.25%). In an ex vivo model KNK437 was used as an inhibition model assay of HSP70, showed dose-dependent inhibition of cell growth and burst formation unit erythroid (BFU-E) in PV and ET, increased apoptosis in the erythroid lineage, and decreased pJAK2 signaling, as well as a specific siRNA for HSP70. These data suggest a key role for HSP70 in proliferation and survival of the erythroid lineage in PV, and may represent a potential therapeutic target in MPN, especially in PV.

  5. Organoid cystogenesis reveals a critical role of microenvironment in human polycystic kidney disease

    Science.gov (United States)

    Cruz, Nelly M.; Song, Xuewen; Czerniecki, Stefan M.; Gulieva, Ramila E.; Churchill, Angela J.; Kim, Yong Kyun; Winston, Kosuke; Tran, Linh M.; Diaz, Marco A.; Fu, Hongxia; Finn, Laura S.; Pei, York; Himmelfarb, Jonathan; Freedman, Benjamin S.

    2017-11-01

    Polycystic kidney disease (PKD) is a life-threatening disorder, commonly caused by defects in polycystin-1 (PC1) or polycystin-2 (PC2), in which tubular epithelia form fluid-filled cysts. A major barrier to understanding PKD is the absence of human cellular models that accurately and efficiently recapitulate cystogenesis. Previously, we have generated a genetic model of PKD using human pluripotent stem cells and derived kidney organoids. Here we show that systematic substitution of physical components can dramatically increase or decrease cyst formation, unveiling a critical role for microenvironment in PKD. Removal of adherent cues increases cystogenesis 10-fold, producing cysts phenotypically resembling PKD that expand massively to 1-centimetre diameters. Removal of stroma enables outgrowth of PKD cell lines, which exhibit defects in PC1 expression and collagen compaction. Cyclic adenosine monophosphate (cAMP), when added, induces cysts in both PKD organoids and controls. These biomaterials establish a highly efficient model of PKD cystogenesis that directly implicates the microenvironment at the earliest stages of the disease.

  6. Prominent Role of Spin-Orbit Coupling in FeSe Revealed by Inelastic Neutron Scattering

    Directory of Open Access Journals (Sweden)

    Mingwei Ma

    2017-05-01

    Full Text Available In most existing theories for iron-based superconductors, spin-orbit coupling (SOC has been assumed to be insignificant. Here, we use spin-polarized inelastic neutron scattering to show that collective low-energy spin excitations in the orthorhombic (or “nematic” phase of FeSe possess nearly no in-plane component. Such spin-space anisotropy is present over an energy range greater than the superconducting gap 2Δ_{sc} and gets fully inherited in the superconducting state, resulting in a c-axis polarized “spin resonance” without any noticeable isotropic spectral-weight rearrangement related to the superconductivity, which is distinct from observations in the superconducting iron pnictides. The contrast between the strong suppression of long-range magnetic order in FeSe and the persisting large spin-space anisotropy, which cannot be explained microscopically by introducing single-ion anisotropy into local-moment spin models, demonstrates the importance of SOC in an itinerant-electron description of the low-energy spin excitations. Our result helps to elucidate the nearby magnetic instabilities and the debated interplay between spin and orbital degrees of freedom in FeSe. The prominent role of SOC also implies a possible unusual nature of the superconducting state.

  7. Continental cichlid radiations: functional diversity reveals the role of changing ecological opportunity in the Neotropics.

    Science.gov (United States)

    Arbour, Jessica Hilary; López-Fernández, Hernán

    2016-08-17

    Adaptive radiations have been hypothesized to contribute broadly to the diversity of organisms. Models of adaptive radiation predict that ecological opportunity and ecological release, the availability of empty ecological niches and the response by adapting lineages to occupy them, respectively, drive patterns of phenotypic and lineage diversification. Adaptive radiations driven by 'ecological opportunity' are well established in island systems; it is less clear if ecological opportunity influences continent-wide diversification. We use Neotropical cichlid fishes to test if variation in rates of functional evolution is consistent with changing ecological opportunity. Across a functional morphological axis associated with ram-suction feeding traits, evolutionary rates declined through time as lineages diversified in South America. Evolutionary rates of ram-suction functional morphology also appear to have accelerated as cichlids colonized Central America and encountered renewed opportunity. Our results suggest that ecological opportunity may play an important role in shaping patterns of morphological diversity of even broadly distributed lineages like Neotropical cichlids. © 2016 The Author(s).

  8. Rigid Residue Scan Simulations Systematically Reveal Residue Entropic Roles in Protein Allostery.

    Directory of Open Access Journals (Sweden)

    Robert Kalescky

    2016-04-01

    Full Text Available Intra-protein information is transmitted over distances via allosteric processes. This ubiquitous protein process allows for protein function changes due to ligand binding events. Understanding protein allostery is essential to understanding protein functions. In this study, allostery in the second PDZ domain (PDZ2 in the human PTP1E protein is examined as model system to advance a recently developed rigid residue scan method combining with configurational entropy calculation and principal component analysis. The contributions from individual residues to whole-protein dynamics and allostery were systematically assessed via rigid body simulations of both unbound and ligand-bound states of the protein. The entropic contributions of individual residues to whole-protein dynamics were evaluated based on covariance-based correlation analysis of all simulations. The changes of overall protein entropy when individual residues being held rigid support that the rigidity/flexibility equilibrium in protein structure is governed by the La Châtelier's principle of chemical equilibrium. Key residues of PDZ2 allostery were identified with good agreement with NMR studies of the same protein bound to the same peptide. On the other hand, the change of entropic contribution from each residue upon perturbation revealed intrinsic differences among all the residues. The quasi-harmonic and principal component analyses of simulations without rigid residue perturbation showed a coherent allosteric mode from unbound and bound states, respectively. The projection of simulations with rigid residue perturbation onto coherent allosteric modes demonstrated the intrinsic shifting of ensemble distributions supporting the population-shift theory of protein allostery. Overall, the study presented here provides a robust and systematic approach to estimate the contribution of individual residue internal motion to overall protein dynamics and allostery.

  9. Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease.

    Science.gov (United States)

    Lewis, Wesley R; Malarkey, Erik B; Tritschler, Douglas; Bower, Raqual; Pasek, Raymond C; Porath, Jonathan D; Birket, Susan E; Saunier, Sophie; Antignac, Corinne; Knowles, Michael R; Leigh, Margaret W; Zariwala, Maimoona A; Challa, Anil K; Kesterson, Robert A; Rowe, Steven M; Drummond, Iain A; Parant, John M; Hildebrandt, Friedhelm; Porter, Mary E; Yoder, Bradley K; Berbari, Nicolas F

    2016-07-01

    Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or 'primary' cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in

  10. Non-Native Metal Ion Reveals the Role of Electrostatics in Synaptotagmin 1-Membrane Interactions.

    Science.gov (United States)

    Katti, Sachin; Nyenhuis, Sarah B; Her, Bin; Srivastava, Atul K; Taylor, Alexander B; Hart, P John; Cafiso, David S; Igumenova, Tatyana I

    2017-06-27

    C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca 2+ -dependent manner. In these cases, membrane association is triggered by Ca 2+ binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd 2+ , in lieu of Ca 2+ to gain insight into the contributions made by long-range Coulombic interactions and direct metal ion-lipid bridging to membrane binding. Using X-ray crystallography, NMR, Förster resonance energy transfer, and vesicle cosedimentation assays, we demonstrate that, although Cd 2+ binds to the loop region of C2A/B domains of synaptotagmin 1 with high affinity, long-range Coulombic interactions are too weak to support membrane binding of individual domains. We attribute this behavior to two factors: the stoichiometry of Cd 2+ binding to the loop regions of the C2A and C2B domains and the impaired ability of Cd 2+ to directly coordinate the lipids. In contrast, electron paramagnetic resonance experiments revealed that Cd 2+ does support membrane binding of the C2 domains in full-length synaptotagmin 1, where the high local lipid concentrations that result from membrane tethering can partially compensate for lack of a full complement of divalent metal ions and specific lipid coordination in Cd 2+ -complexed C2A/B domains. Our data suggest that long-range Coulombic interactions alone can drive the initial association of C2A/B with anionic membranes and that Ca 2+ further augments membrane binding by the formation of metal ion-lipid coordination bonds and additional Ca 2+ ion binding to the C2 domain loop regions.

  11. Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease.

    Directory of Open Access Journals (Sweden)

    Wesley R Lewis

    2016-07-01

    Full Text Available Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or 'primary' cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400. While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8. GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC protein 4 (DRC4 where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR to generate one of these human missense

  12. Distinct role of hydration water in protein misfolding and aggregation revealed by fluctuating thermodynamics analysis.

    Science.gov (United States)

    Chong, Song-Ho; Ham, Sihyun

    2015-04-21

    decrease in solvation free energy, harnessing the monomer solvation free energy earned during the misfolding. The second step, where a compact dimer structure is formed, is driven by direct protein-protein interactions, but again it is accompanied by an increase in solvation free energy. The increased solvation free energy of the dimer will function as the driving force to recruit another Aβ protein in the approach stage of subsequent oligomerizations. The fluctuating thermodynamics analysis of the misfolding and dimerization of the Aβ protein indicates that the interaction of the protein with surrounding water plays a critical role in protein aggregation. Such a water-centric perspective is further corroborated by demonstrating that, for a large number of Aβ mutants and mutants of other protein systems, the change in the experimental aggregation propensity upon mutation has a significant correlation with the protein solvation free energy change. We also find striking discrimination between the positively and negatively charged residues on the protein surface by surrounding water molecules, which is shown to play a crucial role in determining the protein aggregation propensity. We argue that the protein total charge dictates such striking behavior of the surrounding water molecules. Our results provide new insights for understanding and predicting the protein aggregation propensity, thereby offering novel design principles for producing aggregation-resistant proteins for biotherapeutics.

  13. Differential reproductive responses to stress reveal the role of life-history strategies within a species.

    Science.gov (United States)

    Schultner, J; Kitaysky, A S; Gabrielsen, G W; Hatch, S A; Bech, C

    2013-11-22

    Life-history strategies describe that 'slow'- in contrast to 'fast'-living species allocate resources cautiously towards reproduction to enhance survival. Recent evidence suggests that variation in strategies exists not only among species but also among populations of the same species. Here, we examined the effect of experimentally induced stress on resource allocation of breeding seabirds in two populations with contrasting life-history strategies: slow-living Pacific and fast-living Atlantic black-legged kittiwakes. We tested the hypothesis that reproductive responses in kittiwakes under stress reflect their life-history strategies. We predicted that in response to stress, Pacific kittiwakes reduce investment in reproduction compared with Atlantic kittiwakes. We exposed chick-rearing kittiwakes to a short-term (3-day) period of increased exogenous corticosterone (CORT), a hormone that is released during food shortages. We examined changes in baseline CORT levels, parental care and effects on offspring. We found that kittiwakes from the two populations invested differently in offspring when facing stress. In response to elevated CORT, Pacific kittiwakes reduced nest attendance and deserted offspring more readily than Atlantic kittiwakes. We observed lower chick growth, a higher stress response in offspring and lower reproductive success in response to CORT implantation in Pacific kittiwakes, whereas the opposite occurred in the Atlantic. Our findings support the hypothesis that life-history strategies predict short-term responses of individuals to stress within a species. We conclude that behaviour and physiology under stress are consistent with trade-off priorities as predicted by life-history theory. We encourage future studies to consider the pivotal role of life-history strategies when interpreting inter-population differences of animal responses to stressful environmental events.

  14. Transcriptome Analysis of Maize Immature Embryos Reveals the Roles of Cysteine in Improving Agrobacterium Infection Efficiency

    Science.gov (United States)

    Liu, Yan; Zhang, Zhiqiang; Fu, Junjie; Wang, Guoying; Wang, Jianhua; Liu, Yunjun

    2017-01-01

    Maize Agrobacterium-mediated transformation efficiency has been greatly improved in recent years. Antioxidants, such as, cysteine, can significantly improve maize transformation frequency through improving the Agrobacterium infection efficiency. However, the mechanism underlying the transformation improvement after cysteine exposure has not been elucidated. In this study, we showed that the addition of cysteine to the co-cultivation medium significantly increased the Agrobacterium infection efficiency of hybrid HiII and inbred line Z31 maize embryos. Reactive oxygen species contents were higher in embryos treated with cysteine than that without cysteine. We further investigated the mechanism behind cysteine-related infection efficiency increase using transcriptome analysis. The results showed that the cysteine treatment up-regulated 939 genes and down-regulated 549 genes in both Z31 and HiII. Additionally, more differentially expressed genes were found in HiII embryos than those in Z31 embryos, suggesting that HiII was more sensitive to the cysteine treatment than Z31. GO analysis showed that the up-regulated genes were mainly involved in the oxidation reduction process. The up-regulation of these genes could help maize embryos to cope with the oxidative stress stimulated by Agrobacterium infection. The down-regulated genes were mainly involved in the cell wall and membrane metabolism, such as, aquaporin and expansin genes. Decreased expression of these cell wall integrity genes could loosen the cell wall, thereby improving the entry of Agrobacterium into plant cells. This study offers insight into the role of cysteine in improving Agrobacterium-mediated transformation of maize immature embryos. PMID:29089955

  15. Targeted deletion of Atg5 reveals differential roles of autophagy in keratin K5-expressing epithelia

    Energy Technology Data Exchange (ETDEWEB)

    Sukseree, Supawadee [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria); Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok (Thailand); Rossiter, Heidemarie; Mildner, Michael [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria); Pammer, Johannes [Institute of Clinical Pathology, Medical University of Vienna, Vienna (Austria); Buchberger, Maria; Gruber, Florian [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria); Watanapokasin, Ramida [Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok (Thailand); Tschachler, Erwin [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria); Eckhart, Leopold, E-mail: leopold.eckhart@meduniwien.ac.at [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We generated mice lacking Atg5 and autophagy in keratin K5-positive epithelia. Black-Right-Pointing-Pointer Suppression of autophagy in thymic epithelium was not associated with signs of autoimmunity. Black-Right-Pointing-Pointer Autophagy was required for normal terminal differentiation of preputial gland cells. Black-Right-Pointing-Pointer Autophagy-deficient cells of the preputial glands degraded nuclear DNA prematurely. -- Abstract: Autophagy contributes to the homeostasis of many tissues, yet its role in epithelia is incompletely understood. A recent report proposed that Atg5-dependent autophagy in thymic epithelial cells is essential for their function in the negative selection of self-reactive T-cells and, thus, for the suppression of tissue inflammation. Here we crossed mice carrying floxed alleles of the Atg5 gene with mice expressing the Cre recombinase under the control of the keratin K5 promoter to suppress autophagy in all K5-positive epithelia. The efficiency of autophagy abrogation was confirmed by immunoanalyses of LC3, which was converted to the autophagy-associated LC3-II form in normal but not Atg5-deficient cells, and of p62, which accumulated in Atg5-deficient cells. Mice carrying the epithelium-specific deletion of Atg5 showed normal weight gain, absence of tissue inflammation, and a normal morphology of the thymic epithelium. By contrast, autophagy-deficient epithelial cells of the preputial gland showed aberrant eosinophilic staining in histology and premature degradation of nuclear DNA during terminal differentiation. Taken together, the results of this study suggest that autophagy is dispensable for the suppression of autoimmunity by thymic epithelial cells but essential for normal differentiation of the preputial gland in mice.

  16. Comprehensive expression profiling of rice tetraspanin genes reveals diverse roles during development and abiotic stress

    Directory of Open Access Journals (Sweden)

    Balaji eM

    2015-12-01

    Full Text Available Tetraspanin family is comprised of evolutionarily conserved integral membrane proteins. The incredible ability of tetraspanins to form ‘micro domain complexes’ and their preferential targeting to membranes emphasizes their active association with signal recognition and communication with neighboring cells, thus acting as key modulators of signaling cascades. In animals, tetraspanins are associated with multitude of cellular processes. Unlike animals, the biological relevance of tetraspanins in plants has not been well investigated. In Arabidopsis tetraspanins are known to contribute in important plant development processes such as leaf morphogenesis, root and floral organ formation. In the present study we investigated the genomic organization, chromosomal distribution, phylogeny and domain structure of 15 rice tetraspanin proteins (OsTETs. OsTET proteins had similar domain structure and signature ‘GCCK/R’ motif as reported in Arabidopsis. Comprehensive expression profiling of OsTET genes suggested their possible involvement during rice development. While OsTET9 and 10 accumulated predominantly in flowers, OsTET5, 8 and 12 were preferentially expressed in root tissues. Noticeably, seven OsTETs exhibited more than 2-fold up regulation at early stages of flag leaf senescence in rice. Furthermore, several OsTETs were differentially regulated in rice seedlings exposed to abiotic stresses, exogenous treatment of hormones and nutrient deprivation. Transient subcellular localization studies of eight OsTET proteins in tobacco epidermal cells showed that these proteins localized in plasma membrane. The present study provides valuable insights into the possible roles of tetraspanins in regulating development and defining response to abiotic stresses in rice. Targeted proteomic studies would be useful in identification of their interacting partners under different conditions and ultimately their biological function in plants

  17. Depletion of Plasmodium berghei plasmoredoxin reveals a non-essential role for life cycle progression of the malaria parasite.

    Science.gov (United States)

    Buchholz, Kathrin; Rahlfs, Stefan; Schirmer, R Heiner; Becker, Katja; Matuschewski, Kai

    2008-06-25

    Proliferation of the pathogenic Plasmodium asexual blood stages in host erythrocytes requires an exquisite capacity to protect the malaria parasite against oxidative stress. This function is achieved by a complex antioxidant defence system composed of redox-active proteins and low MW antioxidants. Here, we disrupted the P. berghei plasmoredoxin gene that encodes a parasite-specific 22 kDa member of the thioredoxin superfamily. The successful generation of plasmoredoxin knockout mutants in the rodent model malaria parasite and phenotypic analysis during life cycle progression revealed a non-vital role in vivo. Our findings suggest that plasmoredoxin fulfils a specialized and dispensable role for Plasmodium and highlights the need for target validation to inform drug development strategies.

  18. Extending food deprivation reverses the short-term lipolytic response to fasting: role of the triacylglycerol/fatty acid cycle.

    Science.gov (United States)

    Weber, Jean-Michel; Reidy, Shannon P

    2012-05-01

    The effects of short-term food deprivation on lipid metabolism are well documented, but little is known about prolonged fasting. This study monitored the kinetics of glycerol (rate of appearance, R(a) glycerol) and non-esterified fatty acids (R(a) NEFA) in fasting rabbits. Our goals were to determine whether lipolysis is stimulated beyond values seen for short-term fasting, and to characterize the roles of primary (intracellular) and secondary (with transit through the circulation) triacylglycerol/fatty acid cycling (TAG/FA cycling) in regulating fatty acid allocation to oxidation or re-esterification. R(a) glycerol (9.62±0.72 to 15.29±0.96 μmol kg(-1) min(-1)) and R(a) NEFA (18.05±2.55 to 31.25±1.93 μmol kg(-1) min(-1)) were stimulated during the first 2 days of fasting, but returned to baseline after 4 days. An initial increase in TAG/FA cycling was followed by a reduction below baseline after 6 days without food, with primary and secondary cycling contributing to these responses. We conclude that the classic activation of lipolysis caused by short-term fasting is abolished when food deprivation is prolonged. High rates of re-esterification may become impossible to sustain, and TAG/FA cycling could decrease to reduce its cost to 3% of total energy expenditure. Throughout prolonged fasting, fatty acid metabolism gradually shifts towards increased oxidation and reduced re-esterification. Survival is achieved by pressing fuel selection towards the fatty acid dominance of energy metabolism and by slowing substrate cycles to assist metabolic suppression. However, TAG/FA cycling remains active even after prolonged fasting, suggesting that re-esterification is a crucial mechanism that cannot be stopped without harmful consequences.

  19. Novel role for SLPI in MOG-induced EAE revealed by spinal cord expression analysis

    Directory of Open Access Journals (Sweden)

    Aigner Ludwig

    2008-05-01

    suppression of the cell differentiation regulator HES1. Finally, SLPI prevented the degradation of IkappaBalpha, which may explain the suppression of the cell differentiation inhibitor HES1 suggesting a possible mechanism of oligodendroglial differentiation. Conclusion We identified novel features of gene expression in the CNS during EAE, in particular the suppression of genes of cholesterol biosynthesis and a strong upregulation of SLPI, a gene which is for the first time associated with autoimmune inflammation. The capacity of SLPI to increase proliferation of adult NSC and of oligodendroglial differentiation suggests a novel role for SLPI in the promotion of tissue repair, beyond its known functions in the prevention of tissue damages by protease inhibition damage and modulation of inflammatory reactions.

  20. Functional profiles reveal unique ecological roles of various biological soil crust organisms

    Science.gov (United States)

    Bowker, M.A.; Mau, R.L.; Maestre, F.T.; Escolar, C.; Castillo-Monroy, A. P.

    2011-01-01

    1. At the heart of the body of research on biodiversity effects on ecosystem function is the debate over whether different species tend to be functionally singular or redundant. When we consider ecosystem multi-function, the provision of multiple ecosystem functions simultaneously, we may find that seemingly redundant species may in fact play unique roles in ecosystems. 2. Over the last few decades, the significance of biological soil crusts (BSCs) as ecological boundaries and ecosystem engineers, and their multi-functional nature, has become increasingly well documented. We compiled 'functional profiles' of the organisms in this understudied community, to determine whether functional singularity emerges when multiple ecosystem functions are considered. 3. In two data sets, one representing multiple sites around the semi-arid regions of Spain (regional scale), and another from a single site in central Spain (local scale), we examined correlations between the abundance or frequency of BSC species in a community, and multiple surrogates of ecosystem functioning. There was a wide array of apparent effects of species on specific functions. 4. Notably, in gypsiferous soils and at regional scale, we found that indicators of carbon (C) and phosphorus cycling were apparently suppressed and promoted by the lichens Diploschistes diacapsis and Squamarina lentigera, respectively. The moss Pleurochaete squarrosa appears to promote C cycling in calcareous soils at this spatial scale. At the local scale in gypsiferous soils, D. diacapsis positively correlated with carbon cycling, but negatively with nitrogen cycling, whereas numerous lichens exhibited the opposite profile. 5. We found a high degree of functional singularity, i.e. that species were highly individualistic in their effects on multiple functions. Many functional attributes were not easily predictable from existing functional grouping systems based primarily on morphology. 6. Our results suggest that maintaining

  1. Quantitative Proteomics of the Tonoplast Reveals a Role for Glycolytic Enzymes in Salt Tolerance[C][W

    Science.gov (United States)

    Barkla, Bronwyn J.; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

    2009-01-01

    To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na+ sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H+-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H+-pump activity. PMID:20028841

  2. Extending Puppet

    CERN Document Server

    Franceschi, Alessandro

    2014-01-01

    This book is a clear, detailed and practical guide to learn about designing and deploying you puppet architecture, with informative examples to highlight and explain concepts in a focused manner. This book is designed for users who already have good experience with Puppet, and will surprise experienced users with innovative topics that explore how to design, implement, adapt, and deploy a Puppet architecture. The key to extending Puppet is the development of types and providers, for which you must be familiar with Ruby.

  3. The role of the anomaly cancellation mechanism in the evaluation of the radiatively induced Chern-Simons term in extended QED

    International Nuclear Information System (INIS)

    Battistel, O.A.; Dallabona, G.

    2004-01-01

    We consider the possible role played by the anomaly cancellation mechanism in the evaluation of the radiatively induced Chern-Simons (CS) term, arising from the Lorentz and CPT non-invariant fermionic sector, of an extended version of QED. We explicit evaluate the most general mathematical structure associated to the AVV triangle amplitude, closely related to the one involved in the CS term evaluation, using for this purposes an alternative calculational strategy to handle divergences in QFT's. We show that the requirement of consistency with the choices made in the construction of the Standard Model's renormalizability, in the evaluation of the AVV Green function, leave no room for a nonvanishing radiatively induced CS term, independently of the regularization prescription or equivalent philosophy adopted, in accordance with what was previously conjectured by other authors. (orig.)

  4. The Role of Coulomb Interactions for Spin Crossover Behaviors and Crystal Structural Transformation in Novel Anionic Fe(III Complexes from a π-Extended ONO Ligand

    Directory of Open Access Journals (Sweden)

    Suguru Murata

    2016-05-01

    Full Text Available To investigate the π-extension effect on an unusual negative-charged spin crossover (SCO FeIII complex with a weak N2O4 first coordination sphere, we designed and synthesized a series of anionic FeIII complexes from a π-extended naphthalene derivative ligand. Acetonitrile-solvate tetramethylammonium (TMA salt 1 exhibited an SCO conversion, while acetone-solvate TMA salt 2 was in a high-spin state. The crystal structural analysis for 2 revealed that two-leg ladder-like cation-anion arrays derived from π-stacking interactions between π-ligands of the FeIII complex anion and Coulomb interactions were found and the solvated acetone molecules were in one-dimensional channels between the cation-anion arrays. A desolvation-induced single-crystal-to-single-crystal transformation to desolvate compound 2’ may be driven by Coulomb energy gain. Furthermore, the structural comparison between quasi-polymorphic compounds 1 and 2 revealed that the synergy between Coulomb and π-stacking interactions induces a significant distortion of coordination structure of 2.

  5. Analysis of Msx1; Msx2 double mutants reveals multiple roles for Msx genes in limb development.

    Science.gov (United States)

    Lallemand, Yvan; Nicola, Marie-Anne; Ramos, Casto; Bach, Antoine; Cloment, Cécile Saint; Robert, Benoît

    2005-07-01

    The homeobox-containing genes Msx1 and Msx2 are highly expressed in the limb field from the earliest stages of limb formation and, subsequently, in both the apical ectodermal ridge and underlying mesenchyme. However, mice homozygous for a null mutation in either Msx1 or Msx2 do not display abnormalities in limb development. By contrast, Msx1; Msx2 double mutants exhibit a severe limb phenotype. Our analysis indicates that these genes play a role in crucial processes during limb morphogenesis along all three axes. Double mutant limbs are shorter and lack anterior skeletal elements (radius/tibia, thumb/hallux). Gene expression analysis confirms that there is no formation of regions with anterior identity. This correlates with the absence of dorsoventral boundary specification in the anterior ectoderm, which precludes apical ectodermal ridge formation anteriorly. As a result, anterior mesenchyme is not maintained, leading to oligodactyly. Paradoxically, polydactyly is also frequent and appears to be associated with extended Fgf activity in the apical ectodermal ridge, which is maintained up to 14.5 dpc. This results in a major outgrowth of the mesenchyme anteriorly, which nevertheless maintains a posterior identity, and leads to formation of extra digits. These defects are interpreted in the context of an impairment of Bmp signalling.

  6. Characterization of a null allelic mutant of the rice NAL1 gene reveals its role in regulating cell division.

    Directory of Open Access Journals (Sweden)

    Dan Jiang

    Full Text Available Leaf morphology is closely associated with cell division. In rice, mutations in Narrow leaf 1 (NAL1 show narrow leaf phenotypes. Previous studies have shown that NAL1 plays a role in regulating vein patterning and increasing grain yield in indica cultivars, but its role in leaf growth and development remains unknown. In this report, we characterized two allelic mutants of NARROW LEAF1 (NAL1, nal1-2 and nal1-3, both of which showed a 50% reduction in leaf width and length, as well as a dwarf culm. Longitudinal and transverse histological analyses of leaves and internodes revealed that cell division was suppressed in the anticlinal orientation but enhanced in the periclinal orientation in the mutants, while cell size remained unaltered. In addition to defects in cell proliferation, the mutants showed abnormal midrib in leaves. Map-based cloning revealed that nal1-2 is a null allelic mutant of NAL1 since both the whole promoter and a 404-bp fragment in the first exon of NAL1 were deleted, and that a 6-bp fragment was deleted in the mutant nal1-3. We demonstrated that NAL1 functions in the regulation of cell division as early as during leaf primordia initiation. The altered transcript level of G1- and S-phase-specific genes suggested that NAL1 affects cell cycle regulation. Heterogeneous expression of NAL1 in fission yeast (Schizosaccharomyces pombe further supported that NAL1 affects cell division. These results suggest that NAL1 controls leaf width and plant height through its effects on cell division.

  7. Comparative genome analysis of Megasphaera sp. reveals niche specialization and its potential role in the human gut.

    Directory of Open Access Journals (Sweden)

    Sudarshan Anand Shetty

    Full Text Available With increasing number of novel bacteria being isolated from the human gut ecosystem, there is a greater need to study their role in the gut ecosystem and their effect on the host health. In the present study, we carried out in silico genome-wide analysis of two novel Megasphaera sp. isolates NM10 (DSM25563 and BL7 (DSM25562, isolated from feces of two healthy individuals and validated the key features by in vitro studies. The analysis revealed the general metabolic potential, adaptive features and the potential effects of these isolates on the host. The comparative genome analysis of the two human gut isolates NM10 and BL7 with ruminal isolate Megasphaera elsdenii (DSM20460 highlighted the differential adaptive features for their survival in human gut. The key findings include features like bile resistance, presence of various sensory and regulatory systems, stress response systems, membrane transporters and resistance to antibiotics. Comparison of the "glycobiome" based on the genomes of the ruminal isolate with the human gut isolates NM10 and BL revealed the presence of diverse and unique sets of Carbohydrate-Active enzymes (CAZymes amongst these isolates, with a higher collection of CAZymes in the human gut isolates. This could be attributed to the difference in host diet and thereby the environment, consequently suggesting host specific adaptation in these isolates. In silico analysis of metabolic potential predicted the ability of these isolates to produce important metabolites like short chain fatty acids (butyrate, acetate, formate, and caproate, vitamins and essential amino acids, which was further validated by in vitro experiments. The ability of these isolates to produce important metabolites advocates for a potential healthy influence on the host. Further in vivo studies including transcriptomic and proteomic analysis will be required for better understanding the role and impact of these Megasphaera sp. isolates NM10 and BL7 on the

  8. Long-Term Live Cell Imaging Reveals New Roles For Salmonella Effector Proteins SseG and SteA

    Science.gov (United States)

    McQuate, Sarah E.; Young, Alexandra M.; Silva-Herzog, Eugenia; Bunker, Eric; Hernandez, Mateo; de Chaumont, Fabrice; Liu, Xuedong; Detweiler, Corrella S.; Palmer, Amy E.

    2016-01-01

    Summary Salmonella Typhimurium is an intracellular bacterial pathogen that infects both epithelial cells and macrophages. Salmonella effector proteins, which are translocated into the host cell and manipulate host cell components, control the ability to replicate and/or survive in host cells. Due to the complexity and heterogeneity of Salmonella infections, there is growing recognition of the need for single cell and live-cell imaging approaches to identify and characterize the diversity of cellular phenotypes and how they evolve over time. Here we establish a pipeline for long-term (16 hours) live-cell imaging of infected cells and subsequent image analysis methods. We apply this pipeline to track bacterial replication within the Salmonella-containing vacuole in epithelial cells, quantify vacuolar replication versus survival in macrophages, and investigate the role of individual effector proteins in mediating these parameters. This approach revealed that dispersed bacteria can coalesce at later stages of infection, that the effector protein SseG influences the propensity for cytosolic hyperreplication in epithelial cells, and that while SteA only has a subtle effect on vacuolar replication in epithelial cells, it has a profound impact on infection parameters in immunocompetent macrophages, suggesting differential roles for effector proteins in different infection models. PMID:27376507

  9. The Arabidopsis nox mutant lacking carotene hydroxylase activity reveals a critical role for xanthophylls in photosystem I biogenesis.

    Science.gov (United States)

    Dall'Osto, Luca; Piques, Maria; Ronzani, Michela; Molesini, Barbara; Alboresi, Alessandro; Cazzaniga, Stefano; Bassi, Roberto

    2013-02-01

    Carotenes, and their oxygenated derivatives xanthophylls, are essential components of the photosynthetic apparatus. They contribute to the assembly of photosynthetic complexes and participate in light absorption and chloroplast photoprotection. Here, we studied the role of xanthophylls, as distinct from that of carotenes, by characterizing a no xanthophylls (nox) mutant of Arabidopsis thaliana, which was obtained by combining mutations targeting the four carotenoid hydroxylase genes. nox plants retained α- and β-carotenes but were devoid in xanthophylls. The phenotype included depletion of light-harvesting complex (LHC) subunits and impairment of nonphotochemical quenching, two effects consistent with the location of xanthophylls in photosystem II antenna, but also a decreased efficiency of photosynthetic electron transfer, photosensitivity, and lethality in soil. Biochemical analysis revealed that the nox mutant was specifically depleted in photosystem I function due to a severe deficiency in PsaA/B subunits. While the stationary level of psaA/B transcripts showed no major differences between genotypes, the stability of newly synthesized PsaA/B proteins was decreased and translation of psaA/B mRNA was impaired in nox with respect to wild-type plants. We conclude that xanthophylls, besides their role in photoprotection and LHC assembly, are also needed for photosystem I core translation and stability, thus making these compounds indispensable for autotrophic growth.

  10. Consciousness extended

    DEFF Research Database (Denmark)

    Carrara-Augustenborg, Claudia

    2012-01-01

    There is no consensus yet regarding a conceptualization of consciousness able to accommodate all the features of such complex phenomenon. Different theoretical and empirical models lend strength to both the occurrence of a non-accessible informational broadcast, and to the mobilization of specific...... brain areas responsible for the emergence of the individual´s explicit and variable access to given segments of such broadcast. Rather than advocating one model over others, this chapter proposes to broaden the conceptualization of consciousness by letting it embrace both mechanisms. Within...... such extended framework, I propose conceptual and functional distinctions between consciousness (global broadcast of information), awareness (individual´s ability to access the content of such broadcast) and unconsciousness (focally isolated neural activations). My hypothesis is that a demarcation in terms...

  11. Chlorosis caused by two recessively interacting genes reveals a role of RNA helicase in hybrid breakdown in Arabidopsis thaliana.

    Science.gov (United States)

    Plötner, Björn; Nurmi, Markus; Fischer, Axel; Watanabe, Mutsumi; Schneeberger, Korbinian; Holm, Svante; Vaid, Neha; Schöttler, Mark Aurel; Walther, Dirk; Hoefgen, Rainer; Weigel, Detlef; Laitinen, Roosa A E

    2017-07-01

    Hybrids often differ in fitness from their parents. They may be superior, translating into hybrid vigour or heterosis, but they may also be markedly inferior, because of hybrid weakness or incompatibility. The underlying genetic causes for the latter can often be traced back to genes that evolve rapidly because of sexual or host-pathogen conflicts. Hybrid weakness may manifest itself only in later generations, in a phenomenon called hybrid breakdown. We have characterized a case of hybrid breakdown among two Arabidopsis thaliana accessions, Shahdara (Sha, Tajikistan) and Lövvik-5 (Lov-5, Northern Sweden). In addition to chlorosis, a fraction of the F 2 plants have defects in leaf and embryo development, and reduced photosynthetic efficiency. Hybrid chlorosis is due to two major-effect loci, of which one, originating from Lov-5, appears to encode an RNA helicase (AtRH18). To examine the role of the chlorosis allele in the Lövvik area, in addition to eight accessions collected in 2009, we collected another 240 accessions from 15 collections sites, including Lövvik, from Northern Sweden in 2015. Genotyping revealed that Lövvik collection site is separated from the rest. Crosses between 109 accessions from this area and Sha revealed 85 cases of hybrid chlorosis, indicating that the chlorosis-causing allele is common in this area. These results suggest that hybrid breakdown alleles not only occur at rapidly evolving loci, but also at genes that code for conserved processes. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

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

    Science.gov (United States)

    Palermo, Giulia; Miao, Yinglong; Walker, Ross C; Jinek, Martin; McCammon, J Andrew

    2016-10-26

    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.

  13. Genomic analysis reveals a potential role for cell cycle perturbation in HCV-mediated apoptosis of cultured hepatocytes.

    Directory of Open Access Journals (Sweden)

    Kathie-Anne Walters

    2009-01-01

    Full Text Available The mechanisms of liver injury associated with chronic HCV infection, as well as the individual roles of both viral and host factors, are not clearly defined. However, it is becoming increasingly clear that direct cytopathic effects, in addition to immune-mediated processes, play an important role in liver injury. Gene expression profiling during multiple time-points of acute HCV infection of cultured Huh-7.5 cells was performed to gain insight into the cellular mechanism of HCV-associated cytopathic effect. Maximal induction of cell-death-related genes and appearance of activated caspase-3 in HCV-infected cells coincided with peak viral replication, suggesting a link between viral load and apoptosis. Gene ontology analysis revealed that many of the cell-death genes function to induce apoptosis in response to cell cycle arrest. Labeling of dividing cells in culture followed by flow cytometry also demonstrated the presence of significantly fewer cells in S-phase in HCV-infected relative to mock cultures, suggesting HCV infection is associated with delayed cell cycle progression. Regulation of numerous genes involved in anti-oxidative stress response and TGF-beta1 signaling suggest these as possible causes of delayed cell cycle progression. Significantly, a subset of cell-death genes regulated during in vitro HCV infection was similarly regulated specifically in liver tissue from a cohort of HCV-infected liver transplant patients with rapidly progressive fibrosis. Collectively, these data suggest that HCV mediates direct cytopathic effects through deregulation of the cell cycle and that this process may contribute to liver disease progression. This in vitro system could be utilized to further define the cellular mechanism of this perturbation.

  14. Learning-Induced Gene Expression in the Hippocampus Reveals a Role of Neuron -Astrocyte Metabolic Coupling in Long Term Memory

    KAUST Repository

    Tadi, Monika; Allaman, Igor; Lengacher, Sylvain; Grenningloh, Gabriele; Magistretti, Pierre J.

    2015-01-01

    We examined the expression of genes related to brain energy metabolism and particularly those encoding glia (astrocyte)-specific functions in the dorsal hippocampus subsequent to learning. Context-dependent avoidance behavior was tested in mice using the step-through Inhibitory Avoidance (IA) paradigm. Animals were sacrificed 3, 9, 24, or 72 hours after training or 3 hours after retention testing. The quantitative determination of mRNA levels revealed learning-induced changes in the expression of genes thought to be involved in astrocyte-neuron metabolic coupling in a time dependent manner. Twenty four hours following IA training, an enhanced gene expression was seen, particularly for genes encoding monocarboxylate transporters 1 and 4 (MCT1, MCT4), alpha2 subunit of the Na/K-ATPase and glucose transporter type 1. To assess the functional role for one of these genes in learning, we studied MCT1 deficient mice and found that they exhibit impaired memory in the inhibitory avoidance task. Together, these observations indicate that neuron-glia metabolic coupling undergoes metabolic adaptations following learning as indicated by the change in expression of key metabolic genes.

  15. Diuron treatment reveals the different roles of two cyclic electron transfer pathways in photosystem II in Arabidopsis thaliana.

    Science.gov (United States)

    Jin, Yujian; Chen, Si; Fan, Xiaoji; Song, Hao; Li, Xingxing; Xu, Jiahui; Qian, Haifeng

    2017-04-01

    Three ecotypes of Arabidopsis thaliana, ecotype Columbia (Wild type, Wt) and two mutants (pgr5 and ndf4), were used to evaluate the effects of diuron on photosynthetic activity of A. thaliana. It was found that diuron adversely affected the fresh weight and chlorophyll content of the plants. Chlorophyll fluorescence studies determined that the pgr5 mutant was more sensitive to diuron than Wt and the ndf4 mutant. Gene expression analysis revealed different roles for the two cyclic electron transfer (CET) pathways, NAD(P)H dehydrogenase (NDH) and proton gradient regulation (PGR5) pathways, in the plant after diuron treatment. For example, a gene in the NDH pathway, lhca5, was activated in the low dose (LD) group in the pgr5 mutant, but was down-regulated in the moderate dose (MD) group, along with two other NDH-related genes (ppl2 and ndhH). In the PGR5 pathway, the pgr5 gene was functional under conditions of increased stress (MD group), and was up-regulated to a greater extent in the ndf4 mutant than that in the Wt and pgr5 mutant. Our results suggest that the PGR5 pathway in plants is more important than the NDH pathway during resistance to environmental stress. Deficiencies in the PGR5 pathway could not be counteracted by the NDH pathway, but deficiencies in the NDH pathway could be overcome by stimulating PGR5. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Disruption of Msx-1 and Msx-2 reveals roles for these genes in craniofacial, eye, and axial development.

    Science.gov (United States)

    Foerst-Potts, L; Sadler, T W

    1997-05-01

    In mouse embryos, the muscle segment homeobox genes, Msx-1 and Msx-2 are expressed during critical stages of neural tube, neural crest, and craniofacial development, suggesting that these genes play important roles in organogenesis and cell differentiation. Although the patterns of expression are intriguing, little is known about the function of these genes in vertebrate embryonic development. Therefore, the expression of both genes, separately and together, was disrupted using antisense oligodeoxynucleotides and whole embryo culture techniques. Antisense attenuation of Msx-1 during early stages of neurulation produced hypoplasia of the maxillary, mandibular, and frontonasal prominences, eye anomalies, and somite and neural tube abnormalities. Eye defects consisted of enlarged optic vesicles, which may ultimately result in micropthalmia similar to that observed in Small eye mice homozygous for mutations in the Pax-6 gene. Histological sections and SEM analysis revealed a thinning of the neuroepithelium in the diencephalon and optic vesicle and mesenchymal deficiencies in the craniofacial region. Injections of Msx-2 antisense oligodeoxynucleotides produced similar malformations as those targeting Msx-1, with the exception that there was an increase in number and severity of neural tube and somite defects. Embryos injected with the combination of Msx-1 + Msx-2 antisense oligodeoxynucleotides showed no novel abnormalities, suggesting that the genes do not operate in a redundant manner.

  17. Structural Snapshots of an Engineered Cystathionine-γ-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Wupeng; Stone, Everett; Zhang, Yan Jessie

    2017-02-01

    Enzyme therapeutics that can degrade l-methionine (l-Met) are of great interest as numerous malignancies are exquisitely sensitive to l-Met depletion. To exhaust the pool of methionine in human serum, we previously engineered an l-Met-degrading enzyme based on the human cystathionine-γ-lyase scaffold (hCGL-NLV) to circumvent immunogenicity and stability issues observed in the preclinical application of bacterially derived methionine-γ-lyases. To gain further insights into the structure–activity relationships governing the chemistry of the hCGL-NLV lead molecule, we undertook a biophysical characterization campaign that captured crystal structures (2.2 Å) of hCGL-NLV with distinct reaction intermediates, including internal aldimine, substrate-bound, gem-diamine, and external aldimine forms. Curiously, an alternate form of hCGL-NLV that crystallized under higher-salt conditions revealed a locally unfolded active site, correlating with inhibition of activity as a function of ionic strength. Subsequent mutational and kinetic experiments pinpointed that a salt bridge between the phosphate of the essential cofactor pyridoxal 5'-phosphate (PLP) and residue R62 plays an important role in catalyzing β- and γ-eliminations. Our study suggests that solvent ions such as NaCl disrupt electrostatic interactions between R62 and PLP, decreasing catalytic efficiency.

  18. Isolation of Chromatin from Dysfunctional Telomeres Reveals an Important Role for Ring1b in NHEJ-Mediated Chromosome Fusions

    Directory of Open Access Journals (Sweden)

    Cristina Bartocci

    2014-05-01

    Full Text Available When telomeres become critically short, DNA damage response factors are recruited at chromosome ends, initiating a cellular response to DNA damage. We performed proteomic isolation of chromatin fragments (PICh in order to define changes in chromatin composition that occur upon onset of acute telomere dysfunction triggered by depletion of the telomere-associated factor TRF2. This unbiased purification of telomere-associated proteins in functional or dysfunctional conditions revealed the dynamic changes in chromatin composition that take place at telomeres upon DNA damage induction. On the basis of our results, we describe a critical role for the polycomb group protein Ring1b in nonhomologous end-joining (NHEJ-mediated end-to-end chromosome fusions. We show that cells with reduced levels of Ring1b have a reduced ability to repair uncapped telomeric chromatin. Our data represent an unbiased isolation of chromatin undergoing DNA damage and are a valuable resource to map the changes in chromatin composition in response to DNA damage activation.

  19. A whole-body model for glycogen regulation reveals a critical role for substrate cycling in maintaining blood glucose homeostasis.

    Directory of Open Access Journals (Sweden)

    Ke Xu

    2011-12-01

    Full Text Available Timely, and sometimes rapid, metabolic adaptation to changes in food supply is critical for survival as an organism moves from the fasted to the fed state, and vice versa. These transitions necessitate major metabolic changes to maintain energy homeostasis as the source of blood glucose moves away from ingested carbohydrates, through hepatic glycogen stores, towards gluconeogenesis. The integration of hepatic glycogen regulation with extra-hepatic energetics is a key aspect of these adaptive mechanisms. Here we use computational modeling to explore hepatic glycogen regulation under fed and fasting conditions in the context of a whole-body model. The model was validated against previous experimental results concerning glycogen phosphorylase a (active and glycogen synthase a dynamics. The model qualitatively reproduced physiological changes that occur during transition from the fed to the fasted state. Analysis of the model reveals a critical role for the inhibition of glycogen synthase phosphatase by glycogen phosphorylase a. This negative regulation leads to high levels of glycogen synthase activity during fasting conditions, which in turn increases substrate (futile cycling, priming the system for a rapid response once an external source of glucose is restored. This work demonstrates that a mechanistic understanding of the design principles used by metabolic control circuits to maintain homeostasis can benefit from the incorporation of mathematical descriptions of these networks into "whole-body" contextual models that mimic in vivo conditions.

  20. Activation of acid-sensing ion channels by localized proton transient reveals their role in proton signaling.

    Science.gov (United States)

    Zeng, Wei-Zheng; Liu, Di-Shi; Liu, Lu; She, Liang; Wu, Long-Jun; Xu, Tian-Le

    2015-09-15

    Extracellular transients of pH alterations likely mediate signal transduction in the nervous system. Neuronal acid-sensing ion channels (ASICs) act as sensors for extracellular protons, but the mechanism underlying ASIC activation remains largely unknown. Here, we show that, following activation of a light-activated proton pump, Archaerhodopsin-3 (Arch), proton transients induced ASIC currents in both neurons and HEK293T cells co-expressing ASIC1a channels. Using chimera proteins that bridge Arch and ASIC1a by a glycine/serine linker, we found that successful coupling occurred within 15 nm distance. Furthermore, two-cell sniffer patch recording revealed that regulated release of protons through either Arch or voltage-gated proton channel Hv1 activated neighbouring cells expressing ASIC1a channels. Finally, computational modelling predicted the peak proton concentration at the intercellular interface to be at pH 6.7, which is acidic enough to activate ASICs in vivo. Our results highlight the pathophysiological role of proton signalling in the nervous system.

  1. Learning-Induced Gene Expression in the Hippocampus Reveals a Role of Neuron -Astrocyte Metabolic Coupling in Long Term Memory.

    Directory of Open Access Journals (Sweden)

    Monika Tadi

    Full Text Available We examined the expression of genes related to brain energy metabolism and particularly those encoding glia (astrocyte-specific functions in the dorsal hippocampus subsequent to learning. Context-dependent avoidance behavior was tested in mice using the step-through Inhibitory Avoidance (IA paradigm. Animals were sacrificed 3, 9, 24, or 72 hours after training or 3 hours after retention testing. The quantitative determination of mRNA levels revealed learning-induced changes in the expression of genes thought to be involved in astrocyte-neuron metabolic coupling in a time dependent manner. Twenty four hours following IA training, an enhanced gene expression was seen, particularly for genes encoding monocarboxylate transporters 1 and 4 (MCT1, MCT4, alpha2 subunit of the Na/K-ATPase and glucose transporter type 1. To assess the functional role for one of these genes in learning, we studied MCT1 deficient mice and found that they exhibit impaired memory in the inhibitory avoidance task. Together, these observations indicate that neuron-glia metabolic coupling undergoes metabolic adaptations following learning as indicated by the change in expression of key metabolic genes.

  2. Learning-Induced Gene Expression in the Hippocampus Reveals a Role of Neuron -Astrocyte Metabolic Coupling in Long Term Memory

    KAUST Repository

    Tadi, Monika

    2015-10-29

    We examined the expression of genes related to brain energy metabolism and particularly those encoding glia (astrocyte)-specific functions in the dorsal hippocampus subsequent to learning. Context-dependent avoidance behavior was tested in mice using the step-through Inhibitory Avoidance (IA) paradigm. Animals were sacrificed 3, 9, 24, or 72 hours after training or 3 hours after retention testing. The quantitative determination of mRNA levels revealed learning-induced changes in the expression of genes thought to be involved in astrocyte-neuron metabolic coupling in a time dependent manner. Twenty four hours following IA training, an enhanced gene expression was seen, particularly for genes encoding monocarboxylate transporters 1 and 4 (MCT1, MCT4), alpha2 subunit of the Na/K-ATPase and glucose transporter type 1. To assess the functional role for one of these genes in learning, we studied MCT1 deficient mice and found that they exhibit impaired memory in the inhibitory avoidance task. Together, these observations indicate that neuron-glia metabolic coupling undergoes metabolic adaptations following learning as indicated by the change in expression of key metabolic genes.

  3. Gene expression profiles of prohibitin in testes of Octopus tankahkeei (ot-phb) revealing its possible role during spermiogenesis.

    Science.gov (United States)

    Mao, Hai-Tao; Wang, Da-Hui; Lan, Zhou; Zhou, Hong; Yang, Wan-Xi

    2012-05-01

    Prohibitin is essential for intracellular homeostasis and stabilization of mitochondrial respiratory chain complexes. To explore its functions during spermiogenesis of Octopus tankahkeei (O. tankahkeei), we have cloned and sequenced the cDNA of this mammalian PHB homologue (termed ot-PHB) from the testes of O. tankahkeei. The 1165 bp ot-phb cDNA contains a 100 bp 5' UTR, a 882 bp open reading frame and a 183 bp 3' UTR. The putative ot-PHB protein owns a transmembrane domain from 6 to 31 amino acid (aa) and a putative PHB domain from 26 to 178 aa. Protein alignment demonstrated that ot-PHB had 73.3, 73.6, 74.0, 75.1, and 45.4% identity with its homologues in Homo sapiens, Mus muculus, Danio rerio, Xenopus tropicalis and Trypanosoma brucei, respectively. Tissue distribution profile analysis revealed its presence in all the tissues examined. In situ hybridization in spermiogenic cells demonstrated that ot-phb was expressed moderately at the beginning of the spermiogenesis. The abundance of transcripts increased in intermediate spermatids and in drastically remodeling final spermatids. In mature spermatozoa, the residuary transcripts concentrated around the chondriosomal mantle where mitochondria assemble around. In summary, the expression of ot-phb during spermiogenesis implicates a potential function of this protein during mitochondrial ubiquitination. It is the first time to implicate the role of prohibitin in cephalopod spermiogenesis.

  4. An Essential Role for Coagulase in Staphylococcus aureus Biofilm Development Reveals New Therapeutic Possibilities for Device-Related Infections.

    Science.gov (United States)

    Zapotoczna, Marta; McCarthy, Hannah; Rudkin, Justine K; O'Gara, James P; O'Neill, Eoghan

    2015-12-15

    High-level resistance to antimicrobial drugs is a major factor in the pathogenesis of chronic Staphylococcus aureus biofilm-associated, medical device-related infections. Antimicrobial susceptibility analysis revealed that biofilms grown for ≤ 24 hours on biomaterials conditioned with human plasma under venous shear in iron-free cell culture medium were significantly more susceptible to antistaphylococcal antibiotics. Biofilms formed under these physiologically relevant conditions were regulated by SaeRS and dependent on coagulase-catalyzed conversion of fibrinogen into fibrin. In contrast, SarA-regulated biofilms formed on uncoated polystyrene in nutrient-rich bacteriological medium were mediated by the previously characterized biofilm factors poly-N-acetyl glucosamine, fibronectin-binding proteins, or autolytic activity and were antibiotic resistant. Coagulase-mediated biofilms exhibited increased antimicrobial resistance over time (>48 hours) but were always susceptible to dispersal by the fibrinolytic enzymes plasmin or nattokinase. Biofilms recovered from infected central venous catheters in a rat model of device-related infection were dispersed by nattokinase, supporting the important role of the biofilm phenotype and identifying a potentially new therapeutic approach with antimicrobials and fibrinolytic drugs, particularly during the early stages of device-related infection. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  5. Bath Breakfast Project (BBP - Examining the role of extended daily fasting in human energy balance and associated health outcomes: Study protocol for a randomised controlled trial [ISRCTN31521726

    Directory of Open Access Journals (Sweden)

    Jeans Matthew

    2011-07-01

    Full Text Available Abstract Background Current guidance regarding the role of daily breakfast in human health is largely grounded in cross-sectional observations. However, the causal nature of these relationships has not been fully explored and what limited information is emerging from controlled laboratory-based experiments appears inconsistent with much existing data. Further progress in our understanding therefore requires a direct examination of how daily breakfast impacts human health under free-living conditions. Methods/Design The Bath Breakfast Project (BBP is a randomised controlled trial comparing the effects of daily breakfast consumption relative to extended fasting on energy balance and human health. Approximately 70 men and women will undergo extensive laboratory-based assessments of their acute metabolic responses under fasted and post-prandial conditions, to include: resting metabolic rate, substrate oxidation, dietary-induced thermogenesis and systemic concentrations of key metabolites/hormones. Physiological and psychological indices of appetite will also be monitored both over the first few hours of the day (i.e. whether fed or fasted and also following a standardised test lunch used to assess voluntary energy intake under controlled conditions. Baseline measurements of participants' anthropometric characteristics (e.g. DEXA will be recorded prior to intervention, along with an oral glucose tolerance test and acquisition of adipose tissue samples to determine expression of key genes and estimates of tissue-specific insulin action. Participants will then be randomly assigned either to a group prescribed an energy intake of ≥3000 kJ before 1100 each day or a group to extend their overnight fast by abstaining from ingestion of energy-providing nutrients until 1200 each day, with all laboratory-based measurements followed-up 6 weeks later. Free-living assessments of energy intake (via direct weighed food diaries and energy expenditure (via

  6. Mechanistic studies of anticancer aptamer AS1411 reveal a novel role for nucleolin in regulating Rac1 activation.

    Science.gov (United States)

    Reyes-Reyes, E Merit; Šalipur, Francesca R; Shams, Mitra; Forsthoefel, Matthew K; Bates, Paula J

    2015-08-01

    AS1411 is a G-rich quadruplex-forming oligodeoxynucleotide that binds specifically to nucleolin, a protein found on the surface and in the cytoplasm of most malignant cells but absent from the surface/cytoplasm of most normal cells. AS1411 has shown promising clinical activity and is being widely used as a tumor-targeting agent, but its mechanism of action is not fully understood. Previously, we showed that AS1411 is taken up in cancer cells by macropinocytosis (fluid phase endocytosis) and subsequently stimulates further macropinocytosis by a nucleolin-dependent mechanism. In the current study, we have investigated the significance and molecular mechanisms of AS1411-induced macropinocytosis. Our results indicate that the antiproliferative activity of AS1411 in various cell lines correlated with its capacity to stimulate macropinocytosis. In DU145 prostate cancer cells, AS1411 induced activation of EGFR, Akt, p38, and Rac1. Activation of Akt and p38 were not critical for AS1411 activity because Akt activation was not observed in all AS1411-responsive cell lines and knockdown of p38 had no effect on AS1411's ability to inhibit proliferation. On the other hand, activation of EGFR and Rac1 appeared to play a role in AS1411 activity in all cancer cell lines examined (DU145, MDA-MB-468, A549, LNCaP) and their inhibition significantly reduced AS1411-mediated macropinocytosis and AS1411 antiproliferative activity. Interestingly, downregulation of nucleolin expression by siRNA also produced a substantial increase in activated Rac1, revealing a previously unknown role for nucleolin as a negative regulator of Rac1 activation. Our results are consistent with a model whereby AS1411 binding to nucleolin leads to sustained activation of Rac1 and causes methuosis, a novel type of nonapoptotic cell death characterized by hyperstimulation of macropinocytosis. We speculate that methuosis is a tumor/metastasis suppressor mechanism that opposes the malignant functions of Rac1 and that

  7. Effects of CeO2 nanoparticles on sludge aggregation and the role of extracellular polymeric substances – Explanation based on extended DLVO

    International Nuclear Information System (INIS)

    You, Guoxiang; Hou, Jun; Wang, Peifang; Xu, Yi; Wang, Chao; Miao, Lingzhan; Lv, Bowen; Yang, Yangyang; Luo, Hao

    2016-01-01

    The extended DLVO (XDLVO) theory was applied to elucidate the potential effects of CeO 2 nanoparticles (CeO 2 NPs) on sludge aggregation and the role of extracellular polymeric substances (EPS). In this study, seven different concentrations of CeO 2 NPs were added to activated sludge cultured in sequencing batch reactors (SBRs) and compared with a control test that received no CeO 2 NPs. After exposure to 50 mg/L CeO 2 NPs, a negligible change (p>0.1) occurred in the sludge volume index (SVI), whereas the flocculability and aggregation of the sludge decreased by 18.8% and 11.2%, respectively, resulting in a high effluent turbidity. The XDLVO theory demonstrated that the adverse effects of the CeO 2 NPs on sludge aggregation were due to an enhanced barrier energy. Compared to the van der Waals energies (W A ) and the electric double layer (W R ), the acid-base interaction (W AB ) markedly changed for the various concentrations of CeO 2 NPs. The EPS played a decisive role in the sludge surface characteristics, as the removal of EPS equals to the negative effects induced by 5–10 mg/L CeO 2 NPs on the sludge flocculability and aggregation. The presence of CeO 2 NPs induced negative contributions to the tight boundary EPS (TB-EPS) and core bacteria while positive contributions to the total interaction energy of the loose boundary EPS (LB-EPS). - Highlights: • CeO 2 NPs adversely affected the flocculability and aggregation of the sludge. • The presence of CeO 2 NPs increased the energy barrier and led to a stable suspension. • The removal of EPS equals to the negative effects induced by 5–10 mg/L CeO 2 NPs. • The acid-base interaction was dominate and markedly changed for the CeO 2 NPs. • CeO 2 NPs induced negative contributions to the TB-EPS while positive to the LB-EPS.

  8. Effects of CeO{sub 2} nanoparticles on sludge aggregation and the role of extracellular polymeric substances – Explanation based on extended DLVO

    Energy Technology Data Exchange (ETDEWEB)

    You, Guoxiang [Key Laboratory of Integrated Regulation and Resources Development on Shallow Lakes, Ministry of Education, Hohai University, Nanjing 210098 (China); College of Environment, Hohai University, Nanjing 210098 (China); Hou, Jun, E-mail: hhuhjyhj@126.com [Key Laboratory of Integrated Regulation and Resources Development on Shallow Lakes, Ministry of Education, Hohai University, Nanjing 210098 (China); College of Environment, Hohai University, Nanjing 210098 (China); Wang, Peifang, E-mail: pfwang2005@hhu.edu.cn [Key Laboratory of Integrated Regulation and Resources Development on Shallow Lakes, Ministry of Education, Hohai University, Nanjing 210098 (China); College of Environment, Hohai University, Nanjing 210098 (China); Xu, Yi; Wang, Chao; Miao, Lingzhan; Lv, Bowen; Yang, Yangyang; Luo, Hao [Key Laboratory of Integrated Regulation and Resources Development on Shallow Lakes, Ministry of Education, Hohai University, Nanjing 210098 (China); College of Environment, Hohai University, Nanjing 210098 (China)

    2016-11-15

    The extended DLVO (XDLVO) theory was applied to elucidate the potential effects of CeO{sub 2} nanoparticles (CeO{sub 2} NPs) on sludge aggregation and the role of extracellular polymeric substances (EPS). In this study, seven different concentrations of CeO{sub 2} NPs were added to activated sludge cultured in sequencing batch reactors (SBRs) and compared with a control test that received no CeO{sub 2} NPs. After exposure to 50 mg/L CeO{sub 2} NPs, a negligible change (p>0.1) occurred in the sludge volume index (SVI), whereas the flocculability and aggregation of the sludge decreased by 18.8% and 11.2%, respectively, resulting in a high effluent turbidity. The XDLVO theory demonstrated that the adverse effects of the CeO{sub 2} NPs on sludge aggregation were due to an enhanced barrier energy. Compared to the van der Waals energies (W{sub A}) and the electric double layer (W{sub R}), the acid-base interaction (W{sub AB}) markedly changed for the various concentrations of CeO{sub 2} NPs. The EPS played a decisive role in the sludge surface characteristics, as the removal of EPS equals to the negative effects induced by 5–10 mg/L CeO{sub 2} NPs on the sludge flocculability and aggregation. The presence of CeO{sub 2} NPs induced negative contributions to the tight boundary EPS (TB-EPS) and core bacteria while positive contributions to the total interaction energy of the loose boundary EPS (LB-EPS). - Highlights: • CeO{sub 2} NPs adversely affected the flocculability and aggregation of the sludge. • The presence of CeO{sub 2} NPs increased the energy barrier and led to a stable suspension. • The removal of EPS equals to the negative effects induced by 5–10 mg/L CeO{sub 2} NPs. • The acid-base interaction was dominate and markedly changed for the CeO{sub 2} NPs. • CeO{sub 2} NPs induced negative contributions to the TB-EPS while positive to the LB-EPS.

  9. Improved throughput traction microscopy reveals pivotal role for matrix stiffness in fibroblast contractility and TGF-β responsiveness

    Science.gov (United States)

    Marinković, Aleksandar; Mih, Justin D.; Park, Jin-Ah; Liu, Fei

    2012-01-01

    Lung fibroblast functions such as matrix remodeling and activation of latent transforming growth factor-β1 (TGF-β1) are associated with expression of the myofibroblast phenotype and are directly linked to fibroblast capacity to generate force and deform the extracellular matrix. However, the study of fibroblast force-generating capacities through methods such as traction force microscopy is hindered by low throughput and time-consuming procedures. In this study, we improved at the detail level methods for higher-throughput traction measurements on polyacrylamide hydrogels using gel-surface-bound fluorescent beads to permit autofocusing and automated displacement mapping, and transduction of fibroblasts with a fluorescent label to streamline cell boundary identification. Together these advances substantially improve the throughput of traction microscopy and allow us to efficiently compute the forces exerted by lung fibroblasts on substrates spanning the stiffness range present in normal and fibrotic lung tissue. Our results reveal that lung fibroblasts dramatically alter the forces they transmit to the extracellular matrix as its stiffness changes, with very low forces generated on matrices as compliant as normal lung tissue. Moreover, exogenous TGF-β1 selectively accentuates tractions on stiff matrices, mimicking fibrotic lung, but not on physiological stiffness matrices, despite equivalent changes in Smad2/3 activation. Taken together, these results demonstrate a pivotal role for matrix mechanical properties in regulating baseline and TGF-β1-stimulated contraction of lung fibroblasts and suggest that stiff fibrotic lung tissue may promote myofibroblast activation through contractility-driven events, whereas normal lung tissue compliance may protect against such feedback amplification of fibroblast activation. PMID:22659883

  10. Sequential Elution Interactome Analysis of the Mind Bomb 1 Ubiquitin Ligase Reveals a Novel Role in Dendritic Spine Outgrowth*

    Science.gov (United States)

    Mertz, Joseph; Tan, Haiyan; Pagala, Vishwajeeth; Bai, Bing; Chen, Ping-Chung; Li, Yuxin; Cho, Ji-Hoon; Shaw, Timothy; Wang, Xusheng; Peng, Junmin

    2015-01-01

    The mind bomb 1 (Mib1) ubiquitin ligase is essential for controlling metazoan development by Notch signaling and possibly the Wnt pathway. It is also expressed in postmitotic neurons and regulates neuronal morphogenesis and synaptic activity by mechanisms that are largely unknown. We sought to comprehensively characterize the Mib1 interactome and study its potential function in neuron development utilizing a novel sequential elution strategy for affinity purification, in which Mib1 binding proteins were eluted under different stringency and then quantified by the isobaric labeling method. The strategy identified the Mib1 interactome with both deep coverage and the ability to distinguish high-affinity partners from low-affinity partners. A total of 817 proteins were identified during the Mib1 affinity purification, including 56 high-affinity partners and 335 low-affinity partners, whereas the remaining 426 proteins are likely copurified contaminants or extremely weak binding proteins. The analysis detected all previously known Mib1-interacting proteins and revealed a large number of novel components involved in Notch and Wnt pathways, endocytosis and vesicle transport, the ubiquitin-proteasome system, cellular morphogenesis, and synaptic activities. Immunofluorescence studies further showed colocalization of Mib1 with five selected proteins: the Usp9x (FAM) deubiquitinating enzyme, alpha-, beta-, and delta-catenins, and CDKL5. Mutations of CDKL5 are associated with early infantile epileptic encephalopathy-2 (EIEE2), a severe form of mental retardation. We found that the expression of Mib1 down-regulated the protein level of CDKL5 by ubiquitination, and antagonized CDKL5 function during the formation of dendritic spines. Thus, the sequential elution strategy enables biochemical characterization of protein interactomes; and Mib1 analysis provides a comprehensive interactome for investigating its role in signaling networks and neuronal development. PMID:25931508

  11. A Role of Endogenous Progesterone in Stroke Cerebroprotection Revealed by the Neural-Specific Deletion of Its Intracellular Receptors.

    Science.gov (United States)

    Zhu, Xiaoyan; Fréchou, Magalie; Liere, Philippe; Zhang, Shaodong; Pianos, Antoine; Fernandez, Neïké; Denier, Christian; Mattern, Claudia; Schumacher, Michael; Guennoun, Rachida

    2017-11-08

    Treatment with progesterone protects the male and female brain against damage after middle cerebral artery occlusion (MCAO). However, in both sexes, the brain contains significant amounts of endogenous progesterone. It is not known whether endogenously produced progesterone enhances the resistance of the brain to ischemic insult. Here, we used steroid profiling by gas chromatography-tandem mass spectrometry (GC-MS/MS) for exploring adaptive and sex-specific changes in brain levels of progesterone and its metabolites after MCAO. We show that, in the male mouse brain, progesterone is mainly metabolized via 5α-reduction leading to 5α-dihydroprogesterone (5α-DHP), also a progesterone receptor (PR) agonist ligand in neural cells, then to 3α,5α-tetrahydroprogesterone (3α,5α-THP). In the female mouse brain, levels of 5α-DHP and 3α,5α-THP are lower and levels of 20α-DHP are higher than in males. After MCAO, levels of progesterone and 5α-DHP are upregulated rapidly to pregnancy-like levels in the male but not in the female brain. To assess whether endogenous progesterone and 5α-DHP contribute to the resistance of neural cells to ischemic damage, we inactivated PR selectively in the CNS. Deletion of PR in the brain reduced its resistance to MCAO, resulting in increased infarct volumes and neurological deficits in both sexes. Importantly, endogenous PR ligands continue to protect the brain of aging mice. These results uncover the unexpected importance of endogenous progesterone and its metabolites in cerebroprotection. They also reveal that the female reproductive hormone progesterone is an endogenous cerebroprotective neurosteroid in both sexes. SIGNIFICANCE STATEMENT The brain responds to injury with protective signaling and has a remarkable capacity to protect itself. We show here that, in response to ischemic stroke, levels of progesterone and its neuroactive metabolite 5α-dihydroprogesterone are upregulated rapidly in the male mouse brain but not in the

  12. Proteomic analysis of the crayfish gastrolith chitinous extracellular matrix reveals putative protein complexes and a central role for GAP 65.

    Science.gov (United States)

    Glazer, Lilah; Roth, Ziv; Weil, Simy; Aflalo, Eliahu D; Khalaila, Isam; Sagi, Amir

    2015-10-14

    than crystalline calcite. The biological mechanism for the stabilization of a naturally unstable, but at the same time biologically highly available, calcium carbonate polymorph is of great interest from the pharmaceutical point of view. (3) The gastrolith organic matrix is based on a highly structured chitin network that interacts with a variety of substances. This biologically manipulated, biodegradable structure is in itself of biotechnological and pharmaceutical potential. A growing body of evidence indicates that proteins play central roles in all above aspects of gastrolith construction. This study offers the first comprehensive screening of gastrolith proteins, and we believe that the analysis presented in this work can not only help reveal basic biological questions regarding assembly of mineralized and non-mineralized cuticular structures, but may also serve as basis for applied research in the fields of agriculture (e.g. cuticle-based pest management), health (e.g. bioavailable calcium supplements and biodegradable drug carriers) and materials science (e.g. non-toxic scaffolds for water purification). Copyright © 2015. Published by Elsevier B.V.

  13. Review of extended producer responsibility: A case study approach.

    Science.gov (United States)

    Gupt, Yamini; Sahay, Samraj

    2015-07-01

    Principles of extended producer responsibility have been the core of most of the recent policies and legislation dealing with the end-of-life management of recyclable goods. This article makes an exploratory review of 27 cases of extended producer responsibility from developed and developing economies with and without informal recycling, to ascertain the most important aspect of extended producer responsibility. A comparative analysis of the cases with respect to role of stakeholders in the upstream and downstream stages of the extended producer responsibility has been carried out. Further, the study uses exploratory factor analysis to determine the important aspects of the extended producer responsibility in practice using 13 variables identified from the review. Findings of the comparative analysis reveal that financial responsibility of the producers and separate collecting and recycling agencies contributed significantly to the success of the extended producer responsibility-based environmental policies. Regulatory provisions, take-back responsibility and financial flow come out to be the three most important aspects of the extended producer responsibility. Presence of informal sector had a negative impact on the regulatory provisions. The outcomes of this study could serve as a guideline for designing of effective extended producer responsibility-based policies. © The Author(s) 2015.

  14. The Extended Theory of Planned Behaviour and College Grades: The Role of Cognition and Past Behaviour in the Prediction of Students' Academic Intentions and Achievements

    Science.gov (United States)

    Kovac, Velibor Bobo; Cameron, David Lansing; Høigaard, Rune

    2016-01-01

    Understanding the underlying processes influencing college students' academic achievement represents an important goal of educational research. The aim of the present study was to examine the utility of the extended Theory of Planned Behaviour (TPB) and the relative influence of cognitive processes and measures of past behaviour in the prediction…

  15. Extended Theories of Gravity

    International Nuclear Information System (INIS)

    Capozziello, Salvatore; De Laurentis, Mariafelicia

    2011-01-01

    Extended Theories of Gravity can be considered as a new paradigm to cure shortcomings of General Relativity at infrared and ultraviolet scales. They are an approach that, by preserving the undoubtedly positive results of Einstein’s theory, is aimed to address conceptual and experimental problems recently emerged in astrophysics, cosmology and High Energy Physics. In particular, the goal is to encompass, in a self-consistent scheme, problems like inflation, dark energy, dark matter, large scale structure and, first of all, to give at least an effective description of Quantum Gravity. We review the basic principles that any gravitational theory has to follow. The geometrical interpretation is discussed in a broad perspective in order to highlight the basic assumptions of General Relativity and its possible extensions in the general framework of gauge theories. Principles of such modifications are presented, focusing on specific classes of theories like f(R)-gravity and scalar–tensor gravity in the metric and Palatini approaches. The special role of torsion is also discussed. The conceptual features of these theories are fully explored and attention is paid to the issues of dynamical and conformal equivalence between them considering also the initial value problem. A number of viability criteria are presented considering the post-Newtonian and the post-Minkowskian limits. In particular, we discuss the problems of neutrino oscillations and gravitational waves in extended gravity. Finally, future perspectives of extended gravity are considered with possibility to go beyond a trial and error approach.

  16. Extended quantum mechanics

    International Nuclear Information System (INIS)

    Pavel Bona

    2000-01-01

    The work can be considered as an essay on mathematical and conceptual structure of nonrelativistic quantum mechanics which is related here to some other (more general, but also to more special and 'approximative') theories. Quantum mechanics is here primarily reformulated in an equivalent form of a Poisson system on the phase space consisting of density matrices, where the 'observables', as well as 'symmetry generators' are represented by a specific type of real valued (densely defined) functions, namely the usual quantum expectations of corresponding selfjoint operators. It is shown in this paper that inclusion of additional ('nonlinear') symmetry generators (i. e. 'Hamiltonians') into this reformulation of (linear) quantum mechanics leads to a considerable extension of the theory: two kinds of quantum 'mixed states' should be distinguished, and operator - valued functions of density matrices should be used in the role of 'nonlinear observables'. A general framework for physical theories is obtained in this way: By different choices of the sets of 'nonlinear observables' we obtain, as special cases, e.g. classical mechanics on homogeneous spaces of kinematical symmetry groups, standard (linear) quantum mechanics, or nonlinear extensions of quantum mechanics; also various 'quasiclassical approximations' to quantum mechanics are all sub theories of the presented extension of quantum mechanics - a version of the extended quantum mechanics. A general interpretation scheme of extended quantum mechanics extending the usual statistical interpretation of quantum mechanics is also proposed. Eventually, extended quantum mechanics is shown to be (included into) a C * -algebraic (hence linear) quantum theory. Mathematical formulation of these theories is presented. The presentation includes an analysis of problems connected with differentiation on infinite-dimensional manifolds, as well as a solution of some problems connected with the work with only densely defined unbounded

  17. Reconstruction of the yeast Snf1 kinase regulatory network reveals its role as a global energy regulator

    DEFF Research Database (Denmark)

    Usaite, Renata; Jewett, Michael Christopher; Soberano de Oliveira, Ana Paula

    2009-01-01

    Highly conserved among eukaryotic cells, the AMP-activated kinase (AMPK) is a central regulator of carbon metabolism. To map the complete network of interactions around AMPK in yeast (Snf1) and to evaluate the role of its regulatory subunit Snf4, we measured global mRNA, protein and metabolite...

  18. An Oncogenic Role for Alternative NF-κB Signaling in DLBCL Revealed upon Deregulated BCL6 Expression

    Directory of Open Access Journals (Sweden)

    Baochun Zhang

    2015-05-01

    Full Text Available Diffuse large B cell lymphoma (DLBCL is a complex disease comprising diverse subtypes and genetic profiles. Possibly because of the prevalence of genetic alterations activating canonical NF-κB activity, a role for oncogenic lesions that activate the alternative NF-κB pathway in DLBCL has remained elusive. Here, we show that deletion/mutation of TRAF3, a negative regulator of the alternative NF-κB pathway, occurs in ∼15% of DLBCLs and that it often coexists with BCL6 translocation, which prevents terminal B cell differentiation. Accordingly, in a mouse model constitutive activation of the alternative NF-κB pathway cooperates with BCL6 deregulation in DLBCL development. This work demonstrates a key oncogenic role for the alternative NF-κB pathway in DLBCL development.

  19. In trans complementation of lethal factor reveal roles in colonization and dissemination in a murine mouse model.

    Directory of Open Access Journals (Sweden)

    David E Lowe

    Full Text Available Lethal factor (LF is a component of the B. anthracis exotoxin and critical for pathogenesis. The roles of LF in early anthrax pathogenesis, such as colonization and dissemination from the initial site of infection, are poorly understood. In mice models of infection, LF-deficient strains either have altered dissemination patterns or do not colonize, precluding analysis of the role of LF in colonization and dissemination from the portal of entry. Previous reports indicate rabbit and guinea pig models infected with LF-deficient strains have decreased virulence, yet the inability to use bioluminescent imaging techniques to track B. anthracis growth and dissemination in these hosts makes analysis of early pathogenesis challenging. In this study, the roles of LF early in infection were analyzed using bioluminescent signature tagged libraries of B. anthracis with varying ratios of LF-producing and LF-deficient clones. Populations where all clones produced LF and populations where only 40% of clones produce LF were equally virulent. The 40% LF-producing clones trans complimented the LF mutants and permitted them to colonize and disseminate. Decreases of the LF producing strains to 10% or 0.3% of the population led to increased host survival and decreased trans complementation of the LF mutants. A library with 10% LF producing clones could replicate and disseminate, but fewer clones disseminated and the mutant clones were less competitive than wild type. The inoculum with 0.3% LF producing clones could not colonize the host. This strongly suggests that between 10% and 0.3% of the population must produce LF in order to colonize. In total, these findings suggest that a threshold of LF must be produced in order for colonization and dissemination to occur in vivo. These observations suggest that LF has a major role in the early stages of colonization and dissemination.

  20. Reconstruction of the yeast Snf1 kinase regulatory network reveals its role as a global energy regulator

    Science.gov (United States)

    Usaite, Renata; Jewett, Michael C; Oliveira, Ana Paula; Yates, John R; Olsson, Lisbeth; Nielsen, Jens

    2009-01-01

    Highly conserved among eukaryotic cells, the AMP-activated kinase (AMPK) is a central regulator of carbon metabolism. To map the complete network of interactions around AMPK in yeast (Snf1) and to evaluate the role of its regulatory subunit Snf4, we measured global mRNA, protein and metabolite levels in wild type, Δsnf1, Δsnf4, and Δsnf1Δsnf4 knockout strains. Using four newly developed computational tools, including novel DOGMA sub-network analysis, we showed the benefits of three-level ome-data integration to uncover the global Snf1 kinase role in yeast. We for the first time identified Snf1's global regulation on gene and protein expression levels, and showed that yeast Snf1 has a far more extensive function in controlling energy metabolism than reported earlier. Additionally, we identified complementary roles of Snf1 and Snf4. Similar to the function of AMPK in humans, our findings showed that Snf1 is a low-energy checkpoint and that yeast can be used more extensively as a model system for studying the molecular mechanisms underlying the global regulation of AMPK in mammals, failure of which leads to metabolic diseases. PMID:19888214

  1. Repetitive transcranial magnetic stimulation reveals a role for the left inferior parietal lobule in matching observed kinematics during imitation.

    Science.gov (United States)

    Reader, Arran T; Royce, Ben P; Marsh, Jade E; Chivers, Katy-Jayne; Holmes, Nicholas P

    2018-04-01

    Apraxia (a disorder of complex movement) suggests that the left inferior parietal lobule (IPL) plays a role in kinematic or spatial aspects of imitation, which may be particularly important for meaningless (i.e. unfamiliar intransitive) actions. Mirror neuron theories indicate that the IPL is part of a frontoparietal system that can support imitation by linking observed and stored actions through visuomotor matching, and have less to say about different subregions of the left IPL, or how different types of action (i.e. meaningful or meaningless) are processed for imitation. We used repetitive transcranial magnetic stimulation (rTMS) to bridge this gap and better understand the roles of the left supramarginal gyrus (SMG) and left angular gyrus (AG) in imitation. We also examined whether these areas are differentially involved in meaningful and meaningless action imitation. We applied rTMS over the left SMG, over the left AG or during a no-rTMS baseline condition, and then asked participants to imitate a confederate's actions whilst the arm and hand movements of both individuals were motion-tracked. rTMS over both the left SMG and the left AG reduced the velocity of participants' finger movements relative to the actor during imitation of finger gestures, regardless of action meaning. Our results support recent claims in apraxia and confirm a role for the left IPL in kinematic processing during gesture imitation, regardless of action meaning. © 2018 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  2. A Combined Optogenetic-Knockdown Strategy Reveals a Major Role of Tomosyn in Mossy Fiber Synaptic Plasticity

    Directory of Open Access Journals (Sweden)

    Yoav Ben-Simon

    2015-07-01

    Full Text Available Neurotransmitter release probability (Pr largely determines the dynamic properties of synapses. While much is known about the role of presynaptic proteins in transmitter release, their specific contribution to synaptic plasticity is unclear. One such protein, tomosyn, is believed to reduce Pr by interfering with the SNARE complex formation. Tomosyn is enriched at hippocampal mossy fiber-to-CA3 pyramidal cell synapses (MF-CA3, which characteristically exhibit low Pr, strong synaptic facilitation, and pre-synaptic protein kinase A (PKA-dependent long-term potentiation (LTP. To evaluate tomosyn’s role in MF-CA3 function, we used a combined knockdown (KD-optogenetic strategy whereby presynaptic neurons with reduced tomosyn levels were selectively activated by light. Using this approach in mouse hippocampal slices, we found that facilitation, LTP, and PKA-induced potentiation were significantly impaired at tomosyn-deficient synapses. These findings not only indicate that tomosyn is a key regulator of MF-CA3 plasticity but also highlight the power of a combined KD-optogenetic approach to determine the role of presynaptic proteins.

  3. Genetic and Imaging Approaches Reveal Pro-Inflammatory and Immunoregulatory Roles of Mast Cells in Contact Hypersensitivity

    Directory of Open Access Journals (Sweden)

    Nicolas Gaudenzio

    2018-06-01

    Full Text Available Contact hypersensitivity (CHS is a common T cell-mediated skin disease induced by epicutaneous sensitization to haptens. Mast cells (MCs are widely deployed in the skin and can be activated during CHS responses to secrete diverse products, including some with pro-inflammatory and anti-inflammatory functions. Conflicting results have been obtained regarding pathogenic versus protective roles of MCs in CHS, and this has been attributed in part to the limitations of certain models for studying MC functions in vivo. This review discusses recent advances in the development and analysis of mouse models to investigate the roles of MCs and MC-associated products in vivo. Notably, fluorescent avidin-based two-photon imaging approaches enable in vivo selective labeling and simultaneous tracking of MC secretory granules (e.g., during MC degranulation and MC gene activation by real-time longitudinal intravital microscopy in living mice. The combination of such genetic and imaging tools has shed new light on the controversial role played by MCs in mouse models of CHS. On the one hand, they can amplify CHS responses of mild severity while, on the other hand, can limit the inflammation and tissue injury associated with more severe or chronic models, in part by representing an initial source of the anti-inflammatory cytokine IL-10.

  4. Failure to Burrow and Tunnel Reveals Roles for jim lovell in the Growth and Endoreplication of the Drosophila Larval Tracheae.

    Directory of Open Access Journals (Sweden)

    Fanli Zhou

    Full Text Available The Drosophila protein Jim Lovell (Lov is a putative transcription factor of the BTB/POZ (Bric- a-Brac/Tramtrack/Broad/ Pox virus and Zinc finger domain class that is expressed in many elements of the developing larval nervous system. It has roles in innate behaviors such as larval locomotion and adult courtship. In performing tissue-specific knockdown with the Gal4-UAS system we identified a new behavioral phenotype for lov: larvae failed to burrow into their food during their growth phase and then failed to tunnel into an agarose substratum during their wandering phase. We determined that these phenotypes originate in a previously unrecognized role for lov in the tracheae. By using tracheal-specific Gal4 lines, Lov immunolocalization and a lov enhancer trap line, we established that lov is normally expressed in the tracheae from late in embryogenesis through larval life. Using an assay that monitors food burrowing, substrate tunneling and death we showed that lov tracheal knockdown results in tracheal fluid-filling, producing hypoxia that activates the aberrant behaviors and inhibits development. We investigated the role of lov in the tracheae that initiates this sequence of events. We discovered that when lov levels are reduced, the tracheal cells are smaller, more numerous and show lower levels of endopolyploidization. Together our findings indicate that Lov is necessary for tracheal endoreplicative growth and that its loss in this tissue causes loss of tracheal integrity resulting in chronic hypoxia and abnormal burrowing and tunneling behavior.

  5. Genome-wide identification of Bcl11b gene targets reveals role in brain-derived neurotrophic factor signaling.

    Directory of Open Access Journals (Sweden)

    Bin Tang

    Full Text Available B-cell leukemia/lymphoma 11B (Bcl11b is a transcription factor showing predominant expression in the striatum. To date, there are no known gene targets of Bcl11b in the nervous system. Here, we define targets for Bcl11b in striatal cells by performing chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq in combination with genome-wide expression profiling. Transcriptome-wide analysis revealed that 694 genes were significantly altered in striatal cells over-expressing Bcl11b, including genes showing striatal-enriched expression similar to Bcl11b. ChIP-seq analysis demonstrated that Bcl11b bound a mixture of coding and non-coding sequences that were within 10 kb of the transcription start site of an annotated gene. Integrating all ChIP-seq hits with the microarray expression data, 248 direct targets of Bcl11b were identified. Functional analysis on the integrated gene target list identified several zinc-finger encoding genes as Bcl11b targets, and further revealed a significant association of Bcl11b to brain-derived neurotrophic factor/neurotrophin signaling. Analysis of ChIP-seq binding regions revealed significant consensus DNA binding motifs for Bcl11b. These data implicate Bcl11b as a novel regulator of the BDNF signaling pathway, which is disrupted in many neurological disorders. Specific targeting of the Bcl11b-DNA interaction could represent a novel therapeutic approach to lowering BDNF signaling specifically in striatal cells.

  6. Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity

    Science.gov (United States)

    Schoggins, John W.; MacDuff, Donna A.; Imanaka, Naoko; Gainey, Maria D.; Shrestha, Bimmi; Eitson, Jennifer L.; Mar, Katrina B.; Richardson, R. Blake; Ratushny, Alexander V.; Litvak, Vladimir; Dabelic, Rea; Manicassamy, Balaji; Aitchison, John D.; Aderem, Alan; Elliott, Richard M.; García-Sastre, Adolfo; Racaniello, Vincent; Snijder, Eric J.; Yokoyama, Wayne M.; Diamond, Michael S.; Virgin, Herbert W.; Rice, Charles M.

    2014-01-01

    The type I interferon (IFN) response protects cells from viral infection by inducing hundreds of interferon-stimulated genes (ISGs), some of which encode direct antiviral effectors. Recent screening studies have begun to catalogue ISGs with antiviral activity against several RNA and DNA viruses. However, antiviral ISG specificity across multiple distinct classes of viruses remains largely unexplored. Here we used an ectopic expression assay to screen a library of more than 350 human ISGs for effects on 14 viruses representing 7 families and 11 genera. We show that 47 genes inhibit one or more viruses, and 25 genes enhance virus infectivity. Comparative analysis reveals that the screened ISGs target positive-sense single-stranded RNA viruses more effectively than negative-sense single-stranded RNA viruses. Gene clustering highlights the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS, also known as MB21D1) as a gene whose expression also broadly inhibits several RNA viruses. In vitro, lentiviral delivery of enzymatically active cGAS triggers a STING-dependent, IRF3-mediated antiviral program that functions independently of canonical IFN/STAT1 signalling. In vivo, genetic ablation of murine cGAS reveals its requirement in the antiviral response to two DNA viruses, and an unappreciated contribution to the innate control of an RNA virus. These studies uncover new paradigms for the preferential specificity of IFN-mediated antiviral pathways spanning several virus families.

  7. A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function.

    Science.gov (United States)

    Okray, Zeynep; de Esch, Celine E F; Van Esch, Hilde; Devriendt, Koen; Claeys, Annelies; Yan, Jiekun; Verbeeck, Jelle; Froyen, Guy; Willemsen, Rob; de Vrij, Femke M S; Hassan, Bassem A

    2015-04-01

    Loss of function of the FMR1 gene leads to fragile X syndrome (FXS), the most common form of intellectual disability. The loss of FMR1 function is usually caused by epigenetic silencing of the FMR1 promoter leading to expansion and subsequent methylation of a CGG repeat in the 5' untranslated region. Very few coding sequence variations have been experimentally characterized and shown to be causal to the disease. Here, we describe a novel FMR1 mutation and reveal an unexpected nuclear export function for the C-terminus of FMRP. We screened a cohort of patients with typical FXS symptoms who tested negative for CGG repeat expansion in the FMR1 locus. In one patient, we identified a guanine insertion in FMR1 exon 15. This mutation alters the open reading frame creating a short novel C-terminal sequence, followed by a stop codon. We find that this novel peptide encodes a functional nuclear localization signal (NLS) targeting the patient FMRP to the nucleolus in human cells. We also reveal an evolutionarily conserved nuclear export function associated with the endogenous C-terminus of FMRP. In vivo analyses in Drosophila demonstrate that a patient-mimetic mutation alters the localization and function of Dfmrp in neurons, leading to neomorphic neuronal phenotypes. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

  8. A genetic screen for vascular mutants in zebrafish reveals dynamic roles for Vegf/Plcg1 signaling during artery development.

    Science.gov (United States)

    Covassin, L D; Siekmann, A F; Kacergis, M C; Laver, E; Moore, J C; Villefranc, J A; Weinstein, B M; Lawson, N D

    2009-05-15

    In this work we describe a forward genetic approach to identify mutations that affect blood vessel development in the zebrafish. By applying a haploid screening strategy in a transgenic background that allows direct visualization of blood vessels, it was possible to identify several classes of mutant vascular phenotypes. Subsequent characterization of mutant lines revealed that defects in Vascular endothelial growth factor (Vegf) signaling specifically affected artery development. Comparison of phenotypes associated with different mutations within a functional zebrafish Vegf receptor-2 ortholog (referred to as kdr-like, kdrl) revealed surprisingly varied effects on vascular development. In parallel, we identified an allelic series of mutations in phospholipase c gamma 1 (plcg1). Together with in vivo structure-function analysis, our results suggest a requirement for Plcg1 catalytic activity downstream of receptor tyrosine kinases. We further find that embryos lacking both maternal and zygotic plcg1 display more severe defects in artery differentiation but are otherwise similar to zygotic mutants. Finally, we demonstrate through mosaic analysis that plcg1 functions autonomously in endothelial cells. Together our genetic analyses suggest that Vegf/Plcg1 signaling acts at multiple time points and in different signaling contexts to mediate distinct aspects of artery development.

  9. Crystal structures of the CPAP/STIL complex reveal its role in centriole assembly and human microcephaly

    Science.gov (United States)

    Cottee, Matthew A; Muschalik, Nadine; Wong, Yao Liang; Johnson, Christopher M; Johnson, Steven; Andreeva, Antonina; Oegema, Karen; Lea, Susan M; Raff, Jordan W; van Breugel, Mark

    2013-01-01

    Centrioles organise centrosomes and template cilia and flagella. Several centriole and centrosome proteins have been linked to microcephaly (MCPH), a neuro-developmental disease associated with small brain size. CPAP (MCPH6) and STIL (MCPH7) are required for centriole assembly, but it is unclear how mutations in them lead to microcephaly. We show that the TCP domain of CPAP constitutes a novel proline recognition domain that forms a 1:1 complex with a short, highly conserved target motif in STIL. Crystal structures of this complex reveal an unusual, all-β structure adopted by the TCP domain and explain how a microcephaly mutation in CPAP compromises complex formation. Through point mutations, we demonstrate that complex formation is essential for centriole duplication in vivo. Our studies provide the first structural insight into how the malfunction of centriole proteins results in human disease and also reveal that the CPAP–STIL interaction constitutes a conserved key step in centriole biogenesis. DOI: http://dx.doi.org/10.7554/eLife.01071.001 PMID:24052813

  10. Multiple roles of integrin-linked kinase in epidermal development, maturation and pigmentation revealed by molecular profiling.

    Directory of Open Access Journals (Sweden)

    David Judah

    Full Text Available Integrin-linked kinase (ILK is an important scaffold protein that mediates a variety of cellular responses to integrin stimulation by extracellular matrix proteins. Mice with epidermis-restricted inactivation of the Ilk gene exhibit pleiotropic phenotypic defects, including impaired hair follicle morphogenesis, reduced epidermal adhesion to the basement membrane, compromised epidermal integrity, as well as wasting and failure to thrive leading to perinatal death. To better understand the underlying molecular mechanisms that cause such a broad range of alterations, we investigated the impact of Ilk gene inactivation on the epidermis transcriptome. Microarray analysis showed over 700 differentially regulated mRNAs encoding proteins involved in multiple aspects of epidermal function, including keratinocyte differentiation and barrier formation, inflammation, regeneration after injury, and fundamental epidermal developmental pathways. These studies also revealed potential effects on genes not previously implicated in ILK functions, including those important for melanocyte and melanoblast development and function, regulation of cytoskeletal dynamics, and homeobox genes. This study shows that ILK is a critical regulator of multiple aspects of epidermal function and homeostasis, and reveals the previously unreported involvement of ILK not only in epidermal differentiation and barrier formation, but also in melanocyte genesis and function.

  11. Multiple roles of integrin-linked kinase in epidermal development, maturation and pigmentation revealed by molecular profiling.

    Science.gov (United States)

    Judah, David; Rudkouskaya, Alena; Wilson, Ryan; Carter, David E; Dagnino, Lina

    2012-01-01

    Integrin-linked kinase (ILK) is an important scaffold protein that mediates a variety of cellular responses to integrin stimulation by extracellular matrix proteins. Mice with epidermis-restricted inactivation of the Ilk gene exhibit pleiotropic phenotypic defects, including impaired hair follicle morphogenesis, reduced epidermal adhesion to the basement membrane, compromised epidermal integrity, as well as wasting and failure to thrive leading to perinatal death. To better understand the underlying molecular mechanisms that cause such a broad range of alterations, we investigated the impact of Ilk gene inactivation on the epidermis transcriptome. Microarray analysis showed over 700 differentially regulated mRNAs encoding proteins involved in multiple aspects of epidermal function, including keratinocyte differentiation and barrier formation, inflammation, regeneration after injury, and fundamental epidermal developmental pathways. These studies also revealed potential effects on genes not previously implicated in ILK functions, including those important for melanocyte and melanoblast development and function, regulation of cytoskeletal dynamics, and homeobox genes. This study shows that ILK is a critical regulator of multiple aspects of epidermal function and homeostasis, and reveals the previously unreported involvement of ILK not only in epidermal differentiation and barrier formation, but also in melanocyte genesis and function.

  12. The extracellular proteome of Bifidobacterium animalis subsp. lactis BB‐12 reveals proteins with putative roles in probiotic effects

    DEFF Research Database (Denmark)

    Gilad, Ofir; Svensson, Birte; Viborg, Alexander Holm

    2011-01-01

    Probiotics are live microorganisms that exert health‐promoting effects on the human host, as demonstrated for numerous strains of the genus Bifidobacterium. To unravel the proteins involved in the interactions between the host and the extensively used and well‐studied probiotic strain Bifidobacte......Probiotics are live microorganisms that exert health‐promoting effects on the human host, as demonstrated for numerous strains of the genus Bifidobacterium. To unravel the proteins involved in the interactions between the host and the extensively used and well‐studied probiotic strain...... Bifidobacterium animalis subsp. lactis BB‐12, proteins secreted by the bacterium, i.e. belonging to the extracellular proteome present in the culture medium, were identified by 2‐DE coupled with MALDI‐TOF MS. Among the 74 distinct proteins identified, 31 are predicted to carry out their physiological role either...... functions include binding of plasminogen, formation of fimbriae, adhesion to collagen, attachment to mucin and intestinal cells as well as induction of immunomodulative response. These findings suggest a role of the proteins in colonization of the gastrointestinal tract, adhesion to host tissues...

  13. Comprehensive functional characterization of the glycoside hydrolase family 3 enzymes from Cellvibrio japonicus reveals unique metabolic roles in biomass saccharification.

    Science.gov (United States)

    Nelson, Cassandra E; Attia, Mohamed A; Rogowski, Artur; Morland, Carl; Brumer, Harry; Gardner, Jeffrey G

    2017-12-01

    Lignocellulose degradation is central to the carbon cycle and renewable biotechnologies. The xyloglucan (XyG), β(1→3)/β(1→4) mixed-linkage glucan (MLG) and β(1→3) glucan components of lignocellulose represent significant carbohydrate energy sources for saprophytic microorganisms. The bacterium Cellvibrio japonicus has a robust capacity for plant polysaccharide degradation, due to a genome encoding a large contingent of Carbohydrate-Active enZymes (CAZymes), many of whose specific functions remain unknown. Using a comprehensive genetic and biochemical approach, we have delineated the physiological roles of the four C. japonicus glycoside hydrolase family 3 (GH3) members on diverse β-glucans. Despite high protein sequence similarity and partially overlapping activity profiles on disaccharides, these β-glucosidases are not functionally equivalent. Bgl3A has a major role in MLG and sophorose utilization, and supports β(1→3) glucan utilization, while Bgl3B underpins cellulose utilization and supports MLG utilization. Bgl3C drives β(1→3) glucan utilization. Finally, Bgl3D is the crucial β-glucosidase for XyG utilization. This study not only sheds the light on the metabolic machinery of C. japonicus, but also expands the repertoire of characterized CAZymes for future deployment in biotechnological applications. In particular, the precise functional analysis provided here serves as a reference for informed bioinformatics on the genomes of other Cellvibrio and related species. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  14. Global gene expression analysis of canine osteosarcoma stem cells reveals a novel role for COX-2 in tumour initiation.

    Science.gov (United States)

    Pang, Lisa Y; Gatenby, Emma L; Kamida, Ayako; Whitelaw, Bruce A; Hupp, Ted R; Argyle, David J

    2014-01-01

    Osteosarcoma is the most common primary bone tumour of both children and dogs. It is an aggressive tumour in both species with a rapid clinical course leading ultimately to metastasis. In dogs and children distant metastasis occurs in >80% of individuals treated by surgery alone. Both canine and human osteosarcoma has been shown to contain a sub-population of cancer stem cells (CSCs), which may drive tumour growth, recurrence and metastasis, suggesting that naturally occurring canine osteosarcoma could act as a preclinical model for the human disease. Here we report the successful isolation of CSCs from primary canine osteosarcoma, as well as established cell lines. We show that these cells can form tumourspheres, and demonstrate relative resistance to chemotherapy. We demonstrate similar results for the human osteosarcma cell lines, U2OS and SAOS2. Utilizing the Affymetrix canine microarray, we are able to definitively show that there are significant differences in global gene expression profiles of isolated osteosarcoma stem cells and the daughter adherent cells. We identified 13,221 significant differences (p = 0.05), and significantly, COX-2 was expressed 141-fold more in CSC spheres than daughter adherent cells. To study the role of COX-2 expression in CSCs we utilized the COX-2 inhibitors meloxicam and mavacoxib. We found that COX-2 inhibition had no effect on CSC growth, or resistance to chemotherapy. However inhibition of COX-2 in daughter cells prevented sphere formation, indicating a potential significant role for COX-2 in tumour initiation.

  15. Global gene expression analysis of canine osteosarcoma stem cells reveals a novel role for COX-2 in tumour initiation.

    Directory of Open Access Journals (Sweden)

    Lisa Y Pang

    Full Text Available Osteosarcoma is the most common primary bone tumour of both children and dogs. It is an aggressive tumour in both species with a rapid clinical course leading ultimately to metastasis. In dogs and children distant metastasis occurs in >80% of individuals treated by surgery alone. Both canine and human osteosarcoma has been shown to contain a sub-population of cancer stem cells (CSCs, which may drive tumour growth, recurrence and metastasis, suggesting that naturally occurring canine osteosarcoma could act as a preclinical model for the human disease. Here we report the successful isolation of CSCs from primary canine osteosarcoma, as well as established cell lines. We show that these cells can form tumourspheres, and demonstrate relative resistance to chemotherapy. We demonstrate similar results for the human osteosarcma cell lines, U2OS and SAOS2. Utilizing the Affymetrix canine microarray, we are able to definitively show that there are significant differences in global gene expression profiles of isolated osteosarcoma stem cells and the daughter adherent cells. We identified 13,221 significant differences (p = 0.05, and significantly, COX-2 was expressed 141-fold more in CSC spheres than daughter adherent cells. To study the role of COX-2 expression in CSCs we utilized the COX-2 inhibitors meloxicam and mavacoxib. We found that COX-2 inhibition had no effect on CSC growth, or resistance to chemotherapy. However inhibition of COX-2 in daughter cells prevented sphere formation, indicating a potential significant role for COX-2 in tumour initiation.

  16. Comprehensive functional characterization of the Glycoside Hydrolase Family 3 enzymes from Cellvibrio japonicus reveals unique metabolic roles in biomass saccharification

    International Nuclear Information System (INIS)

    Nelson, Cassandra E.; Attia, Mohamed A.; Rogowski, Artur; Morland, Carl; Brumer, Harry; Gardner, Jeffrey G.

    2017-01-01

    Here, lignocellulose degradation is central to the carbon cycle and renewable biotechnologies. The xyloglucan (XyG), β(1!3)/β(1!4) mixed-linkage glucan (MLG), and β(1!3) glucan components of lignocellulose represent significant carbohydrate energy sources for saprophytic microorganisms. The bacterium Cellvibrio japonicus has a robust capacity for plant polysaccharide degradation, due to a genome encoding a large contingent of Carbohydrate-Active Enzymes (CAZymes), many of whose specific functions remain unknown. Using a comprehensive genetic and biochemical approach we have delineated the physiological roles of the four C. japonicus Glycoside Hydrolase Family 3 (GH3) members on diverse β-glucans. Despite high protein sequence similarity and partially overlapping activity profiles on disaccharides, these β-glucosidases are not functionally equivalent. Bgl3A has a major role in MLG and sophorose utilization, and supports β(1!3) glucan utilization, while Bgl3B underpins cellulose utilization and supports MLG utilization. Bgl3C drives β(1!3) glucan utilization. Finally, Bgl3D is the crucial β-glucosidase for XyG utilization. This study not only sheds the light on the metabolic machinery of C. japonicus, but also expands the repertoire of characterized CAZymes for future deployment in biotechnological applications. In particular, the precise functional analysis provided here serves as a reference for informed bioinformatics on the genomes of other Cellvibrio and related species.

  17. Polynomial algebra reveals diverging roles of the unfolded protein response in endothelial cells during ischemia-reperfusion injury.

    Science.gov (United States)

    Le Pape, Sylvain; Dimitrova, Elena; Hannaert, Patrick; Konovalov, Alexander; Volmer, Romain; Ron, David; Thuillier, Raphaël; Hauet, Thierry

    2014-08-25

    The unfolded protein response (UPR)--the endoplasmic reticulum stress response--is found in various pathologies including ischemia-reperfusion injury (IRI). However, its role during IRI is still unclear. Here, by combining two different bioinformatical methods--a method based on ordinary differential equations (Time Series Network Inference) and an algebraic method (probabilistic polynomial dynamical systems)--we identified the IRE1α-XBP1 and the ATF6 pathways as the main UPR effectors involved in cell's adaptation to IRI. We validated these findings experimentally by assessing the impact of their knock-out and knock-down on cell survival during IRI. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  18. Metagenomic binning reveals the functional roles of core abundant microorganisms in twelve full-scale biogas plants

    DEFF Research Database (Denmark)

    Campanaro, Stefano; Treu, Laura; Kougias, Panagiotis

    2018-01-01

    and environmental factors on MAGs abundance and to investigate the methanogenic performance of the biogas plants. Prediction of the functional properties of MAGs was obtained analyzing their KEGG pathways and their carbohydrate active domains. Network analysis allowed investigation of species-species associations......The aim of this work was to elucidate the microbial ecology in twelve mesophilic and thermophilic full-scale biogas plants using a genome-centric metagenomic approach. In this study both biogas plants treating manure and those treating sludge from waste water treatment plants were considered...... and shed light on syntrophic interactions between members belonging to the anaerobic digestion dark matter (phylum Fermentibacteria). By stratifying and comparing different levels of information, it was predicted that some MAGs have a crucial role in the manure-supplemented thermophilic biogas plants...

  19. Molecular Characterization of Down Syndrome Embryonic Stem Cells Reveals a Role for RUNX1 in Neural Differentiation

    Directory of Open Access Journals (Sweden)

    Tomer Halevy

    2016-10-01

    Full Text Available Down syndrome (DS is the leading genetic cause of mental retardation and is caused by a third copy of human chromosome 21. The different pathologies of DS involve many tissues with a distinct array of neural phenotypes. Here we characterize embryonic stem cell lines with DS (DS-ESCs, and focus on the neural aspects of the disease. Our results show that neural progenitor cells (NPCs differentiated from five independent DS-ESC lines display increased apoptosis and downregulation of forehead developmental genes. Analysis of differentially expressed genes suggested RUNX1 as a key transcription regulator in DS-NPCs. Using genome editing we were able to disrupt all three copies of RUNX1 in DS-ESCs, leading to downregulation of several RUNX1 target developmental genes accompanied by reduced apoptosis and neuron migration. Our work sheds light on the role of RUNX1 and the importance of dosage balance in the development of neural phenotypes in DS.

  20. Functional Roles of Neural Preparatory Processes in a Cued Stroop Task Revealed by Linking Electrophysiology with Behavioral Performance.

    Science.gov (United States)

    Wang, Chao; Ding, Mingzhou; Kluger, Benzi M

    2015-01-01

    It is well established that cuing facilitates behavioral performance and that different aspects of instructional cues evoke specific neural preparatory processes in cued task-switching paradigms. To deduce the functional role of these neural preparatory processes the majority of studies vary aspects of the experimental paradigm and describe how these variations alter markers of neural preparatory processes. Although these studies provide important insights, they also have notable limitations, particularly in terms of understanding the causal or functional relationship of neural markers to cognitive and behavioral processes. In this study, we sought to address these limitations and uncover the functional roles of neural processes by examining how variability in the amplitude of neural preparatory processes predicts behavioral performance to subsequent stimuli. To achieve this objective 16 young adults were recruited to perform a cued Stroop task while their brain activity was measured using high-density electroencephalography. Four temporally overlapping but functionally and topographically distinct cue-triggered event related potentials (ERPs) were identified: 1) A left-frontotemporal negativity (250-700 ms) that was positively associated with word-reading performance; 2) a midline-frontal negativity (450-800 ms) that was positively associated with color-naming and incongruent performance; 3) a left-frontal negativity (450-800 ms) that was positively associated with switch trial performance; and 4) a centroparietal positivity (450-800 ms) that was positively associated with performance for almost all trial types. These results suggest that at least four dissociable cognitive processes are evoked by instructional cues in the present task, including: 1) domain-specific task facilitation; 2) switch-specific task-set reconfiguration; 3) preparation for response conflict; and 4) proactive attentional control. Examining the relationship between ERPs and behavioral

  1. Functional Roles of Neural Preparatory Processes in a Cued Stroop Task Revealed by Linking Electrophysiology with Behavioral Performance.

    Directory of Open Access Journals (Sweden)

    Chao Wang

    Full Text Available It is well established that cuing facilitates behavioral performance and that different aspects of instructional cues evoke specific neural preparatory processes in cued task-switching paradigms. To deduce the functional role of these neural preparatory processes the majority of studies vary aspects of the experimental paradigm and describe how these variations alter markers of neural preparatory processes. Although these studies provide important insights, they also have notable limitations, particularly in terms of understanding the causal or functional relationship of neural markers to cognitive and behavioral processes. In this study, we sought to address these limitations and uncover the functional roles of neural processes by examining how variability in the amplitude of neural preparatory processes predicts behavioral performance to subsequent stimuli. To achieve this objective 16 young adults were recruited to perform a cued Stroop task while their brain activity was measured using high-density electroencephalography. Four temporally overlapping but functionally and topographically distinct cue-triggered event related potentials (ERPs were identified: 1 A left-frontotemporal negativity (250-700 ms that was positively associated with word-reading performance; 2 a midline-frontal negativity (450-800 ms that was positively associated with color-naming and incongruent performance; 3 a left-frontal negativity (450-800 ms that was positively associated with switch trial performance; and 4 a centroparietal positivity (450-800 ms that was positively associated with performance for almost all trial types. These results suggest that at least four dissociable cognitive processes are evoked by instructional cues in the present task, including: 1 domain-specific task facilitation; 2 switch-specific task-set reconfiguration; 3 preparation for response conflict; and 4 proactive attentional control. Examining the relationship between ERPs and behavioral

  2. Dissection of the complex phenotype in cuticular mutants of Arabidopsis reveals a role of SERRATE as a mediator.

    Directory of Open Access Journals (Sweden)

    Derry Voisin

    2009-10-01

    Full Text Available Mutations in LACERATA (LCR, FIDDLEHEAD (FDH, and BODYGUARD (BDG cause a complex developmental syndrome that is consistent with an important role for these Arabidopsis genes in cuticle biogenesis. The genesis of their pleiotropic phenotypes is, however, poorly understood. We provide evidence that neither distorted depositions of cutin, nor deficiencies in the chemical composition of cuticular lipids, account for these features, instead suggesting that the mutants alleviate the functional disorder of the cuticle by reinforcing their defenses. To better understand how plants adapt to these mutations, we performed a genome-wide gene expression analysis. We found that apparent compensatory transcriptional responses in these mutants involve the induction of wax, cutin, cell wall, and defense genes. To gain greater insight into the mechanism by which cuticular mutations trigger this response in the plants, we performed an overlap meta-analysis, which is termed MASTA (MicroArray overlap Search Tool and Analysis, of differentially expressed genes. This suggested that different cell integrity pathways are recruited in cesA cellulose synthase and cuticular mutants. Using MASTA for an in silico suppressor/enhancer screen, we identified SERRATE (SE, which encodes a protein of RNA-processing multi-protein complexes, as a likely enhancer. In confirmation of this notion, the se lcr and se bdg double mutants eradicate severe leaf deformations as well as the organ fusions that are typical of lcr and bdg and other cuticular mutants. Also, lcr does not confer resistance to Botrytis cinerea in a se mutant background. We propose that there is a role for SERRATE-mediated RNA signaling in the cuticle integrity pathway.

  3. Development of short questionnaire to measure an extended set of role expectation conflict, coworker support and work-life balance: The new job stress scale

    Directory of Open Access Journals (Sweden)

    Abhishek Shukla

    2016-12-01

    Full Text Available This study aimed to investigate the reliability and validity of a new version of job stress scale, which measures the extended set of psychosocial stressors by adding new scales to the current version of the job stress scale. Additional scales were extensively collected from theoretical job stress models and similar questionnaire from different countries. Items were tested in workplace and refined through a pilot survey (n = 400 to examine the reliability and construct validity. Most scales showed acceptable levels of internal consistency, intra-class reliability, and test–retest reliability. Factor analysis and correlation analysis showed that these scales fit the theoretical expectations. These findings provided enough evidences that the new job stress scale is reliable and valid. Although confirmatory analysis should be examined in future studies. The new job stress scale is a useful instrument for organization and academicians to evaluate job stress in modern Indian workplace.

  4. Metabolomics Reveals New Mechanisms for Pathogenesis in Barth Syndrome and Introduces Novel Roles for Cardiolipin in Cellular Function.

    Directory of Open Access Journals (Sweden)

    Yana Sandlers

    Full Text Available Barth Syndrome is the only known Mendelian disorder of cardiolipin remodeling, with characteristic clinical features of cardiomyopathy, skeletal myopathy, and neutropenia. While the primary biochemical defects of reduced mature cardiolipin and increased monolysocardiolipin are well-described, much of the downstream biochemical dysregulation has not been uncovered, and biomarkers are limited. In order to further expand upon the knowledge of the biochemical abnormalities in Barth Syndrome, we analyzed metabolite profiles in plasma from a cohort of individuals with Barth Syndrome compared to age-matched controls via 1H nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry. A clear distinction between metabolite profiles of individuals with Barth Syndrome and controls was observed, and was defined by an array of metabolite classes including amino acids and lipids. Pathway analysis of these discriminating metabolites revealed involvement of mitochondrial and extra-mitochondrial biochemical pathways including: insulin regulation of fatty acid metabolism, lipid metabolism, biogenic amine metabolism, amino acid metabolism, endothelial nitric oxide synthase signaling, and tRNA biosynthesis. Taken together, this data indicates broad metabolic dysregulation in Barth Syndrome with wide cellular effects.

  5. High-throughput cell-based screening reveals a role for ZNF131 as a repressor of ERalpha signaling

    Directory of Open Access Journals (Sweden)

    Du Peige

    2008-10-01

    Full Text Available Abstract Background Estrogen receptor α (ERα is a transcription factor whose activity is affected by multiple regulatory cofactors. In an effort to identify the human genes involved in the regulation of ERα, we constructed a high-throughput, cell-based, functional screening platform by linking a response element (ERE with a reporter gene. This allowed the cellular activity of ERα, in cells cotransfected with the candidate gene, to be quantified in the presence or absence of its cognate ligand E2. Results From a library of 570 human cDNA clones, we identified zinc finger protein 131 (ZNF131 as a repressor of ERα mediated transactivation. ZNF131 is a typical member of the BTB/POZ family of transcription factors, and shows both ubiquitous expression and a high degree of sequence conservation. The luciferase reporter gene assay revealed that ZNF131 inhibits ligand-dependent transactivation by ERα in a dose-dependent manner. Electrophoretic mobility shift assay clearly demonstrated that the interaction between ZNF131 and ERα interrupts or prevents ERα binding to the estrogen response element (ERE. In addition, ZNF131 was able to suppress the expression of pS2, an ERα target gene. Conclusion We suggest that the functional screening platform we constructed can be applied for high-throughput genomic screening candidate ERα-related genes. This in turn may provide new insights into the underlying molecular mechanisms of ERα regulation in mammalian cells.

  6. Genome-wide mapping of infection-induced SINE RNAs reveals a role in selective mRNA export.

    Science.gov (United States)

    Karijolich, John; Zhao, Yang; Alla, Ravi; Glaunsinger, Britt

    2017-06-02

    Short interspersed nuclear elements (SINEs) are retrotransposons evolutionarily derived from endogenous RNA Polymerase III RNAs. Though SINE elements have undergone exaptation into gene regulatory elements, how transcribed SINE RNA impacts transcriptional and post-transcriptional regulation is largely unknown. This is partly due to a lack of information regarding which of the loci have transcriptional potential. Here, we present an approach (short interspersed nuclear element sequencing, SINE-seq), which selectively profiles RNA Polymerase III-derived SINE RNA, thereby identifying transcriptionally active SINE loci. Applying SINE-seq to monitor murine B2 SINE expression during a gammaherpesvirus infection revealed transcription from 28 270 SINE loci, with ∼50% of active SINE elements residing within annotated RNA Polymerase II loci. Furthermore, B2 RNA can form intermolecular RNA-RNA interactions with complementary mRNAs, leading to nuclear retention of the targeted mRNA via a mechanism involving p54nrb. These findings illuminate a pathway for the selective regulation of mRNA export during stress via retrotransposon activation. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Transcriptomic analysis of Portunus trituberculatus reveals a critical role for WNT4 and WNT signalling in limb regeneration.

    Science.gov (United States)

    Liu, Lei; Fu, Yuanyuan; Zhu, Fang; Mu, Changkao; Li, Ronghua; Song, Weiwei; Shi, Ce; Ye, Yangfang; Wang, Chunlin

    2018-06-05

    The swimming crab (Portunus trituberculatus) is among the most economically important seawater crustacean species in Asia. Despite its commercial importance and being well-studied status, genomic and transcriptomic data are scarce for this crab species. In the present study, limb bud tissue was collected at different developmental stages post amputation for transcriptomic analysis. Illumina RNA-sequencing was applied to characterise the limb regeneration transcriptome and identify the most characteristic genes. A total of 289,018 transcripts were obtained by clustering and assembly of clean reads, producing 150,869 unigenes with an average length of 956 bp. Subsequent analysis revealed WNT signalling as the key pathway involved in limb regeneration, with WNT4 a key mediator. Overall, limb regeneration appears to be regulated by multiple signalling pathways, with numerous cell differentiation, muscle growth, moult, metabolism, and immune-related genes upregulated, including WNT4, LAMA, FIP2, FSTL5, TNC, HUS1, SWI5, NCGL, SLC22, PLA2, Tdc2, SMOX, GDH, and SMPD4. This is the first experimental study done on regenerating claws of P. trituberculatus. These findings expand existing sequence resources for crab species, and will likely accelerate research into regeneration and development in crustaceans, particularly functional studies on genes involved in limb regeneration. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. High-throughput deep sequencing reveals that microRNAs play important roles in salt tolerance of euhalophyte Salicornia europaea.

    Science.gov (United States)

    Feng, Juanjuan; Wang, Jinhui; Fan, Pengxiang; Jia, Weitao; Nie, Lingling; Jiang, Ping; Chen, Xianyang; Lv, Sulian; Wan, Lichuan; Chang, Sandra; Li, Shizhong; Li, Yinxin

    2015-02-26

    microRNAs (miRNAs) are implicated in plant development processes and play pivotal roles in plant adaptation to environmental stresses. Salicornia europaea, a salt mash euhalophyte, is a suitable model plant to study salt adaptation mechanisms. S. europaea is also a vegetable, forage, and oilseed that can be used for saline land reclamation and biofuel precursor production on marginal lands. Despite its importance, no miRNA has been identified from S. europaea thus far. Deep sequencing was performed to investigate small RNA transcriptome of S. europaea. Two hundred and ten conserved miRNAs comprising 51 families and 31 novel miRNAs (including seven miRNA star sequences) belonging to 30 families were identified. About half (13 out of 31) of the novel miRNAs were only detected in salt-treated samples. The expression of 43 conserved and 13 novel miRNAs significantly changed in response to salinity. In addition, 53 conserved and 13 novel miRNAs were differentially expressed between the shoots and roots. Furthermore, 306 and 195 S. europaea unigenes were predicted to be targets of 41 conserved and 29 novel miRNA families, respectively. These targets encoded a wide range of proteins, and genes involved in transcription regulation constituted the largest category. Four of these genes encoding laccase, F-box family protein, SAC3/GANP family protein, and NADPH cytochrome P-450 reductase were validated using 5'-RACE. Our results indicate that specific miRNAs are tightly regulated by salinity in the shoots and/or roots of S. europaea, which may play important roles in salt tolerance of this euhalophyte. The S. europaea salt-responsive miRNAs and miRNAs that target transcription factors, nucleotide binding site-leucine-rich repeat proteins and enzymes involved in lignin biosynthesis as well as carbon and nitrogen metabolism may be applied in genetic engineering of crops with high stress tolerance, and genetic modification of biofuel crops with high biomass and regulatable

  9. Genome-wide siRNA Screening at Biosafety Level 4 Reveals a Crucial Role for Fibrillarin in Henipavirus Infection.

    Directory of Open Access Journals (Sweden)

    Celine Deffrasnes

    2016-03-01

    Full Text Available Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections.

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

    Science.gov (United States)

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

    2016-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Debashis Barik

    2016-12-01

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

  12. Small RNA profiling reveals important roles for miRNAs in Arabidopsis response to Bacillus velezensis FZB42.

    Science.gov (United States)

    Xie, Shanshan; Jiang, Haiyang; Xu, Zhilan; Xu, Qianqian; Cheng, Beijiu

    2017-09-20

    Bacillus velezensis FZB42 (previously classified as Bacillus amyloliquefaciens FZB42) has been confirmed to successfully colonize plant roots and enhance defense response against pathogen infection. This study indicated that FZB42 inoculation enhanced Arabidopsis defense response against Pseudomonas syringae DC3000 through inducing the expression of PR1, PDF1.2 and stomata closure. To further clarify the induced defense response at miRNA level, sRNA libraries from Arabidopsis roots inoculated with FZB42 and control were constructed and sequenced. The reads of 21nt and 24nt in length were the most abundant groups in FZB42-treated library and control library, respectively. 234 known miRNAs and 16 novel miRNAs were identified. Among them, 11 known miRNAs and 4 novel miRNAs were differentially expressed after FZB42 inoculation. Moreover cis-elements (TC-rich repeats, TCA-element and CGTCA-motif) associated with plant defense were also found in the promoters of these miRNAs. Additionally, 141 mRNAs were predicted as potential targets of these differentially expressed miRNAs. GO annotations of the target genes indicated their potential roles in polyamine biosynthetic process and intracellular protein transport biological process, which may contribute to increased defense response. Our findings indicated that Bacillus velezensis FZB42 inoculation altered the expression of Arabidopsis miRNAs and their target genes, which were associated with defense response. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. A Global Analysis of Kinase Function in Candida albicans Hyphal Morphogenesis Reveals a Role for the Endocytosis Regulator Akl1

    Directory of Open Access Journals (Sweden)

    Hagit Bar-Yosef

    2018-02-01

    Full Text Available The human pathogenic fungus Candida albicans can switch between yeast and hyphal morphologies as a function of environmental conditions and cellular physiology. The yeast-to-hyphae morphogenetic switch is activated by well-established, kinase-based signal transduction pathways that are induced by extracellular stimuli. In order to identify possible inhibitory pathways of the yeast-to-hyphae transition, we interrogated a collection of C. albicans protein kinases and phosphatases ectopically expressed under the regulation of the TETon promoter. Proportionately more phosphatases than kinases were identified that inhibited hyphal morphogenesis, consistent with the known role of protein phosphorylation in hyphal induction. Among the kinases, we identified AKL1 as a gene that significantly suppressed hyphal morphogenesis in serum. Akl1 specifically affected hyphal elongation rather than initiation: overexpression of AKL1 repressed hyphal growth, and deletion of AKL1 resulted in acceleration of the rate of hyphal elongation. Akl1 suppressed fluid-phase endocytosis, probably via Pan1, a putative clathrin-mediated endocytosis scaffolding protein. In the absence of Akl1, the Pan1 patches were delocalized from the sub-apical region, and fluid-phase endocytosis was intensified. These results underscore the requirement of an active endocytic pathway for hyphal morphogenesis. Furthermore, these results suggest that under standard conditions, endocytosis is rate-limiting for hyphal elongation.

  14. A Global Analysis of Kinase Function in Candida albicans Hyphal Morphogenesis Reveals a Role for the Endocytosis Regulator Akl1.

    Science.gov (United States)

    Bar-Yosef, Hagit; Gildor, Tsvia; Ramírez-Zavala, Bernardo; Schmauch, Christian; Weissman, Ziva; Pinsky, Mariel; Naddaf, Rawi; Morschhäuser, Joachim; Arkowitz, Robert A; Kornitzer, Daniel

    2018-01-01

    The human pathogenic fungus Candida albicans can switch between yeast and hyphal morphologies as a function of environmental conditions and cellular physiology. The yeast-to-hyphae morphogenetic switch is activated by well-established, kinase-based signal transduction pathways that are induced by extracellular stimuli. In order to identify possible inhibitory pathways of the yeast-to-hyphae transition, we interrogated a collection of C. albicans protein kinases and phosphatases ectopically expressed under the regulation of the TETon promoter. Proportionately more phosphatases than kinases were identified that inhibited hyphal morphogenesis, consistent with the known role of protein phosphorylation in hyphal induction. Among the kinases, we identified AKL1 as a gene that significantly suppressed hyphal morphogenesis in serum. Akl1 specifically affected hyphal elongation rather than initiation: overexpression of AKL1 repressed hyphal growth, and deletion of AKL1 resulted in acceleration of the rate of hyphal elongation. Akl1 suppressed fluid-phase endocytosis, probably via Pan1, a putative clathrin-mediated endocytosis scaffolding protein. In the absence of Akl1, the Pan1 patches were delocalized from the sub-apical region, and fluid-phase endocytosis was intensified. These results underscore the requirement of an active endocytic pathway for hyphal morphogenesis. Furthermore, these results suggest that under standard conditions, endocytosis is rate-limiting for hyphal elongation.

  15. A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor

    Energy Technology Data Exchange (ETDEWEB)

    Datta, Supratim; Koutmos, Markos; Pattridge, Katherine A.; Ludwig, Martha L.; Matthews, Rowena G. (Michigan)

    2008-07-08

    B{sub 12}-dependent methionine synthase (MetH) from Escherichia coli is a large modular protein that is alternately methylated by methyltetrahydrofolate to form methylcobalamin and demethylated by homocysteine to form cob(I)alamin. Major domain rearrangements are required to allow cobalamin to react with three different substrates: homocysteine, methyltetrahydrofolate, and S-adenosyl-l-methionine (AdoMet). These same rearrangements appear to preclude crystallization of the wild-type enzyme. Disulfide cross-linking was used to lock a C-terminal fragment of the enzyme into a unique conformation. Cysteine point mutations were introduced at Ile-690 and Gly-743. These cysteine residues span the cap and the cobalamin-binding module and form a cross-link that reduces the conformational space accessed by the enzyme, facilitating protein crystallization. Here, we describe an x-ray structure of the mutant fragment in the reactivation conformation; this conformation enables the transfer of a methyl group from AdoMet to the cobalamin cofactor. In the structure, the axial ligand to the cobalamin, His-759, dissociates from the cobalamin and forms intermodular contacts with residues in the AdoMet-binding module. This unanticipated intermodular interaction is expected to play a major role in controlling the distribution of conformers required for the catalytic and the reactivation cycles of the enzyme.

  16. Comparative proteome analysis between C . briggsae embryos and larvae reveals a role of chromatin modification proteins in embryonic cell division

    KAUST Repository

    An, Xiaomeng

    2017-06-21

    Caenorhabditis briggsae has emerged as a model for comparative biology against model organism C. elegans. Most of its cell fate specifications are completed during embryogenesis whereas its cell growth is achieved mainly in larval stages. The molecular mechanism underlying the drastic developmental changes is poorly understood. To gain insights into the molecular changes between the two stages, we compared the proteomes between the two stages using iTRAQ. We identified a total of 2,791 proteins in the C. briggsae embryos and larvae, 247 of which undergo up- or down-regulation between the two stages. The proteins that are upregulated in the larval stages are enriched in the Gene Ontology categories of energy production, protein translation, and cytoskeleton; whereas those upregulated in the embryonic stage are enriched in the categories of chromatin dynamics and posttranslational modification, suggesting a more active chromatin modification in the embryos than in the larva. Perturbation of a subset of chromatin modifiers followed by cell lineage analysis suggests their roles in controlling cell division pace. Taken together, we demonstrate a general molecular switch from chromatin modification to metabolism during the transition from C. briggsae embryonic to its larval stages using iTRAQ approach. The switch might be conserved across metazoans.

  17. A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate.

    Science.gov (United States)

    Loch, Christian M; Strickler, James E

    2012-11-01

    Substrate ubiquitylation is a reversible process critical to cellular homeostasis that is often dysregulated in many human pathologies including cancer and neurodegeneration. Elucidating the mechanistic details of this pathway could unlock a large store of information useful to the design of diagnostic and therapeutic interventions. Proteomic approaches to the questions at hand have generally utilized mass spectrometry (MS), which has been successful in identifying both ubiquitylation substrates and profiling pan-cellular chain linkages, but is generally unable to connect the two. Interacting partners of the deubiquitylating enzymes (DUBs) have also been reported by MS, although substrates of catalytically competent DUBs generally cannot be. Where they have been used towards the study of ubiquitylation, protein microarrays have usually functioned as platforms for the identification of substrates for specific E3 ubiquitin ligases. Here, we report on the first use of protein microarrays to identify substrates of DUBs, and in so doing demonstrate the first example of microarray proteomics involving multiple (i.e., distinct, sequential and opposing) enzymatic activities. This technique demonstrates the selectivity of DUBs for both substrate and type (mono- versus poly-) of ubiquitylation. This work shows that the vast majority of DUBs are monoubiquitylated in vitro, and are incapable of removing this modification from themselves. This work also underscores the critical role of utilizing both ubiquitin chains and substrates when attempting to characterize DUBs. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. A combination of genomic approaches reveals the role of FOXO1a in regulating an oxidative stress response pathway.

    Directory of Open Access Journals (Sweden)

    Paola de Candia

    2008-02-01

    Full Text Available While many of the phenotypic differences between human and chimpanzee may result from changes in gene regulation, only a handful of functionally important regulatory differences are currently known. As a first step towards identifying transcriptional pathways that have been remodeled in the human lineage, we focused on a transcription factor, FOXO1a, which we had previously found to be up-regulated in the human liver compared to that of three other primate species. We concentrated on this gene because of its known role in the regulation of metabolism and in longevity.Using a combination of expression profiling following siRNA knockdown and chromatin immunoprecipitation in a human liver cell line, we identified eight novel direct transcriptional targets of FOXO1a. This set includes the gene for thioredoxin-interacting protein (TXNIP, the expression of which is directly repressed by FOXO1a. The thioredoxin-interacting protein is known to inhibit the reducing activity of thioredoxin (TRX, thereby hindering the cellular response to oxidative stress and affecting life span.Our results provide an explanation for the repeated observations that differences in the regulation of FOXO transcription factors affect longevity. Moreover, we found that TXNIP is down-regulated in human compared to chimpanzee, consistent with the up-regulation of its direct repressor FOXO1a in humans, and with differences in longevity between the two species.

  19. Priming can affect naming colours using the study-test procedure. Revealing the role of task conflict.

    Science.gov (United States)

    Sharma, Dinkar

    2016-11-14

    The Stroop paradigm has been widely used to study attention whilst its use to explore implicit memory have been mixed. Using the non-colour word Stroop task we tested contrasting predictions from the proactive-control/task-conflict model (Kalanthroff, Avnit, Henik, Davelaar & Usher, 2015) that implicate response conflict and task conflict for the priming effects. Using the study-test procedure 60 native English speakers were tested to determine whether priming effects from words that had previously been studied would cause interference when presented in a colour naming task. The results replicate a finding by MacLeod (1996) who showed no differences between the response latencies to studied and unstudied words. However, this pattern was predominately in the first half of the study where it was also found that both studied and unstudied words in a mixed block were slower to respond to than a block of pure unstudied words. The second half of the study showed stronger priming interference effects as well as a sequential modulation effect in which studied words slowed down the responses of studied words on the next trial. We discuss the role of proactive and reactive control processes and conclude that task conflict best explains the pattern of priming effects reported. Copyright © 2016. Published by Elsevier B.V.

  20. Stochastic kinetics reveal imperative role of anisotropic interfacial tension to determine morphology and evolution of nucleated droplets in nematogenic films

    Science.gov (United States)

    Bhattacharjee, Amit Kumar

    2017-01-01

    For isotropic fluids, classical nucleation theory predicts the nucleation rate, barrier height and critical droplet size by ac- counting for the competition between bulk energy and interfacial tension. The nucleation process in liquid crystals is less understood. We numerically investigate nucleation in monolayered nematogenic films using a mesoscopic framework, in par- ticular, we study the morphology and kinetic pathway in spontaneous formation and growth of droplets of the stable phase in the metastable background. The parameter κ that quantifies the anisotropic elastic energy plays a central role in determining the geometric structure of the droplets. Noncircular nematic droplets with homogeneous director orientation are nucleated in a background of supercooled isotropic phase for small κ. For large κ, noncircular droplets with integer topological charge, accompanied by a biaxial ring at the outer surface, are nucleated. The isotropic droplet shape in a superheated nematic background is found to depend on κ in a similar way. Identical growth laws are found in the two cases, although an unusual two-stage mechanism is observed in the nucleation of isotropic droplets. Temporal distributions of successive events indi- cate the relevance of long-ranged elasticity-mediated interactions within the isotropic domains. Implications for a theoretical description of nucleation in anisotropic fluids are discussed.

  1. Double-lock ratchet mechanism revealing the role of  SER-344 in FoF1 ATP synthase

    KAUST Repository

    Beke-Somfai, T.

    2011-03-07

    In a majority of living organisms, FoF1 ATP synthase performs the fundamental process of ATP synthesis. Despite the simple net reaction formula, ADP+Pi→ATP+H2O, the detailed step-by-step mechanism of the reaction yet remains to be resolved owing to the complexity of this multisubunit enzyme. Based on quantum mechanical computations using recent high resolution X-ray structures, we propose that during ATP synthesis the enzyme first prepares the inorganic phosphate for the γP-OADP bond-forming step via a double-proton transfer. At this step, the highly conserved αS344 side chain plays a catalytic role. The reaction thereafter progresses through another transition state (TS) having a planar ion configuration to finally form ATP. These two TSs are concluded crucial for ATP synthesis. Using stepwise scans and several models of the nucleotide-bound active site, some of the most important conformational changes were traced toward direction of synthesis. Interestingly, as the active site geometry progresses toward the ATP-favoring tight binding site, at both of these TSs, a dramatic increase in barrier heights is observed for the reverse direction, i.e., hydrolysis of ATP. This change could indicate a "ratchet" mechanism for the enzyme to ensure efficacy of ATP synthesis by shifting residue conformation and thus locking access to the crucial TSs.

  2. Ion channel profile of TRPM8 cold receptors reveals a novel role of TASK-3 potassium channels in thermosensation

    Science.gov (United States)

    Morenilla-Palao, Cruz; Luis, Enoch; Fernández-Peña, Carlos; Quintero, Eva; Weaver, Janelle L.; Bayliss, Douglas A.; Viana, Félix

    2017-01-01

    Summary Animals sense cold ambient temperatures through the activation of peripheral thermoreceptors that express TRPM8, a cold- and menthol-activated ion channel. These receptors can discriminate a very wide range of temperatures from innocuous to noxious. The molecular mechanism responsible for the variable sensitivity of individual cold receptors to temperature is unclear. To address this question, we performed a detailed ion channel expression analysis of cold sensitive neurons, combining BAC transgenesis with a molecular profiling approach in FACS purified TRPM8 neurons. We found that TASK-3 leak potassium channels are highly enriched in a subpopulation of these sensory neurons. The thermal threshold of TRPM8 cold neurons is decreased during TASK-3 blockade and in mice lacking TASK-3 and, most importantly, these mice display hypersensitivity to cold. Our results demonstrate a novel role of TASK-3 channels in thermosensation, showing that a channel-based combinatorial strategy in TRPM8 cold thermoreceptors leads to molecular specialization and functional diversity. PMID:25199828

  3. Deletion of GLUT1 and GLUT3 Reveals Multiple Roles for Glucose Metabolism in Platelet and Megakaryocyte Function

    Directory of Open Access Journals (Sweden)

    Trevor P. Fidler

    2017-07-01

    Full Text Available Anucleate platelets circulate in the blood to facilitate thrombosis and diverse immune functions. Platelet activation leading to clot formation correlates with increased glycogenolysis, glucose uptake, glucose oxidation, and lactic acid production. Simultaneous deletion of glucose transporter (GLUT 1 and GLUT3 (double knockout [DKO] specifically in platelets completely abolished glucose uptake. In DKO platelets, mitochondrial oxidative metabolism of non-glycolytic substrates, such as glutamate, increased. Thrombosis and platelet activation were decreased through impairment at multiple activation nodes, including Ca2+ signaling, degranulation, and integrin activation. DKO mice developed thrombocytopenia, secondary to impaired pro-platelet formation from megakaryocytes, and increased platelet clearance resulting from cytosolic calcium overload and calpain activation. Systemic treatment with oligomycin, inhibiting mitochondrial metabolism, induced rapid clearance of platelets, with circulating counts dropping to zero in DKO mice, but not wild-type mice, demonstrating an essential role for energy metabolism in platelet viability. Thus, substrate metabolism is essential for platelet production, activation, and survival.

  4. Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation*

    Science.gov (United States)

    Lalo, Ulyana; Roberts, Jonathan A.; Evans, Richard J.

    2011-01-01

    P2X1 receptors are ATP-gated ion channels expressed by smooth muscle and blood cells. Carboxyl-terminally His-FLAG-tagged human P2X1 receptors were stably expressed in HEK293 cells and co-purified with cytoskeletal proteins including actin. Disruption of the actin cytoskeleton with cytochalasin D inhibited P2X1 receptor currents with no effect on the time course of the response or surface expression of the receptor. Stabilization of the cytoskeleton with jasplakinolide had no effect on P2X1 receptor currents but decreased receptor mobility. P2X2 receptor currents were unaffected by cytochalasin, and P2X1/2 receptor chimeras were used to identify the molecular basis of actin sensitivity. These studies showed that the intracellular amino terminus accounts for the inhibitory effects of cytoskeletal disruption similar to that shown for lipid raft/cholesterol sensitivity. Stabilization of the cytoskeleton with jasplakinolide abolished the inhibitory effects of cholesterol depletion on P2X1 receptor currents, suggesting that lipid rafts may regulate the receptor through stabilization of the cytoskeleton. These studies show that the cytoskeleton plays an important role in P2X1 receptor regulation. PMID:21757694

  5. Characterization of the Dioscorin Gene Family in Dioscorea alata Reveals a Role in Tuber Development and Environmental Response.

    Science.gov (United States)

    Liu, Linya; Huang, Yacheng; Huang, Xiaolong; Yang, Jianghua; Wu, Wenqiang; Xu, Yun; Cong, Ziwen; Xie, Jun; Xia, Wei; Huang, Dongyi

    2017-07-20

    Dioscorin is one of the major soluble proteins in yam tubers. Unlike other well-known plant storage proteins, such as patatin and sporamin, dioscorin is argued for its function as storage proteins, and the molecular mechanisms underlying its expressional complexity are little understood. In this study, we isolated five dioscorin genes from Dioscorea alata L., comprising three class A ( Da-dio1 , - 3 and - 4 ) and two class B ( Da-dio2 and - 5 ) isoforms. Expressions of all dioscorin genes gradually decreased in mother tubers during yam sprouting and regrowth. On the other hand, all dioscorin genes accumulated transcripts progressively with tuber development in new tubers, with Da-dio5 being the most prominent isoform. In yam leaves, the expressions of Da-dio 5 were up-regulated by the treatments of five phytohormones (gibberellic acid, salicylic acid, indole-3-acetic acid, abscisic acid, and ethylene), and three abiotic stresses (high-temperature, low-temperature and drought). To further elucidate the regulatory mechanisms of Da-dio5 expressions, transgenic Arabidopsis plants harboring the Da-dio5 promoter-β-glucuronidase (GUS) fusion were generated. GUS staining showed that expressions of the Da-dio5 promoter were detected mainly in the shoot apical meristem (SAM) and hypocotyls, and enhanced by the treatments of the five hormones, and the three abiotic stresses mentioned above. These results suggest diverse roles of Da-dio5 in yam sprouting, regrowth, and tuberization, as well as in response to enviromental cues.

  6. Inhibition of HMG CoA reductase reveals an unexpected role for cholesterol during PGC migration in the mouse

    Directory of Open Access Journals (Sweden)

    Ewing Andrew G

    2008-12-01

    Full Text Available Abstract Background Primordial germ cells (PGCs are the embryonic precursors of the sperm and eggs. Environmental or genetic defects that alter PGC development can impair fertility or cause formation of germ cell tumors. Results We demonstrate a novel role for cholesterol during germ cell migration in mice. Cholesterol was measured in living tissue dissected from mouse embryos and was found to accumulate within the developing gonads as germ cells migrate to colonize these structures. Cholesterol synthesis was blocked in culture by inhibiting the activity of HMG CoA reductase (HMGCR resulting in germ cell survival and migration defects. These defects were rescued by co-addition of isoprenoids and cholesterol, but neither compound alone was sufficient. In contrast, loss of the last or penultimate enzyme in cholesterol biosynthesis did not alter PGC numbers or position in vivo. However embryos that lack these enzymes do not exhibit cholesterol defects at the stage at which PGCs are migrating. This demonstrates that during gestation, the cholesterol required for PGC migration can be supplied maternally. Conclusion In the mouse, cholesterol is required for PGC survival and motility. It may act cell-autonomously by regulating clustering of growth factor receptors within PGCs or non cell-autonomously by controlling release of growth factors required for PGC guidance and survival.

  7. Transcriptome Analysis Reveals Novel Entry Mechanisms and a Central Role of SRC in Host Defense during High Multiplicity Mycobacterial Infection.

    Directory of Open Access Journals (Sweden)

    Jay Zhang

    Full Text Available Mycobacterium tuberculosis (MTB infects an estimated one-third of the global population and is one of the main causes of mortality from an infectious agent. The characteristics of macrophages challenged by MTB with a high multiplicity of infection (MOI, which mimics both clinical disseminated infection and granuloma formation, are distinct from macrophages challenged with a low MOI. To better understand the cross talk between macrophage host cells and mycobacteria, we compared the transcription patterns of mouse macrophages infected with bacille Calmette-Guérin, H37Ra and M. smegmatis. Attention was focused on the changes in the abundance of transcripts related to immune system function. From the results of a transcriptome profiling study with a high mycobacterial MOI, we defined a pathogen-specific host gene expression pattern. The present study suggests that two integrins, ITGA5 and ITGAV, are novel cell surface receptors mediating mycobacterium entry into macrophages challenged with high MOI. Our results indicate that SRC likely plays a central role in regulating multiple unique signaling pathways activated by MTB infection. The integrated results increase our understanding of the molecular networks behind the host innate immune response and identify important targets that might be useful for the development of tuberculosis therapy.

  8. Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication.

    Directory of Open Access Journals (Sweden)

    Denis Avey

    2015-07-01

    Full Text Available Kaposi's Sarcoma-Associated Herpesvirus (KSHV is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK mitogen-activated protein kinase (MAPK pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5' UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45

  9. Structure-Related Roles for the Conservation of the HIV-1 Fusion Peptide Sequence Revealed by Nuclear Magnetic Resonance.

    Science.gov (United States)

    Serrano, Soraya; Huarte, Nerea; Rujas, Edurne; Andreu, David; Nieva, José L; Jiménez, María Angeles

    2017-10-17

    Despite extensive characterization of the human immunodeficiency virus type 1 (HIV-1) hydrophobic fusion peptide (FP), the structure-function relationships underlying its extraordinary degree of conservation remain poorly understood. Specifically, the fact that the tandem repeat of the FLGFLG tripeptide is absolutely conserved suggests that high hydrophobicity may not suffice to unleash FP function. Here, we have compared the nuclear magnetic resonance (NMR) structures adopted in nonpolar media by two FP surrogates, wtFP-tag and scrFP-tag, which had equal hydrophobicity but contained wild-type and scrambled core sequences LFLGFLG and FGLLGFL, respectively. In addition, these peptides were tagged at their C-termini with an epitope sequence that folded independently, thereby allowing Western blot detection without interfering with FP structure. We observed similar α-helical FP conformations for both specimens dissolved in the low-polarity medium 25% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), but important differences in contact with micelles of the membrane mimetic dodecylphosphocholine (DPC). Thus, whereas wtFP-tag preserved a helix displaying a Gly-rich ridge, the scrambled sequence lost in great part the helical structure upon being solubilized in DPC. Western blot analyses further revealed the capacity of wtFP-tag to assemble trimers in membranes, whereas membrane oligomers were not observed in the case of the scrFP-tag sequence. We conclude that, beyond hydrophobicity, preserving sequence order is an important feature for defining the secondary structures and oligomeric states adopted by the HIV FP in membranes.

  10. Elucidation of the regulatory role of the fructose operon reveals a novel target for enhancing the NADPH supply in Corynebacterium glutamicum

    DEFF Research Database (Denmark)

    Wang, Zhihao; Chan, Siu Hung Joshua; Sudarsan, Suresh

    2016-01-01

    is linked to redox and to the general metabolism. We here provide new insights into the regulation of the metabolism of this important platform organism by systematically characterizing mutants carrying various lesions in the fructose operon. Initially, we found that a strain where the dedicated fructose...... uptake system had been inactivated (KO-ptsF) was hampered in growth on sucrose minimal medium, and suppressor mutants appeared readily. Comparative genomic analysis in conjunction with enzymatic assays revealed that suppression was linked to inactivation of the pfkB gene, encoding a fructose-1-phosphate...... kinase. Detailed characterization of KO-ptsF, KO-pfkB and double knock-out (DKO) derivatives revealed a strong role for sugar-phosphates, especially fructose-1-phosphate (F1P), in governing sugar as well as redox metabolism due to effects on transcriptional regulation of key genes. These findings allowed...

  11. A new in vivo model of pantothenate kinase-associated neurodegeneration reveals a surprising role for transcriptional regulation in pathogenesis.

    Directory of Open Access Journals (Sweden)

    Varun ePandey

    2013-09-01

    Full Text Available Pantothenate Kinase-Associated Neurodegeneration (PKAN is a neurodegenerative disorder with a poorly understood molecular mechanism. It is caused by mutations in Pantothenate Kinase, the first enzyme in the Coenzyme A (CoA biosynthetic pathway. Here, we developed a Drosophila model of PKAN (tim-fbl flies that allows us to continuously monitor the modeled disease in the brain. In tim-fbl flies, downregulation of fumble, the Drosophila PanK homologue in the cells containing a circadian clock results in characteristic features of PKAN such as developmental lethality, hypersensitivity to oxidative stress, and diminished life span. Despite quasi-normal circadian transcriptional rhythms, tim-fbl flies display brain-specific aberrant circadian locomotor rhythms, and a unique transcriptional signature. Comparison with expression data from flies exposed to paraquat demonstrates that, as previously suggested, pathways others than oxidative stress are affected by PANK downregulation. Surprisingly we found a significant decrease in the expression of key components of the photoreceptor recycling pathways, which could lead to retinal degeneration, a hallmark of PKAN. Importantly, these defects are not accompanied by changes in structural components in eye genes suggesting that changes in gene expression in the eye precede and may cause the retinal degeneration. Indeed tim-fbl flies have diminished response to light transitions, and their altered day/night patterns of activity demonstrates defects in light perception. This suggest that retinal lesions are not solely due to oxidative stress and demonstrates a role for the transcriptional response to CoA deficiency underlying the defects observed in dPanK deficient flies. Moreover, in the present study we developed a new fly model that can be applied to other diseases and that allows the assessment of neurodegeneration in the brains of living flies.

  12. Spheroid Culture of Head and Neck Cancer Cells Reveals an Important Role of EGFR Signalling in Anchorage Independent Survival.

    Science.gov (United States)

    Braunholz, Diana; Saki, Mohammad; Niehr, Franziska; Öztürk, Merve; Borràs Puértolas, Berta; Konschak, Robert; Budach, Volker; Tinhofer, Ingeborg

    2016-01-01

    In solid tumours millions of cells are shed into the blood circulation each day. Only a subset of these circulating tumour cells (CTCs) survive, many of them presumable because of their potential to form multi-cellular clusters also named spheroids. Tumour cells within these spheroids are protected from anoikis, which allows them to metastasize to distant organs or re-seed at the primary site. We used spheroid cultures of head and neck squamous cell carcinoma (HNSCC) cell lines as a model for such CTC clusters for determining the role of the epidermal growth factor receptor (EGFR) in cluster formation ability and cell survival after detachment from the extra-cellular matrix. The HNSCC cell lines FaDu, SCC-9 and UT-SCC-9 (UT-SCC-9P) as well as its cetuximab (CTX)-resistant sub-clone (UT-SCC-9R) were forced to grow in an anchorage-independent manner by coating culture dishes with the anti-adhesive polymer poly-2-hydroxyethylmethacrylate (poly-HEMA). The extent of apoptosis, clonogenic survival and EGFR signalling under such culture conditions was evaluated. The potential of spheroid formation in suspension culture was found to be positively correlated with the proliferation rate of HNSCC cell lines as well as their basal EGFR expression levels. CTX and gefitinib blocked, whereas the addition of EGFR ligands promoted anchorage-independent cell survival and spheroid formation. Increased spheroid formation and growth were associated with persistent activation of EGFR and its downstream signalling component (MAPK/ERK). Importantly, HNSCC cells derived from spheroid cultures retained their clonogenic potential in the absence of cell-matrix contact. Addition of CTX under these conditions strongly inhibited colony formation in CTX-sensitive cell lines but not their resistant subclones. Altogether, EGFR activation was identified as crucial factor for anchorage-independent survival of HNSCC cells. Targeting EGFR in CTC cluster formation might represent an attractive anti

  13. Systems reconsolidation reveals a selective role for the anterior cingulate cortex in generalized contextual fear memory expression.

    Science.gov (United States)

    Einarsson, Einar Ö; Pors, Jennifer; Nader, Karim

    2015-01-01

    After acquisition, hippocampus-dependent memories undergo a systems consolidation process, during which they become independent of the hippocampus and dependent on the anterior cingulate cortex (ACC) for memory expression. However, consolidated remote memories can become transiently hippocampus-dependent again following memory reactivation. How this systems reconsolidation affects the role of the ACC in remote memory expression is not known. Using contextual fear conditioning, we show that the expression of 30-day-old remote memory can transiently be supported by either the ACC or the dorsal hippocampus following memory reactivation, and that the ACC specifically mediates expression of remote generalized contextual fear memory. We found that suppression of neural activity in the ACC with the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) impaired the expression of remote, but not recent, contextual fear memory. Fear expression was not affected by this treatment if preceded by memory reactivation 6 h earlier, nor was it affected by suppression of neural activity in the dorsal hippocampus with the GABA-receptor agonist muscimol. However, simultaneous targeting of both the ACC and the dorsal hippocampus 6 h after memory reactivation disrupted contextual fear memory expression. Second, we observed that expression of a 30-day-old generalized contextual fear memory in a novel context was not affected by memory reactivation 6 h earlier. However, intra-ACC CNQX infusion before testing impaired contextual fear expression in the novel context, but not the original training context. Together, these data suggest that although the dorsal hippocampus may be recruited during systems reconsolidation, the ACC remains necessary for the expression of generalized contextual fear memory.

  14. Knockdown expression and hepatic deficiency reveal anatheroprotective role for SR-BI in liver and peripheral tissues

    Energy Technology Data Exchange (ETDEWEB)

    Huby, Thierry; Doucet, Chantal; Dachet, Christiane; Ouzilleau,Betty; Ueda, Yukihiko; Afzal, Veena; Rubin, Edward; Chapman, M. John; Lesnik, Philippe

    2006-07-18

    Scavenger receptor SR-BI has been implicated inHDL-dependent atheroprotective mechanisms. We report the generation of anSR-BI conditional knockout mouse model in which SR-BI gene targeting byloxP site insertion produced a hypomorphic allele (hypomSR-BI).Attenuated SR-BI expression in hypomSR-BI mice resulted in 2-foldelevation in plasma total cholesterol (TC) levels. Cre-mediated SR-BIgene inactivation of the hypomorphic SR-BI allele in hepatocytes(hypomSR-BI-KOliver) was associated with high plasma TC concentrations,increased plasma free cholesterol/TC (FC/TC) ratio, and alipoprotein-cholesterol profile typical of SR-BI-/- mice. Plasma TClevels were increased 2-fold in hypomSR-BI and control mice fed anatherogenic diet, whereas hypomSR-BI-KOliver and SR-BI-/- mice developedsevere hypercholesterolemia due to accumulation of FC-rich, VLDL-sizedparticles. Atherosclerosis in hypomSR-BI mice was enhanced (2.5-fold)compared with that in controls, but to a much lower degree than inhypomSR-BI-KOliver (32-fold) and SR-BI-/- (48-fold) mice. The lattermodels did not differ in either plasma lipid levels or in the capacity ofVLDL-sized lipoproteins to induce macrophage cholesterol loading.However, reduced atherosclerosis in hypomSR-BI-KOliver mice wasassociated with decreased lesional macrophage content as compared withthat in SR-BI-/- mice. These data imply that, in addition to its majoratheroprotective role in liver, SR-BI may exert an antiatherogenic rolein extrahepatic tissues.

  15. RNAi screen reveals a role of SPHK2 in dengue virus-mediated apoptosis in hepatic cell lines.

    Directory of Open Access Journals (Sweden)

    Atthapan Morchang

    Full Text Available Hepatic dysfunction is a feature of dengue virus (DENV infection. Hepatic biopsy specimens obtained from fatal cases of DENV infection show apoptosis, which relates to the pathogenesis of DENV infection. However, how DENV induced liver injury is not fully understood. In this study, we aim to identify the factors that influence cell death by employing an apoptosis-related siRNA library screening. Our results show the effect of 558 gene silencing on caspase 3-mediated apoptosis in DENV-infected Huh7 cells. The majority of genes that contributed to apoptosis were the apoptosis-related kinase enzymes. Tumor necrosis factor superfamily member 12 (TNFSF12, and sphingosine kinase 2 (SPHK2, were selected as the candidate genes to further validate their influences on DENV-induced apoptosis. Transfection of siRNA targeting SPHK2 but not TNFSF12 genes reduced apoptosis determined by Annexin V/PI staining. Knockdown of SPHK2 did not reduce caspase 8 activity; however, did significantly reduce caspase 9 activity, suggesting its involvement of SPHK2 in the intrinsic pathway of apoptosis. Treatment of ABC294649, an inhibitor of SPHK2, reduced the caspase 3 activity, suggesting the involvement of its kinase activity in apoptosis. Knockdown of SPHK2 significantly reduced caspase 3 activity not only in DENV-infected Huh7 cells but also in DENV-infected HepG2 cells. Our results were consistent across all of the four serotypes of DENV infection, which supports the pro-apoptotic role of SPHK2 in DENV-infected liver cells.

  16. In situ SUMOylation analysis reveals a modulatory role of RanBP2 in the nuclear rim and PML bodies

    International Nuclear Information System (INIS)

    Saitoh, Noriko; Uchimura, Yasuhiro; Tachibana, Taro; Sugahara, Satoko; Saitoh, Hisato; Nakao, Mitsuyoshi

    2006-01-01

    SUMO modification plays a critical role in a number of cellular functions including nucleocytoplasmic transport, gene expression, cell cycle and formation of subnuclear structures such as promyelocytic leukemia (PML) bodies. In order to identify the sites where SUMOylation takes place in the cell, we developed an in situ SUMOylation assay using a semi-intact cell system and subsequently combined it with siRNA-based knockdown of nucleoporin RanBP2, also known as Nup358, which is one of the known SUMO E3 proteins. With the in situ SUMOylation assay, we found that both nuclear rim and PML bodies, besides mitotic apparatuses, are major targets for active SUMOylation. The ability to analyze possible SUMO conjugation sites would be a valuable tool to investigate where SUMO E3-like activities and/or SUMO substrates exist in the cell. Specific knockdown of RanBP2 completely abolished SUMOylation along the nuclear rim and dislocated RanGAP1 from the nuclear pore complexes. Interestingly, the loss of RanBP2 markedly reduced the number of PML bodies, in contrast to other, normal-appearing nuclear compartments including the nuclear lamina, nucleolus and chromatin, suggesting a novel link between RanBP2 and PML bodies. SUMOylation facilitated by RanBP2 at the nuclear rim may be a key step for the formation of a particular subnuclear organization. Our data imply that SUMO E3 proteins like RanBP2 facilitate spatio-temporal SUMOylation for certain nuclear structure and function

  17. A zebrafish model for Waardenburg syndrome type IV reveals diverse roles for Sox10 in the otic vesicle.

    Science.gov (United States)

    Dutton, Kirsten; Abbas, Leila; Spencer, Joanne; Brannon, Claire; Mowbray, Catriona; Nikaido, Masataka; Kelsh, Robert N; Whitfield, Tanya T

    2009-01-01

    In humans, mutations in the SOX10 gene are a cause of the auditory-pigmentary disorder Waardenburg syndrome type IV (WS4) and related variants. SOX10 encodes an Sry-related HMG box protein essential for the development of the neural crest; deafness in WS4 and other Waardenburg syndromes is usually attributed to loss of neural-crest-derived melanocytes in the stria vascularis of the cochlea. However, SOX10 is strongly expressed in the developing otic vesicle and so direct roles for SOX10 in the otic epithelium might also be important. Here, we examine the otic phenotype of zebrafish sox10 mutants, a model for WS4. As a cochlea is not present in the fish ear, the severe otic phenotype in these mutants cannot be attributed to effects on this tissue. In zebrafish sox10 mutants, we see abnormalities in all otic placodal derivatives. Gene expression studies indicate deregulated expression of several otic genes, including fgf8, in sox10 mutants. Using a combination of mutant and morphant data, we show that the three sox genes belonging to group E (sox9a, sox9b and sox10) provide a link between otic induction pathways and subsequent otic patterning: they act redundantly to maintain sox10 expression throughout otic tissue and to restrict fgf8 expression to anterior macula regions. Single-cell labelling experiments indicate a small and transient neural crest contribution to the zebrafish ear during normal development, but this is unlikely to account for the strong defects seen in the sox10 mutant. We discuss the implication that the deafness in WS4 patients with SOX10 mutations might reflect a haploinsufficiency for SOX10 in the otic epithelium, resulting in patterning and functional abnormalities in the inner ear.

  18. 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

    exogenous tryptophan or indole. Conclusions Biofilm growth of S. Typhimurium causes distinct changes in gene and protein expression. Our results show that aromatic amino acids make an important contribution to biofilm formation and reveal a link between SPI2 expression and surface-associated growth in S. Typhimurium.

  19. Optogenetic Inhibition Reveals Distinct Roles for Basolateral Amygdala Activity at Discrete Time Points during Risky Decision Making.

    Science.gov (United States)

    Orsini, Caitlin A; Hernandez, Caesar M; Singhal, Sarthak; Kelly, Kyle B; Frazier, Charles J; Bizon, Jennifer L; Setlow, Barry

    2017-11-29

    in which inhibition occurs. BLA inhibition during a period of deliberation between small, safe and large, risky outcomes decreased risky choice. In contrast, BLA inhibition during receipt of the large, punished outcome increased risky choice. These findings highlight the importance of temporally targeted approaches to understand neural substrates underlying complex cognitive processes. More importantly, they reveal novel information about dynamic BLA modulation of risky choice. Copyright © 2017 the authors 0270-6474/17/3711537-12$15.00/0.

  20. An extended version of the theory of planned behaviour: the role of self-efficacy and past behaviour in predicting the physical activity of Chinese adolescents.

    Science.gov (United States)

    Wang, Lijuan; Zhang, Ying

    2016-01-01

    This study aimed to use an extended theory of planned behaviour (TPB), which incorporated additional self-efficacy and past behaviour, to predict the intention to engage in moderate-to-vigorous physical activity (MVPA) and the MVPA level of Chinese adolescents. Questionnaires that focused on MVPA, attitude, subjective norm, perceived behavioural control (PBC), self-efficacy and past behaviour related to the MVPA engagement were administered to a sample of 488 young people. Multiple regression analyses provided moderate support for TPB. Three TPB constructs predicted 28.7% of the variance in intentions to engage in MVPA, and that PBC, but not intention, explained 3.4% of the variance in MVPA. Self-efficacy significantly affected intention and behaviour over and above the influence of TPB. Past behaviour had a small but significant improvement in the prediction of intention, but no improvement in the prediction of MVPA. Based on the results, interventions should target adolescent self-efficacy and PBC in physical activity participation.

  1. Identification of nucleolus-associated chromatin domains reveals the role of the nucleolus in the 3D organisation of the A. thaliana genome

    Science.gov (United States)

    Pontvianne, Frédéric; Carpentier, Marie-Christine; Durut, Nathalie; Pavlištová, Veronika; Jaške, Karin; Schořová, Šárka; Parrinello, Hugues; Rohmer, Marine; Pikaard, Craig S; Fojtová, Miloslava; Fajkus, Jiří; Saez-Vasquez, Julio

    2017-01-01

    The nucleolus is the site of ribosomal RNA (rRNA) gene transcription, rRNA processing and ribosome biogenesis. However, the nucleolus also plays additional roles in the cell. We isolated nucleoli by Fluorescence Activated Cell Sorting (FACS) and identified Nucleolus-Associated Chromatin Domains (NADs) by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein, NUCLEOLIN 1 (NUC1). NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs), sub-telomeric regions and mostly inactive protein-coding genes. However, NADs also include active ribosomal RNA genes, and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance. PMID:27477271

  2. Development of a highly metastatic model that reveals a crucial role of fibronectin in lung cancer cell migration and invasion

    Directory of Open Access Journals (Sweden)

    He Xianghuo

    2010-07-01

    Full Text Available Abstract Background The formation of metastasis is the most common cause of death in patients with lung cancer. A major implement to understand the molecular mechanisms involved in lung cancer metastasis has been the lack of suitable models to address it. In this study, we aimed at establishing a highly metastatic model of human lung cancer and characterizing its metastatic properties and underlying mechanisms. Methods The human lung adeno-carcinoma SPC-A-1 cell line was used as parental cells for developing of highly metastatic cells by in vivo selection in NOD/SCID mice. After three rounds of selection, a new SPC-A-1sci cell line was established from pulmonary metastatic lesions. Subsequently, the metastatic properties of this cell line were analyzed, including optical imaging of in vivo metastasis, immunofluorescence and immunohistochemical analysis of several epithelial mesenchymal transition (EMT makers and trans-well migration and invasion assays. Finally, the functional roles of fibronectin in the invasive and metastatic potentials of SPC-A-1sci cells were determined by shRNA analysis. Results A spontaneously pulmonary metastatic model of human lung adeno-carcinoma was established in NOD/SCID mice, from which a new lung cancer cell line, designated SPC-A-1sci, was isolated. Initially, the highly metastatic behavior of this cell line was validated by optical imaging in mice models. Further analyses showed that this cell line exhibit phenotypic and molecular alterations consistent with EMT. Compared with its parent cell line SPC-A-1, SPC-A-1sci was more aggressive in vitro, including increased potentials for cell spreading, migration and invasion. Importantly, fibronectin, a mesenchymal maker of EMT, was found to be highly expressed in SPC-A-1sci cells and down-regulation of it can decrease the in vitro and in vivo metastatic abilities of this cell line. Conclusions We have successfully established a new human lung cancer cell line with

  3. Development of a highly metastatic model that reveals a crucial role of fibronectin in lung cancer cell migration and invasion

    International Nuclear Information System (INIS)

    Jia, Deshui; Yao, Ming; Yan, Mingxia; Wang, Xiaomin; Hao, Xiangfang; Liang, Linhui; Liu, Lei; Kong, Hanwei; He, Xianghuo; Li, Jinjun

    2010-01-01

    The formation of metastasis is the most common cause of death in patients with lung cancer. A major implement to understand the molecular mechanisms involved in lung cancer metastasis has been the lack of suitable models to address it. In this study, we aimed at establishing a highly metastatic model of human lung cancer and characterizing its metastatic properties and underlying mechanisms. The human lung adeno-carcinoma SPC-A-1 cell line was used as parental cells for developing of highly metastatic cells by in vivo selection in NOD/SCID mice. After three rounds of selection, a new SPC-A-1sci cell line was established from pulmonary metastatic lesions. Subsequently, the metastatic properties of this cell line were analyzed, including optical imaging of in vivo metastasis, immunofluorescence and immunohistochemical analysis of several epithelial mesenchymal transition (EMT) makers and trans-well migration and invasion assays. Finally, the functional roles of fibronectin in the invasive and metastatic potentials of SPC-A-1sci cells were determined by shRNA analysis. A spontaneously pulmonary metastatic model of human lung adeno-carcinoma was established in NOD/SCID mice, from which a new lung cancer cell line, designated SPC-A-1sci, was isolated. Initially, the highly metastatic behavior of this cell line was validated by optical imaging in mice models. Further analyses showed that this cell line exhibit phenotypic and molecular alterations consistent with EMT. Compared with its parent cell line SPC-A-1, SPC-A-1sci was more aggressive in vitro, including increased potentials for cell spreading, migration and invasion. Importantly, fibronectin, a mesenchymal maker of EMT, was found to be highly expressed in SPC-A-1sci cells and down-regulation of it can decrease the in vitro and in vivo metastatic abilities of this cell line. We have successfully established a new human lung cancer cell line with highly metastatic potentials, which is subject to EMT and possibly

  4. Suicide gene reveals the myocardial neovascularization role of mesenchymal stem cells overexpressing CXCR4 (MSC(CXCR4.

    Directory of Open Access Journals (Sweden)

    Jialiang Liang

    Full Text Available BACKGROUND: Our previous studies indicated that MSC(CXCR4 improved cardiac function after myocardial infarction (MI. This study was aimed to investigate the specific role of MSC(CXCR4 in neovascularization of infarcted myocardium using a suicide gene approach. METHODS: MSCs were transduced with either lentivirus-null vector/GFP (MSC(Null as control or vector encoding for overexpressing CXCR4/GFP. The MSC derived-endothelial cell (EC differentiation was assessed by a tube formation assay, Dil-ac-LDL uptake, EC marker expression, and VE-cadherin promoter activity assay. Gene expression was analyzed by quantitative RT-PCR or Western blot. The suicide gene approach was under the control of VE-cadherin promoter. In vivo studies: Cell patches containing MSC(Null or MSC(CXCR4 were transduced with suicide gene and implanted into the myocardium of MI rat. Rats received either ganciclovir (GCV or vehicle after cell implantation. After one month, the cardiac functional changes and neovascularization were assessed by echocardiography, histological analysis, and micro-CT imaging. RESULTS: The expression of VEGF-A and HIF-1α was significantly higher in MSC(CXCR4 as compared to MSC(Null under hypoxia. Additionally, MSC(CXCR4 enhanced new vessel formation and EC differentiation, as well as STAT3 phosphorylation under hypoxia. STAT3 participated in the transcription of VE-cadherin in MSC(CXCR4 under hypoxia, which was inhibited by WP1066 (a STAT3 inhibitor. In addition, GCV specifically induced death of ECs with suicide gene activation. In vivo studies: MSC(CXCR4 implantation promoted cardiac functional restoration, reduced infarct size, improved cardiac remodeling, and enhanced neovascularization in ischemic heart tissue. New vessels derived from MSC(CXCR4 were observed at the injured heart margins and communicated with native coronary arteries. However, the derived vessel networks were reduced by GCV, reversing improvement of cardiac function. CONCLUSION: The

  5. Proteomic Analysis Revealed the Important Role of Vimentin in Human Cervical Carcinoma HeLa Cells Treated With Gambogic Acid*

    Science.gov (United States)

    Yue, Qingxi; Feng, Lixing; Cao, Biyin; Liu, Miao; Zhang, Dongmei; Wu, Wanying; Jiang, Baohong; Yang, Min; Liu, Xuan; Guo, Dean

    2016-01-01

    Gambogic acid (GA) is an anticancer agent in phase IIb clinical trial in China. In HeLa cells, GA inhibited cell proliferation, induced cell cycle arrest at G2/M phase and apoptosis, as showed by results of MTT assay and flow cytometric analysis. Possible target-related proteins of GA were searched using comparative proteomic analysis (2-DE) and nine proteins at early (3 h) stage together with nine proteins at late (24 h) stage were found. Vimentin was the only target-related protein found at both early and late stage. Results of both 2-DE analysis and Western blotting assay suggested cleavage of vimentin induced by GA. MS/MS analysis of cleaved vimentin peptides indicated possible cleavage sites of vimentin at or near ser51 and glu425. Results of targeted proteomic analysis showed that GA induced change in phosphorylation state of the vimentin head domain (aa51–64). Caspase inhibitors could not abrogate GA-induced cleavage of vimentin. Over-expression of vimentin ameliorated cytotoxicity of GA in HeLa cells. The GA-activated signal transduction, from p38 MAPK, heat shock protein 27 (HSP27), vimentin, dysfunction of cytoskeleton, to cell death, was predicted and then confirmed. Results of animal study showed that GA treatment inhibited tumor growth in HeLa tumor-bearing mice and cleavage of vimentin could be observed in tumor xenografts of GA-treated animals. Results of immunohistochemical staining also showed down-regulated vimentin level in tumor xenografts of GA-treated animals. Furthermore, compared with cytotoxicity of GA in HeLa cells, cytotoxicity of GA in MCF-7 cells with low level of vimentin was weaker whereas cytotoxicity of GA in MG-63 cells with high level of vimentin was stronger. These results indicated the important role of vimentin in the cytotoxicity of GA. The effects of GA on vimentin and other epithelial-to-mesenchymal transition (EMT) markers provided suggestion for better usage of GA in clinic. PMID:26499837

  6. Analysis of Large Seeds from Three Different Medicago truncatula Ecotypes Reveals a Potential Role of Hormonal Balance in Final Size Determination of Legume Grains

    Directory of Open Access Journals (Sweden)

    Kaustav Bandyopadhyay

    2016-09-01

    Full Text Available Legume seeds are important as protein and oil source for human diet. Understanding how their final seed size is determined is crucial to improve crop yield. In this study, we analyzed seed development of three accessions of the model legume, Medicago truncatula, displaying contrasted seed size. By comparing two large seed accessions to the reference accession A17, we described mechanisms associated with large seed size determination and potential factors modulating the final seed size. We observed that early events during embryogenesis had a major impact on final seed size and a delayed heart stage embryo development resulted to large seeds. We also observed that the difference in seed growth rate was mainly due to a difference in embryo cell number, implicating a role of cell division rate. Large seed accessions could be explained by an extended period of cell division due to a longer embryogenesis phase. According to our observations and recent reports, we observed that auxin (IAA and abscisic acid (ABA ratio could be a key determinant of cell division regulation at the end of embryogenesis. Overall, our study highlights that timing of events occurring during early seed development play decisive role for final seed size determination.

  7. Imaging a memory trace over half a life-time in the medial temporal lobe reveals a time-limited role of CA3 neurons in retrieval

    Science.gov (United States)

    Lux, Vanessa; Atucha, Erika; Kitsukawa, Takashi; Sauvage, Magdalena M

    2016-01-01

    Whether retrieval still depends on the hippocampus as memories age or relies then on cortical areas remains a major controversy. Despite evidence for a functional segregation between CA1, CA3 and parahippocampal areas, their specific role within this frame is unclear. Especially, the contribution of CA3 is questionable as very remote memories might be too degraded to be used for pattern completion. To identify the specific role of these areas, we imaged brain activity in mice during retrieval of recent, early remote and very remote fear memories by detecting the immediate-early gene Arc. Investigating correlates of the memory trace over an extended period allowed us to report that, in contrast to CA1, CA3 is no longer recruited in very remote retrieval. Conversely, we showed that parahippocampal areas are then maximally engaged. These results suggest a shift from a greater contribution of the trisynaptic loop to the temporoammonic pathway for retrieval. DOI: http://dx.doi.org/10.7554/eLife.11862.001 PMID:26880561

  8. The role of hyaluronan as a drug carrier to enhance the bioavailability of extended release ophthalmic formulations. Hyaluronan-timolol ionic complexes as a model case.

    Science.gov (United States)

    Battistini, F D; Tártara, L I; Boiero, C; Guzmán, M L; Luciani-Giaccobbe, L C; Palma, S D; Allemandi, D A; Manzo, R H; Olivera, M E

    2017-07-15

    The aim of this work was to obtain information concerning the properties of ophthalmic formulations based on hyaluronic-drug ionic complexes, to identify the factors that determine the onset, intensity and duration of the pharmacotherapeutic effect. Dispersions of a complex of 0.5% w/v of sodium hyaluronate (HyNa) loaded with 0.5% w/v of timolol maleate (TM) were obtained and presented a counterionic condensation higher than 75%. For comparison a similar complex obtained with hyaluronic acid (HyH) was also prepared. Although the viscosity of HyNa-TM was significantly higher than that of HyH-TM, in vitro release of TM from both complexes showed a similar extended drug release profile (20-31% over 5h) controlled by diffusion and ionic exchange. Ocular pharmacokinetic study performed in normotensive rabbits showed that HyNa-TM complex exhibited attractive bioavailability properties in the aqueous humor (AUC and Cmax significantly higher and later Tmax) compared to commercial TM eye-drops. Moreover, a more prolonged period of lowered intra-ocular pressure (10h) and a more intense hypotensive activity was observed after instillation of a drop of HyNa-TM as compared to the eye-drops. Such behavior was related to the longer pre-corneal residence times (400%) observed with HyNa-TM complex. No significant changes in rabbit transcorneal permeation were detected upon complexation. These results demonstrate that the ability of HyNa to modulate TM release, together with its mucoadhesiveness related to the viscosity, affected both the pharmacokinetic and pharmacodynamic parameters. The HyNa-TM complex is a potentially useful carrier for ocular drug delivery, which could improve the TM efficacy and reduce the frequency of administration to improve patient compliance. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Extended Enterprise performance Management

    NARCIS (Netherlands)

    Bobbink, Maria Lammerdina; Hartmann, Andreas

    2014-01-01

    The allegiance of partnering organisations and their employees to an Extended Enterprise performance is its proverbial sword of Damocles. Literature on Extended Enterprises focuses on collaboration, inter-organizational integration and learning to avoid diminishing or missing allegiance becoming an

  10. Combined Transcriptomic Analysis Revealed AKR1B10 Played an Important Role in Psoriasis through the Dysregulated Lipid Pathway and Overproliferation of Keratinocyte

    Directory of Open Access Journals (Sweden)

    Yunlu Gao

    2017-01-01

    Full Text Available RNA-seq has enabled in-depth analysis of the pathogenesis of psoriasis on the transcriptomic level, and many biomarkers have been discovered to be related to the immune response, lipid metabolism, and keratinocyte proliferation. However, few studies have combined analysis from various datasets. In this study, we integrated different psoriasis RNA-seq datasets to reveal the pathogenesis of psoriasis through the analysis of differentially expressed genes (DEGs, pathway analysis, and functional annotation. The revealed biomarkers were further validated through proliferation phenotypes. The results showed that DEGs were functionally related to lipid metabolism and keratinocyte differentiation dysregulation. The results also showed new biomarkers, such as AKR1B10 and PLA2G gene families, as well as pathways that include the PPAR signaling pathway, cytokine-cytokine receptor interaction, alpha-linoleic acid metabolism, and glycosphingolipid biosynthesis. Using siRNA knockdown assays, we further validated the role that the AKR1B10 gene plays in proliferation. Our study demonstrated not only the dysfunction of the AKR1B10 gene in lipid metabolizing but also its important role in the overproliferation and migration of keratinocyte, which provided evidence for further therapeutic uses for psoriasis.

  11. The glossyhead1 allele of acc1 reveals a principal role for multidomain acetyl-coenzyme a carboxylase in the biosynthesis of cuticular waxes by Arabidopsis

    KAUST Repository

    Lu, Shiyou

    2011-09-23

    A novel mutant of Arabidopsis (Arabidopsis thaliana), having highly glossy inflorescence stems, postgenital fusion in floral organs, and reduced fertility, was isolated from an ethyl methanesulfonate-mutagenized population and designated glossyhead1 (gsd1). The gsd1 locus was mapped to chromosome 1, and the causal gene was identified as a new allele of Acetyl-Coenzyme A Carboxylase1 (ACC1), a gene encoding the main enzyme in cytosolic malonyl-coenzyme A synthesis. This, to our knowledge, is the first mutant allele of ACC1 that does not cause lethality at the seed or early germination stage, allowing for the first time a detailed analysis of ACC1 function in mature tissues. Broad lipid profiling of mature gsd1 organs revealed a primary role for ACC1 in the biosynthesis of the very-long-chain fatty acids (C 20:0 or longer) associated with cuticular waxes and triacylglycerols. Unexpectedly, transcriptome analysis revealed that gsd1 has limited impact on any lipid metabolic networks but instead has a large effect on environmental stress-responsive pathways, especially senescence and ethylene synthesis determinants, indicating a possible role for the cytosolic malonyl-coenzyme A-derived lipids in stress response signaling. © 2011 American Society of Plant Biologists. All Rights Reserved.

  12. Epigenomic Co-localization and Co-evolution Reveal a Key Role for 5hmC as a Communication Hub in the Chromatin Network of ESCs

    Directory of Open Access Journals (Sweden)

    David Juan

    2016-02-01

    Full Text Available Summary: Epigenetic communication through histone and cytosine modifications is essential for gene regulation and cell identity. Here, we propose a framework that is based on a chromatin communication model to get insight on the function of epigenetic modifications in ESCs. The epigenetic communication network was inferred from genome-wide location data plus extensive manual annotation. Notably, we found that 5-hydroxymethylcytosine (5hmC is the most-influential hub of this network, connecting DNA demethylation to nucleosome remodeling complexes and to key transcription factors of pluripotency. Moreover, an evolutionary analysis revealed a central role of 5hmC in the co-evolution of chromatin-related proteins. Further analysis of regions where 5hmC co-localizes with specific interactors shows that each interaction points to chromatin remodeling, stemness, differentiation, or metabolism. Our results highlight the importance of cytosine modifications in the epigenetic communication of ESCs. : 5-hydroxymethylcytosine (5hmC plays a key role in the epigenomic communication network of embryonic stem cells. Juan et al. build a communication network based in co-localization of epigenomic data and literature. The analysis of the network and its components reveals that proteins reading and editing 5hmC co-evolve and serve as links between diverse molecular processes.

  13. Perspectives on extended Deterrence

    International Nuclear Information System (INIS)

    Tertrais, Bruno; Yost, David S.; Bunn, Elaine; Lee, Seok-soo; Levite, Ariel e.; Russell, James A.; Hokayem, Emile; Kibaroglu, Mustafa; Schulte, Paul; Thraenert, Oliver; Kulesa, Lukasz

    2010-05-01

    In November 2009, the Foundation for Strategic Research (Fondation pour la recherche strategique, FRS) convened a workshop on 'The Future of extended Deterrence', which included the participation of some of the best experts of this topic, from the United States, Europe, the Middle East and East Asia, as well as French and NATO officials. This document brings together the papers prepared for this seminar. Several of them were updated after the publication in April 2010 of the US Nuclear Posture Review. The seminar was organized with the support of the French Atomic energy Commission (Commissariat a l'energie atomique - CEA). Content: 1 - The future of extended deterrence: a brainstorming paper (Bruno Tertrais); 2 - US extended deterrence in NATO and North-East Asia (David S. Yost); 3 - The future of US extended deterrence (Elaine Bunn); 4 - The future of extended deterrence: a South Korean perspective (Seok-soo Lee); 5 - Reflections on extended deterrence in the Middle East (Ariel e. Levite); 6 - extended deterrence, security guarantees and nuclear weapons: US strategic and policy conundrums in the Gulf (James A. Russell); 7 - extended deterrence in the Gulf: a bridge too far? (Emile Hokayem); 8 - The future of extended deterrence: the case of Turkey (Mustafa Kibaroglu); 9 - The future of extended deterrence: a UK view (Paul Schulte); 10 - NATO and extended deterrence (Oliver Thraenert); 11 - extended deterrence and assurance in Central Europe (Lukasz Kulesa)

  14. Genome-Wide Survey of Pseudomonas aeruginosa PA14 Reveals a Role for the Glyoxylate Pathway and Extracellular Proteases in the Utilization of Mucin

    Science.gov (United States)

    Flynn, Jeffrey M.; Phan, Chi

    2017-01-01

    ABSTRACT Chronic airway infections by the opportunistic pathogen Pseudomonas aeruginosa are a major cause of mortality in cystic fibrosis (CF) patients. Although this bacterium has been extensively studied for its virulence determinants, biofilm growth, and immune evasion mechanisms, comparatively little is known about the nutrient sources that sustain its growth in vivo. Respiratory mucins represent a potentially abundant bioavailable nutrient source, although we have recently shown that canonical pathogens inefficiently use these host glycoproteins as a growth substrate. However, given that P. aeruginosa, particularly in its biofilm mode of growth, is thought to grow slowly in vivo, the inefficient use of mucin glycoproteins may be relevant to its persistence within the CF airways. To this end, we used whole-genome fitness analysis, combining transposon mutagenesis with high-throughput sequencing, to identify genetic determinants required for P. aeruginosa growth using intact purified mucins as a sole carbon source. Our analysis reveals a biphasic growth phenotype, during which the glyoxylate pathway and amino acid biosynthetic machinery are required for mucin utilization. Secondary analyses confirmed the simultaneous liberation and consumption of acetate during mucin degradation and revealed a central role for the extracellular proteases LasB and AprA. Together, these studies describe a molecular basis for mucin-based nutrient acquisition by P. aeruginosa and reveal a host-pathogen dynamic that may contribute to its persistence within the CF airways. PMID:28507068

  15. Identification of Novel Proteins Co-Purifying with Cockayne Syndrome Group B (CSB Reveals Potential Roles for CSB in RNA Metabolism and Chromatin Dynamics.

    Directory of Open Access Journals (Sweden)

    Serena Nicolai

    Full Text Available The CSB protein, a member of the SWI/SNF ATP dependent chromatin remodeling family of proteins, plays a role in a sub-pathway of nucleotide excision repair (NER known as transcription coupled repair (TCR. CSB is frequently mutated in Cockayne syndrome group B, a segmental progeroid human autosomal recessive disease characterized by growth failure and degeneration of multiple organs. Though initially classified as a DNA repair protein, recent studies have demonstrated that the loss of CSB results in pleiotropic effects. Identification of novel proteins belonging to the CSB interactome may be useful not only for predicting the molecular basis for diverse pathological symptoms of CS-B patients but also for unraveling the functions of CSB in addition to its authentic role in DNA repair. In this study, we performed tandem affinity purification (TAP technology coupled with mass spectrometry and co-immunoprecipitation studies to identify and characterize the proteins that potentially interact with CSB-TAP. Our approach revealed 33 proteins that were not previously known to interact with CSB. These newly identified proteins indicate potential roles for CSB in RNA metabolism involving repression and activation of transcription process and in the maintenance of chromatin dynamics and integrity.

  16. Extending extant models of the pathogenesis of borderline personality disorder to childhood borderline personality symptoms: the roles of affective dysfunction, disinhibition, and self- and emotion-regulation deficits.

    Science.gov (United States)

    Gratz, Kim L; Tull, Matthew T; Reynolds, Elizabeth K; Bagge, Courtney L; Latzman, Robert D; Daughters, Stacey B; Lejuez, C W

    2009-01-01

    Although research has been conducted on the course, consequences, and correlates of borderline personality disorder (BPD), little is known about its emergence in childhood, and no studies have examined the extent to which theoretical models of the pathogenesis of BPD in adults are applicable to the correlates of borderline personality symptoms in children. The goal of this study was to examine the interrelationships between two BPD-relevant personality traits (affective dysfunction and disinhibition), self- and emotion-regulation deficits, and childhood borderline personality symptoms among 263 children aged 9 to 13. We predicted that affective dysfunction, disinhibition, and their interaction would be associated with childhood borderline personality symptoms, and that self- and emotion-regulation deficits would mediate these relationships. Results provided support for the roles of both affective dysfunction and disinhibition (in the form of sensation seeking) in childhood borderline personality symptoms, as well as their hypothesized interaction. Further, both self- and emotion-regulation deficits partially mediated the relationship between affective dysfunction and childhood borderline personality symptoms. Finally, results provided evidence of different gender-based pathways to childhood borderline personality symptoms, suggesting that models of BPD among adults are more relevant to understanding the factors associated with borderline personality symptoms among girls than boys.

  17. ISC1-dependent metabolic adaptation reveals an indispensable role for mitochondria in induction of nuclear genes during the diauxic shift in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kitagaki, Hiroshi; Cowart, L Ashley; Matmati, Nabil; Montefusco, David; Gandy, Jason; de Avalos, Silvia Vaena; Novgorodov, Sergei A; Zheng, Jim; Obeid, Lina M; Hannun, Yusuf A

    2009-04-17

    Growth of Saccharomyces cerevisiae following glucose depletion (the diauxic shift) depends on a profound metabolic adaptation accompanied by a global reprogramming of gene expression. In this study, we provide evidence for a heretofore unsuspected role for Isc1p in mediating this reprogramming. Initial studies revealed that yeast cells deleted in ISC1, the gene encoding inositol sphingolipid phospholipase C, which resides in mitochondria in the post-diauxic phase, showed defective aerobic respiration in the post-diauxic phase but retained normal intrinsic mitochondrial functions, including intact mitochondrial DNA, normal oxygen consumption, and normal mitochondrial polarization. Microarray analysis revealed that the Deltaisc1 strain failed to up-regulate genes required for nonfermentable carbon source metabolism during the diauxic shift, thus suggesting a mechanism for the defective supply of respiratory substrates into mitochondria in the post-diauxic phase. This defect in regulating nuclear gene induction in response to a defect in a mitochondrial enzyme raised the possibility that mitochondria may initiate diauxic shift-associated regulation of nucleus-encoded genes. This was established by demonstrating that in respiratory-deficient petite cells these genes failed to be up-regulated across the diauxic shift in a manner similar to the Deltaisc1 strain. Isc1p- and mitochondrial function-dependent genes significantly overlapped with Adr1p-, Snf1p-, and Cat8p-dependent genes, suggesting some functional link among these factors. However, the retrograde response was not activated in Deltaisc1, suggesting that the response of Deltaisc1 cannot be simply attributed to mitochondrial dysfunction. These results suggest a novel role for Isc1p in allowing the reprogramming of gene expression during the transition from anaerobic to aerobic metabolism.

  18. Extended icosahedral structures

    CERN Document Server

    Jaric, Marko V

    1989-01-01

    Extended Icosahedral Structures discusses the concepts about crystal structures with extended icosahedral symmetry. This book is organized into six chapters that focus on actual modeling of extended icosahedral crystal structures. This text first presents a tiling approach to the modeling of icosahedral quasiperiodic crystals. It then describes the models for icosahedral alloys based on random connections between icosahedral units, with particular emphasis on diffraction properties. Other chapters examine the glassy structures with only icosahedral orientational order and the extent of tra

  19. Extending Database Integration Technology

    National Research Council Canada - National Science Library

    Buneman, Peter

    1999-01-01

    Formal approaches to the semantics of databases and database languages can have immediate and practical consequences in extending database integration technologies to include a vastly greater range...

  20. Identification of Nucleolus-Associated Chromatin Domains Reveals a Role for the Nucleolus in 3D Organization of the A. thaliana Genome

    Directory of Open Access Journals (Sweden)

    Frédéric Pontvianne

    2016-08-01

    Full Text Available The nucleolus is the site of rRNA gene transcription, rRNA processing, and ribosome biogenesis. However, the nucleolus also plays additional roles in the cell. We isolated nucleoli using fluorescence-activated cell sorting (FACS and identified nucleolus-associated chromatin domains (NADs by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein NUCLEOLIN 1 (NUC1. NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs, sub-telomeric regions, and mostly inactive protein-coding genes. However, NADs also include active rRNA genes and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased, and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance.

  1. miR-132/212 knockout mice reveal roles for these miRNAs in regulating cortical synaptic transmission and plasticity.

    Directory of Open Access Journals (Sweden)

    Judit Remenyi

    Full Text Available miR-132 and miR-212 are two closely related miRNAs encoded in the same intron of a small non-coding gene, which have been suggested to play roles in both immune and neuronal function. We describe here the generation and initial characterisation of a miR-132/212 double knockout mouse. These mice were viable and fertile with no overt adverse phenotype. Analysis of innate immune responses, including TLR-induced cytokine production and IFNβ induction in response to viral infection of primary fibroblasts did not reveal any phenotype in the knockouts. In contrast, the loss of miR-132 and miR-212, while not overtly affecting neuronal morphology, did affect synaptic function. In both hippocampal and neocortical slices miR-132/212 knockout reduced basal synaptic transmission, without affecting paired-pulse facilitation. Hippocampal long-term potentiation (LTP induced by tetanic stimulation was not affected by miR-132/212 deletion, whilst theta burst LTP was enhanced. In contrast, neocortical theta burst-induced LTP was inhibited by loss of miR-132/212. Together these results indicate that miR-132 and/or miR-212 play a significant role in synaptic function, possibly by regulating the number of postsynaptic AMPA receptors under basal conditions and during activity-dependent synaptic plasticity.

  2. Identification of Nucleolus-Associated Chromatin Domains Reveals a Role for the Nucleolus in 3D Organization of the A. thaliana Genome.

    Science.gov (United States)

    Pontvianne, Frédéric; Carpentier, Marie-Christine; Durut, Nathalie; Pavlištová, Veronika; Jaške, Karin; Schořová, Šárka; Parrinello, Hugues; Rohmer, Marine; Pikaard, Craig S; Fojtová, Miloslava; Fajkus, Jiří; Sáez-Vásquez, Julio

    2016-08-09

    The nucleolus is the site of rRNA gene transcription, rRNA processing, and ribosome biogenesis. However, the nucleolus also plays additional roles in the cell. We isolated nucleoli using fluorescence-activated cell sorting (FACS) and identified nucleolus-associated chromatin domains (NADs) by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein NUCLEOLIN 1 (NUC1). NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs), sub-telomeric regions, and mostly inactive protein-coding genes. However, NADs also include active rRNA genes and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased, and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  3. The Arabidopsis nox Mutant Lacking Carotene Hydroxylase Activity Reveals a Critical Role for Xanthophylls in Photosystem I Biogenesis[C][W

    Science.gov (United States)

    Dall’Osto, Luca; Piques, Maria; Ronzani, Michela; Molesini, Barbara; Alboresi, Alessandro; Cazzaniga, Stefano; Bassi, Roberto

    2013-01-01

    Carotenes, and their oxygenated derivatives xanthophylls, are essential components of the photosynthetic apparatus. They contribute to the assembly of photosynthetic complexes and participate in light absorption and chloroplast photoprotection. Here, we studied the role of xanthophylls, as distinct from that of carotenes, by characterizing a no xanthophylls (nox) mutant of Arabidopsis thaliana, which was obtained by combining mutations targeting the four carotenoid hydroxylase genes. nox plants retained α- and β-carotenes but were devoid in xanthophylls. The phenotype included depletion of light-harvesting complex (LHC) subunits and impairment of nonphotochemical quenching, two effects consistent with the location of xanthophylls in photosystem II antenna, but also a decreased efficiency of photosynthetic electron transfer, photosensitivity, and lethality in soil. Biochemical analysis revealed that the nox mutant was specifically depleted in photosystem I function due to a severe deficiency in PsaA/B subunits. While the stationary level of psaA/B transcripts showed no major differences between genotypes, the stability of newly synthesized PsaA/B proteins was decreased and translation of psaA/B mRNA was impaired in nox with respect to wild-type plants. We conclude that xanthophylls, besides their role in photoprotection and LHC assembly, are also needed for photosystem I core translation and stability, thus making these compounds indispensable for autotrophic growth. PMID:23396829

  4. The lethal giant larvae Gene in Tribolium castaneum: Molecular Properties and Roles in Larval and Pupal Development as Revealed by RNA Interference

    Directory of Open Access Journals (Sweden)

    Da Xiao

    2014-04-01

    Full Text Available We identified and characterized the TcLgl gene putatively encoding lethal giant larvae (Lgl protein from the red flour beetle (Tribolium castaneum. Analyses of developmental stage and tissue-specific expression patterns revealed that TcLgl was constitutively expressed. To examine the role of TcLgl in insect development, RNA interference was performed in early (1-day larvae, late (20-day larvae, and early (1-day pupae. The early larvae injected with double-stranded RNA of TcLgl (dsTcLgl at 100, 200, and 400 ng/larva failed to pupate, and 100% mortality was achieved within 20 days after the injection or before the pupation. The late larvae injected with dsTcLgl at these doses reduced the pupation rates to only 50.3%, 36.0%, and 18.2%, respectively. The un-pupated larvae gradually died after one week, and visually unaffected pupae failed to emerge into adults and died during the pupal stage. Similarly, when early pupae were injected with dsTcLgl at these doses, the normal eclosion rates were reduced to only 22.5%, 18.0%, and 11.2%, respectively, on day 7 after the injection, and all the adults with abnormal eclosion died in two days after the eclosion. These results indicate that TcLgl plays an essential role in insect development, especially during their metamorphosis.

  5. Extended family medicine training

    Science.gov (United States)

    Slade, Steve; Ross, Shelley; Lawrence, Kathrine; Archibald, Douglas; Mackay, Maria Palacios; Oandasan, Ivy F.

    2016-01-01

    Abstract Objective To examine trends in family medicine training at a time when substantial pedagogic change is under way, focusing on factors that relate to extended family medicine training. Design Aggregate-level secondary data analysis based on the Canadian Post-MD Education Registry. Setting Canada. Participants All Canadian citizens and permanent residents who were registered in postgraduate family medicine training programs within Canadian faculties of medicine from 1995 to 2013. Main outcome measures Number and proportion of family medicine residents exiting 2-year and extended (third-year and above) family medicine training programs, as well as the types and numbers of extended training programs offered in 2015. Results The proportion of family medicine trainees pursuing extended training almost doubled during the study period, going from 10.9% in 1995 to 21.1% in 2013. Men and Canadian medical graduates were more likely to take extended family medicine training. Among the 5 most recent family medicine exit cohorts (from 2009 to 2013), 25.9% of men completed extended training programs compared with 18.3% of women, and 23.1% of Canadian medical graduates completed extended training compared with 13.6% of international medical graduates. Family medicine programs vary substantially with respect to the proportion of their trainees who undertake extended training, ranging from a low of 12.3% to a high of 35.1% among trainees exiting from 2011 to 2013. Conclusion New initiatives, such as the Triple C Competency-based Curriculum, CanMEDS–Family Medicine, and Certificates of Added Competence, have emerged as part of family medicine education and credentialing. In acknowledgment of the potential effect of these initiatives, it is important that future research examine how pedagogic change and, in particular, extended training shapes the care family physicians offer their patients. As part of that research it will be important to measure the breadth and uptake of

  6. Analysis of the LIV system of Campylobacter jejuni reveals alternative roles for LivJ and LivK in commensalism beyond branched-chain amino acid transport.

    Science.gov (United States)

    Ribardo, Deborah A; Hendrixson, David R

    2011-11-01

    Campylobacter jejuni is a leading cause of diarrheal disease in humans and an intestinal commensal in poultry and other agriculturally important animals. These zoonotic infections result in significant amounts of C. jejuni present in the food supply to contribute to disease in humans. We previously found that a transposon insertion in Cjj81176_1038, encoding a homolog of the Escherichia coli LivJ periplasmic binding protein of the leucine, isoleucine, and valine (LIV) branched-chain amino acid transport system, reduced the commensal colonization capacity of C. jejuni 81-176 in chicks. Cjj81176_1038 is the first gene of a six-gene locus that encodes homologous components of the E. coli LIV system. By analyzing mutants with in-frame deletions of individual genes or pairs of genes, we found that this system constitutes a LIV transport system in C. jejuni responsible for a high level of leucine acquisition and, to a lesser extent, isoleucine and valine acquisition. Despite each LIV protein being required for branched-chain amino acid transport, only the LivJ and LivK periplasmic binding proteins were required for wild-type levels of commensal colonization of chicks. All LIV permease and ATPase components were dispensable for in vivo growth. These results suggest that the biological functions of LivJ and LivK for colonization are more complex than previously hypothesized and extend beyond a role for binding and acquiring branched-chain amino acids during commensalism. In contrast to other studies indicating a requirement and utilization of other specific amino acids for colonization, acquisition of branched-chain amino acids does not appear to be a determinant for C. jejuni during commensalism.

  7. The Role of Chaperone-subunit Usher Domain Interactions in the Mechanism of Bacterial Pilus Biogenesis Revealed by ESI-MS*

    Science.gov (United States)

    Morrissey, Bethny; Leney, Aneika C.; Toste Rêgo, Ana; Phan, Gilles; Allen, William J.; Verger, Denis; Waksman, Gabriel; Ashcroft, Alison E.; Radford, Sheena E.

    2012-01-01

    The PapC usher is a β-barrel outer membrane protein essential for assembly and secretion of P pili that are required for adhesion of pathogenic E. coli, which cause the development of pyelonephritis. Multiple protein subunits form the P pilus, the highly specific assembly of which is coordinated by the usher. Despite a wealth of structural knowledge, how the usher catalyzes subunit polymerization and orchestrates a correct and functional order of subunit assembly remain unclear. Here, the ability of the soluble N-terminal (UsherN), C-terminal (UsherC2), and Plug (UsherP) domains of the usher to bind different chaperone-subunit (PapDPapX) complexes is investigated using noncovalent electrospray ionization mass spectrometry. The results reveal that each usher domain is able to bind all six PapDPapX complexes, consistent with an active role of all three usher domains in pilus biogenesis. Using collision induced dissociation, combined with competition binding experiments and dissection of the adhesin subunit, PapG, into separate pilin and adhesin domains, the results reveal why PapG has a uniquely high affinity for the usher, which is consistent with this subunit always being displayed at the pilus tip. In addition, we show how the different soluble usher domains cooperate to coordinate and control efficient pilus assembly at the usher platform. As well as providing new information about the protein-protein interactions that determine pilus biogenesis, the results highlight the power of noncovalent MS to interrogate biological mechanisms, especially in complex mixtures of species. PMID:22371487

  8. The role of chaperone-subunit usher domain interactions in the mechanism of bacterial pilus biogenesis revealed by ESI-MS.

    Science.gov (United States)

    Morrissey, Bethny; Leney, Aneika C; Toste Rêgo, Ana; Phan, Gilles; Allen, William J; Verger, Denis; Waksman, Gabriel; Ashcroft, Alison E; Radford, Sheena E

    2012-07-01

    The PapC usher is a β-barrel outer membrane protein essential for assembly and secretion of P pili that are required for adhesion of pathogenic E. coli, which cause the development of pyelonephritis. Multiple protein subunits form the P pilus, the highly specific assembly of which is coordinated by the usher. Despite a wealth of structural knowledge, how the usher catalyzes subunit polymerization and orchestrates a correct and functional order of subunit assembly remain unclear. Here, the ability of the soluble N-terminal (UsherN), C-terminal (UsherC2), and Plug (UsherP) domains of the usher to bind different chaperone-subunit (PapDPapX) complexes is investigated using noncovalent electrospray ionization mass spectrometry. The results reveal that each usher domain is able to bind all six PapDPapX complexes, consistent with an active role of all three usher domains in pilus biogenesis. Using collision induced dissociation, combined with competition binding experiments and dissection of the adhesin subunit, PapG, into separate pilin and adhesin domains, the results reveal why PapG has a uniquely high affinity for the usher, which is consistent with this subunit always being displayed at the pilus tip. In addition, we show how the different soluble usher domains cooperate to coordinate and control efficient pilus assembly at the usher platform. As well as providing new information about the protein-protein interactions that determine pilus biogenesis, the results highlight the power of noncovalent MS to interrogate biological mechanisms, especially in complex mixtures of species.

  9. Elucidation of the regulatory role of the fructose operon reveals a novel target for enhancing the NADPH supply in Corynebacterium glutamicum.

    Science.gov (United States)

    Wang, Zhihao; Chan, Siu Hung Joshua; Sudarsan, Suresh; Blank, Lars M; Jensen, Peter Ruhdal; Solem, Christian

    2016-11-01

    The performance of Corynebacterium glutamicum cell factories producing compounds which rely heavily on NADPH has been reported to depend on the sugar being metabolized. While some aspects of this phenomenon have been elucidated, there are still many unresolved questions as to how sugar metabolism is linked to redox and to the general metabolism. We here provide new insights into the regulation of the metabolism of this important platform organism by systematically characterizing mutants carrying various lesions in the fructose operon. Initially, we found that a strain where the dedicated fructose uptake system had been inactivated (KO-ptsF) was hampered in growth on sucrose minimal medium, and suppressor mutants appeared readily. Comparative genomic analysis in conjunction with enzymatic assays revealed that suppression was linked to inactivation of the pfkB gene, encoding a fructose-1-phosphate kinase. Detailed characterization of KO-ptsF, KO-pfkB and double knock-out (DKO) derivatives revealed a strong role for sugar-phosphates, especially fructose-1-phosphate (F1P), in governing sugar as well as redox metabolism due to effects on transcriptional regulation of key genes. These findings allowed us to propose a simple model explaining the correlation between sugar phosphate concentration, gene expression and ultimately the observed phenotype. To guide us in our analysis and help us identify bottlenecks in metabolism we debugged an existing genome-scale model onto which we overlaid the transcriptome data. Based on the results obtained we managed to enhance the NADPH supply and transform the wild-type strain into delivering the highest yield of lysine ever obtained on sucrose and fructose, thus providing a good example of how regulatory mechanisms can be harnessed for bioproduction. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  10. The goya mouse mutant reveals distinct newly identified roles for MAP3K1 in the development and survival of cochlear sensory hair cells

    Directory of Open Access Journals (Sweden)

    Andrew Parker

    2015-12-01

    Full Text Available Mitogen-activated protein kinase, MAP3K1, plays an important role in a number of cellular processes, including epithelial migration during eye organogenesis. In addition, studies in keratinocytes indicate that MAP3K1 signalling through JNK is important for actin stress fibre formation and cell migration. However, MAP3K1 can also act independently of JNK in the regulation of cell proliferation and apoptosis. We have identified a mouse mutant, goya, which exhibits the eyes-open-at-birth and microphthalmia phenotypes. In addition, these mice also have hearing loss. The goya mice carry a splice site mutation in the Map3k1 gene. We show that goya and kinase-deficient Map3k1 homozygotes initially develop supernumerary cochlear outer hair cells (OHCs that subsequently degenerate, and a progressive profound hearing loss is observed by 9 weeks of age. Heterozygote mice also develop supernumerary OHCs, but no cellular degeneration or hearing loss is observed. MAP3K1 is expressed in a number of inner-ear cell types, including outer and inner hair cells, stria vascularis and spiral ganglion. Investigation of targets downstream of MAP3K1 identified an increase in p38 phosphorylation (Thr180/Tyr182 in multiple cochlear tissues. We also show that the extra OHCs do not arise from aberrant control of proliferation via p27KIP1. The identification of the goya mutant reveals a signalling molecule involved with hair-cell development and survival. Mammalian hair cells do not have the ability to regenerate after damage, which can lead to irreversible sensorineural hearing loss. Given the observed goya phenotype, and the many diverse cellular processes that MAP3K1 is known to act upon, further investigation of this model might help to elaborate upon the mechanisms underlying sensory hair cell specification, and pathways important for their survival. In addition, MAP3K1 is revealed as a new candidate gene for human sensorineural hearing loss.

  11. Metabolite profiling reveals a role for atypical cinnamyl alcohol dehydrogenase CAD1 in the synthesis of coniferyl alcohol in tobacco xylem.

    Science.gov (United States)

    Damiani, Isabelle; Morreel, Kris; Danoun, Saïda; Goeminne, Geert; Yahiaoui, Nabila; Marque, Christiane; Kopka, Joachim; Messens, Eric; Goffner, Deborah; Boerjan, Wout; Boudet, Alain-Michel; Rochange, Soizic

    2005-11-01

    In angiosperms, lignin is built from two main monomers, coniferyl and sinapyl alcohol, which are incorporated respectively as G and S units in the polymer. The last step of their synthesis has so far been considered to be performed by a family of dimeric cinnamyl alcohol dehydrogenases (CAD2). However, previous studies on Eucalyptus gunnii xylem showed the presence of an additional, structurally unrelated, monomeric CAD form named CAD1. This form reduces coniferaldehyde to coniferyl alcohol, but is inactive on sinapaldehyde. In this paper, we report the functional characterization of CAD1 in tobacco (Nicotiana tabacum L.). Transgenic tobacco plants with reduced CAD1 expression were obtained through an RNAi strategy. These plants displayed normal growth and development, and detailed biochemical studies were needed to reveal a role for CAD1. Lignin analyses showed that CAD1 down-regulation does not affect Klason lignin content, and has a moderate impact on G unit content of the non-condensed lignin fraction. However, comparative metabolic profiling of the methanol-soluble phenolic fraction from basal xylem revealed significant differences between CAD1 down-regulated and wild-type plants. Eight compounds were less abundant in CAD1 down-regulated lines, five of which were identified as dimers or trimers of monolignols, each containing at least one moiety derived from coniferyl alcohol. In addition, 3-trans-caffeoyl quinic acid accumulated in the transgenic plants. Together, our results support a significant contribution of CAD1 to the synthesis of coniferyl alcohol in planta, along with the previously characterized CAD2 enzymes.

  12. Proteomic and physiological analyses reveal the role of exogenous spermidine on cucumber roots in response to Ca(NO3)2 stress.

    Science.gov (United States)

    Du, Jing; Guo, Shirong; Sun, Jin; Shu, Sheng

    2018-05-01

    The mechanism of exogenous Spd-induced Ca(NO 3 ) 2 stress tolerance in cucumber was studied by proteomics and physiological analyses. Protein-protein interaction network revealed 13 key proteins involved in Spd-induced Ca(NO 3 ) 2 stress resistance. Ca(NO 3 ) 2 stress is one of the major reasons for secondary salinization that limits cucumber plant development in greenhouse. The conferred protective role of exogenous Spd on cucumber in response to Ca(NO 3 ) 2 stress cues involves changes at the cellular and physiological levels. To investigate the molecular foundation of exogenous Spd in Ca(NO 3 ) 2 stress tolerance, a proteomic approach was performed in our work. After a 9 days period of Ca(NO 3 ) 2 stress and/or exogenous Spd, 71 differential protein spots were confidently identified. The resulting proteins were enriched in seven different categories of biological processes, including protein metabolism, carbohydrate and energy metabolism, ROS homeostasis and stress defense, cell wall related, transcription, others and unknown. Protein metabolism (31.2%), carbohydrate and energy metabolism (15.6%), ROS homeostasis and stress defense (32.5%) were the three largest functional categories in cucumber root and most of them were significantly increased by exogenous Spd. The Spd-responsive protein interaction network revealed 13 key proteins, whose accumulation changes could be critical for Spd-induced resistance; all 13 proteins were upregulated by Spd at transcriptional and protein levels in response to Ca(NO 3 ) 2 stress. Furthermore, accumulation of antioxidant enzymes, non-enzymatic antioxidant and polyamines, along with reduction of H 2 O 2 and MDA, were detected after exogenous Spd application during Ca(NO 3 ) 2 stress. The results of these proteomic and physiological analyses in cucumber root may facilitate a better understanding of the underlying mechanism of Ca(NO 3 ) 2 stress tolerance mediated by exogenous Spd.

  13. The Extended Enterprise concept

    DEFF Research Database (Denmark)

    Larsen, Lars Bjørn; Vesterager, Johan; Gobbi, Chiara

    1999-01-01

    This paper provides an overview of the work that has been done regarding the Extended Enterprise concept in the Common Concept team of Globeman 21 including references to results deliverables concerning the development of the Extended Enterprise concept. The first section presents the basic concept...... picture from Globeman21, which illustrates the Globeman21 way of realising the Extended Enterprise concept. The second section presents the Globeman21 EE concept in a life cycle perspective, which to a large extent is based on the thoughts and ideas behind GERAM (ISO/DIS 15704)....

  14. STAT2 Knockout Syrian Hamsters Support Enhanced Replication and Pathogenicity of Human Adenovirus, Revealing an Important Role of Type I Interferon Response in Viral Control.

    Directory of Open Access Journals (Sweden)

    Karoly Toth

    2015-08-01

    Full Text Available Human adenoviruses have been studied extensively in cell culture and have been a model for studies in molecular, cellular, and medical biology. However, much less is known about adenovirus replication and pathogenesis in vivo in a permissive host because of the lack of an adequate animal model. Presently, the most frequently used permissive immunocompetent animal model for human adenovirus infection is the Syrian hamster. Species C human adenoviruses replicate in these animals and cause pathology that is similar to that seen with humans. Here, we report findings with a new Syrian hamster strain in which the STAT2 gene was functionally knocked out by site-specific gene targeting. Adenovirus-infected STAT2 knockout hamsters demonstrated an accentuated pathology compared to the wild-type control animals, and the virus load in the organs of STAT2 knockout animals was 100- to 1000-fold higher than that in wild-type hamsters. Notably, the adaptive immune response to adenovirus is not adversely affected in STAT2 knockout hamsters, and surviving hamsters cleared the infection by 7 to 10 days post challenge. We show that the Type I interferon pathway is disrupted in these hamsters, revealing the critical role of interferon-stimulated genes in controlling adenovirus infection. This is the first study to report findings with a genetically modified Syrian hamster infected with a virus. Further, this is the first study to show that the Type I interferon pathway plays a role in inhibiting human adenovirus replication in a permissive animal model. Besides providing an insight into adenovirus infection in humans, our results are also interesting from the perspective of the animal model: STAT2 knockout Syrian hamster may also be an important animal model for studying other viral infections, including Ebola-, hanta-, and dengue viruses, where Type I interferon-mediated innate immunity prevents wild type hamsters from being effectively infected to be used as

  15. An extended technicolor model

    International Nuclear Information System (INIS)

    Appelquist, T.; Terning, J.

    1994-01-01

    An extended technicolor model is constructed. Quark and lepton masses, spontaneous CP violation, and precision electroweak measurements are discussed. Dynamical symmetry breaking is analyzed using the concept of the big MAC (most attractive channel)

  16. Extending mine life

    International Nuclear Information System (INIS)

    Anon.

    1984-01-01

    Mine layouts, new machines and techniques, research into problem areas of ground control and so on, are highlighted in this report on extending mine life. The main resources taken into account are coal mining, uranium mining, molybdenum and gold mining

  17. Rational extended thermodynamics

    CERN Document Server

    Müller, Ingo

    1998-01-01

    Ordinary thermodynamics provides reliable results when the thermodynamic fields are smooth, in the sense that there are no steep gradients and no rapid changes. In fluids and gases this is the domain of the equations of Navier-Stokes and Fourier. Extended thermodynamics becomes relevant for rapidly varying and strongly inhomogeneous processes. Thus the propagation of high­ frequency waves, and the shape of shock waves, and the regression of small-scale fluctuation are governed by extended thermodynamics. The field equations of ordinary thermodynamics are parabolic while extended thermodynamics is governed by hyperbolic systems. The main ingredients of extended thermodynamics are • field equations of balance type, • constitutive quantities depending on the present local state and • entropy as a concave function of the state variables. This set of assumptions leads to first order quasi-linear symmetric hyperbolic systems of field equations; it guarantees the well-posedness of initial value problems and f...

  18. The dialogically extended mind

    DEFF Research Database (Denmark)

    Fusaroli, Riccardo; Gangopadhyay, Nivedita; Tylén, Kristian

    2014-01-01

    A growing conceptual and empirical literature is advancing the idea that language extends our cognitive skills. One of the most influential positions holds that language – qua material symbols – facilitates individual thought processes by virtue of its material properties. Extending upon this model...... relate our approach to other ideas about collective minds and review a number of empirical studies to identify the mechanisms enabling the constitution of interpersonal cognitive systems....

  19. Extending Mondrian Memory Protection

    Science.gov (United States)

    2010-11-01

    a kernel semaphore is locked or unlocked. In addition, we extended the system call interface to receive notifications about user-land locking...operations (such as calls to the mutex and semaphore code provided by the C library). By patching the dynamically loadable GLibC5, we are able to test... semaphores , and spinlocks. RTO-MP-IST-091 10- 9 Extending Mondrian Memory Protection to loading extension plugins. This prevents any untrusted code

  20. Extended Life Coolant Testing

    Science.gov (United States)

    2016-06-06

    number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE (DD-MM-YYYY) 06-06-2016 2. REPORT TYPE Interim Report 3. DATES COVERED ... Corrosion Testing of Traditional and Extended Life Coolants 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Hansen, Gregory A. T...providing vehicle specific coolants. Several laboratory corrosion tests were performed according to ASTM D1384 and D2570, but with a 2.5x extended time

  1. Proteomic analysis reveals the important roles of alpha-5-collagen and ATP5β during skin ulceration syndrome progression of sea cucumber Apostichopus japonicus.

    Science.gov (United States)

    Zhao, Zelong; Jiang, Jingwei; Pan, Yongjia; Sun, Hongjuan; Guan, Xiaoyan; Gao, Shan; Chen, Zhong; Dong, Ying; Zhou, Zunchun

    2018-03-20

    Apostichopus japonicus is one of the most important aquaculture species in China. Skin ulceration syndrome (SUS) of sea cucumber is a common and serious disease affected the development of A. japonicus culture industry. To better understand the response mechanisms of A. japonicus during SUS progression, the protein variations in the body wall of A. japonicus at different stages of SUS were investigated by a comparative proteomic approach based on isobaric tags for relative and absolute quantification. A total of 1449 proteins were identified from the samples at different SUS stages. Among these proteins, 145 proteins were differentially expressed in the SUS-related samples compared to those of healthy A. japonicus. These differentially expressed proteins involved a wide range of functions. Among these differentially expressed proteins, only two proteins, alpha-5-collagen and an unknown function protein, were differentially expressed during the whole progression of SUS compared with healthy A. japonicus. In addition, ATP synthase subunit beta (ATP5β) interacted with a variety of proteins with different functions during the SUS progression. These results implied that alpha-5-collagen and ATP5β could play important roles during the SUS progression of A. japonicus. Our study provided a new sight to understand the molecular responses of sea cucumber during the SUS progression and accumulated data for the prevention of SUS in sea cucumber aquaculture. The current study aimed to reveal how the body wall of Apostichopus japonicus response to skin ulceration syndrome (SUS). To the best of our knowledge, this is the first proteomic study analyzing the differences in protein profile of sea cucumber during the whole SUS progression. By analyzing the expression differences of the proteome via isobaric labeling-based quantitative proteomic, we identified some proteins which may play important roles during the SUS progression. According to the enrichment analyses of these

  2. Genetic activation, inactivation and deletion reveal a limited and nuanced role for somatostatin-containing basal forebrain neurons in behavioral state control.

    Science.gov (United States)

    Anaclet, Christelle; De Luca, Roberto; Venner, Anne; Malyshevskaya, Olga; Lazarus, Michael; Arrigoni, Elda; Fuller, Patrick M

    2018-05-07

    Recent studies have identified an especially important role for basal forebrain GABAergic (BF VGAT ) neurons in the regulation of behavioral waking and fast cortical rhythms associated with cognition. However, BF VGAT neurons comprise several neurochemically and anatomically distinct sub-populations, including parvalbumin- and somatostatin-containing BF VGAT neurons (BF Parv and BF SOM ), and it was recently reported that optogenetic activation of BF SOM neurons increases the probability of a wakefulness to non-rapid-eye movement (NREM) sleep transition when stimulated during the animal's rest period. This finding was unexpected given that most BF SOM neurons are not NREM sleep active and that central administration of the synthetic SOM analog, octreotide, suppresses NREM sleep or increases REM sleep. Here we employed a combination of genetically-driven chemogenetic and optogenetic activation, chemogenetic inhibition and ablation approaches to further explore the in vivo role of BF SOM neurons in arousal control. Our findings indicate that acute activation or inhibition of BF SOM neurons is neither wakefulness- nor NREM sleep-promoting, is without significant effect on the EEG, and that chronic loss of these neurons is without effect on total 24h sleep amounts, although a small but significant increase in waking was observed in the lesioned mice during the early active period. Our in vitro cell recordings further reveal electrophysiological heterogeneity in BF SOM neurons, specifically suggesting at least two distinct sub-populations. Taken together our data support the more nuanced view that BF SOM are electrically heterogeneous and are not NREM sleep- or wake-promoting per se , but may exert, in particular during the early active period, a modest inhibitory influence on arousal circuitry. SIGNIFICANCE STATEMENT The cellular basal forebrain (BF) is a highly complex area of the brain that is implicated in a wide-range of higher-level neurobiological processes

  3. 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.

  4. In situ camera observations reveal major role of zooplankton in modulating marine snow formation during an upwelling-induced plankton bloom

    Science.gov (United States)

    Taucher, Jan; Stange, Paul; Algueró-Muñiz, María; Bach, Lennart T.; Nauendorf, Alice; Kolzenburg, Regina; Büdenbender, Jan; Riebesell, Ulf

    2018-05-01

    Particle aggregation and the consequent formation of marine snow alter important properties of biogenic particles (size, sinking rate, degradability), thus playing a key role in controlling the vertical flux of organic matter to the deep ocean. However, there are still large uncertainties about rates and mechanisms of particle aggregation, as well as the role of plankton community structure in modifying biomass transfer from small particles to large fast-sinking aggregates. Here we present data from a high-resolution underwater camera system that we used to observe particle size distributions and formation of marine snow (aggregates >0.5 mm) over the course of a 9-week in situ mesocosm experiment in the Eastern Subtropical North Atlantic. After an oligotrophic phase of almost 4 weeks, addition of nutrient-rich deep water (650 m) initiated the development of a pronounced diatom bloom and the subsequent formation of large marine snow aggregates in all 8 mesocosms. We observed a substantial time lag between the peaks of chlorophyll a and marine snow biovolume of 9-12 days, which is much longer than previously reported and indicates a marked temporal decoupling of phytoplankton growth and marine snow formation during our study. Despite this time lag, our observations revealed substantial transfer of biomass from small particle sizes (single phytoplankton cells and chains) to marine snow aggregates of up to 2.5 mm diameter (ESD), with most of the biovolume being contained in the 0.5-1 mm size range. Notably, the abundance and community composition of mesozooplankton had a substantial influence on the temporal development of particle size spectra and formation of marine snow aggregates: While higher copepod abundances were related to reduced aggregate formation and biomass transfer towards larger particle sizes, the presence of appendicularia and doliolids enhanced formation of large marine snow. Furthermore, we combined in situ particle size distributions with

  5. 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

    2015-02-01

    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.

  6. Gcn4 misregulation reveals a direct role for the evolutionary conserved EKC/KEOPS in the t6A modification of tRNAs.

    Science.gov (United States)

    Daugeron, Marie-Claire; Lenstra, Tineke L; Frizzarin, Martina; El Yacoubi, Basma; Liu, Xipeng; Baudin-Baillieu, Agnès; Lijnzaad, Philip; Decourty, Laurence; Saveanu, Cosmin; Jacquier, Alain; Holstege, Frank C P; de Crécy-Lagard, Valérie; van Tilbeurgh, Herman; Libri, Domenico

    2011-08-01

    The EKC/KEOPS complex is universally conserved in Archaea and Eukarya and has been implicated in several cellular processes, including transcription, telomere homeostasis and genomic instability. However, the molecular function of the complex has remained elusive so far. We analyzed the transcriptome of EKC/KEOPS mutants and observed a specific profile that is highly enriched in targets of the Gcn4p transcriptional activator. GCN4 expression was found to be activated at the translational level in mutants via the defective recognition of the inhibitory upstream ORFs (uORFs) present in its leader. We show that EKC/KEOPS mutants are defective for the N6-threonylcarbamoyl adenosine modification at position 37 (t(6)A(37)) of tRNAs decoding ANN codons, which affects initiation at the inhibitory uORFs and provokes Gcn4 de-repression. Structural modeling reveals similarities between Kae1 and bacterial enzymes involved in carbamoylation reactions analogous to t(6)A(37) formation, supporting a direct role for the EKC in tRNA modification. These findings are further supported by strong genetic interactions of EKC mutants with a translation initiation factor and with threonine biosynthesis genes. Overall, our data provide a novel twist to understanding the primary function of the EKC/KEOPS and its impact on several essential cellular functions like transcription and telomere homeostasis.

  7. Evolutionary and polymorphism analyses reveal the central role of BTN3A2 in the concerted evolution of the BTN3 gene family.

    Science.gov (United States)

    Afrache, Hassnae; Pontarotti, Pierre; Abi-Rached, Laurent; Olive, Daniel

    2017-06-01

    The butyrophilin 3 (BTN3) receptors are implicated in the T lymphocytes regulation and present a wide plasticity in mammals. In order to understand how these genes have been diversified, we studied their evolution and show that the three human BTN3 are the result of two successive duplications in Primates and that the three genes are present in Hominoids and the Old World Monkey groups. A thorough phylogenetic analysis reveals a concerted evolution of BTN3 characterized by a strong and recurrent homogenization of the region encoding the signal peptide and the immunoglobulin variable (IgV) domain in Hominoids, where the sequences of BTN3A1 or BTN3A3 are replaced by BTN3A2 sequence. In human, the analysis of the diversity of these genes in 1683 individuals representing 26 worldwide populations shows that the three genes are polymorphic, with more than 46 alleles for each gene, and marked by extreme homogenization of the IgV sequences. The same analysis performed for the BTN2 genes shows also a concerted evolution; however, it is not as strong and recurrent as for BTN3. This study shows that BTN3 receptors are marked by extreme concerted evolution at the IgV domain and that BTN3A2 plays a central role in this evolution.

  8. SUMO modification through rapamycin-mediated heterodimerization reveals a dual role for Ubc9 in targeting RanGAP1 to nuclear pore complexes

    International Nuclear Information System (INIS)

    Zhu Shanshan; Zhang Hong; Matunis, Michael J.

    2006-01-01

    SUMOs (small ubiquitin-related modifiers) are eukaryotic proteins that are covalently conjugated to other proteins and thereby regulate a wide range of important cellular processes. The molecular mechanisms by which SUMO modification influences the functions of most target proteins and cellular processes, however, remain poorly defined. A major obstacle to investigating the effects of SUMO modification is the availability of a system for selectively inducing the modification or demodification of an individual protein. To address this problem, we have developed a procedure using the rapamycin heterodimerizer system. This procedure involves co-expression of rapamycin-binding domain fusion proteins of SUMO and candidate SUMO substrates in living cells. Treating cells with rapamycin induces a tight association between SUMO and a single SUMO substrate, thereby allowing specific downstream effects to be analyzed. Using RanGAP1 as a model SUMO substrate, the heterodimerizer system was used to investigate the molecular mechanism by which SUMO modification targets RanGAP1 from the cytoplasm to nuclear pore complexes (NPCs). Our results revealed a dual role for Ubc9 in targeting RanGAP1 to NPCs: In addition to conjugating SUMO-1 to RanGAP1, Ubc9 is also required to form a stable ternary complex with SUMO-1 modified RanGAP1 and Nup358. As illustrated by our studies, the rapamycin heterodimerizer system represents a novel tool for studying the molecular effects of SUMO modification

  9. SWATH label-free proteomics analyses revealed the roles of oxidative stress and antioxidant defensing system in sclerotia formation of Polyporus umbellatus

    Science.gov (United States)

    Li, Bing; Tian, Xiaofang; Wang, Chunlan; Zeng, Xu; Xing, Yongmei; Ling, Hong; Yin, Wanqiang; Tian, Lixia; Meng, Zhixia; Zhang, Jihui; Guo, Shunxing

    2017-01-01

    Understanding the initiation and maturing mechanisms is important for rational manipulating sclerotia differentiation and growth from hypha of Polyporus umbellatus. Proteomes in P. umbellatus sclerotia and hyphae at initial, developmental and mature phases were studied. 1391 proteins were identified by nano-liquid chromatograph-mass spectrometry (LC-MS) in Data Dependant Acquisition mode, and 1234 proteins were quantified successfully by Sequential Window Acquisition of all THeoretical fragment ion spectra-MS (SWATH-MS) technology. There were 347 differentially expressed proteins (DEPs) in sclerotia at initial phase compared with those in hypha, and the DEP profiles were dynamically changing with sclerotia growth. Oxidative stress (OS) in sclerotia at initial phase was indicated by the repressed proteins of respiratory chain, tricarboxylic acid cycle and the activation of glycolysis/gluconeogenesis pathways were determined based on DEPs. The impact of glycolysis/gluconeogenesis on sclerotium induction was further verified by glycerol addition assays, in which 5% glycerol significantly increased sclerotial differentiation rate and biomass. It can be speculated that OS played essential roles in triggering sclerotia differentiation from hypha of P. umbellatus, whereas antioxidant activity associated with glycolysis is critical for sclerotia growth. These findings reveal a mechanism for sclerotial differentiation in P. umbellatus, which may also be applicable for other fungi.

  10. Haplotype Analysis of the Pre-harvest Sprouting Resistance Locus Phs-A1 Reveals a Causal Role of TaMKK3-A in Global Germplasm.

    Science.gov (United States)

    Shorinola, Oluwaseyi; Balcárková, Barbara; Hyles, Jessica; Tibbits, Josquin F G; Hayden, Matthew J; Holušova, Katarina; Valárik, Miroslav; Distelfeld, Assaf; Torada, Atsushi; Barrero, Jose M; Uauy, Cristobal

    2017-01-01

    Pre-harvest sprouting (PHS) is an important cause of quality loss in many cereal crops and is particularly prevalent and damaging in wheat. Resistance to PHS is therefore a valuable target trait in many breeding programs. The Phs-A1 locus on wheat chromosome arm 4AL has been consistently shown to account for a significant proportion of natural variation to PHS in diverse mapping populations. However, the deployment of sprouting resistance is confounded by the fact that different candidate genes, including the tandem duplicated Plasma Membrane 19 ( PM19 ) genes and the mitogen-activated protein kinase kinase 3 ( TaMKK3-A) gene, have been proposed to underlie Phs-A1 . To further define the Phs-A1 locus, we constructed a physical map across this interval in hexaploid and tetraploid wheat. We established close proximity of the proposed candidate genes which are located within a 1.2 Mb interval. Genetic characterization of diverse germplasm used in previous genetic mapping studies suggests that TaMKK3-A , and not PM19 , is the major gene underlying the Phs-A1 effect in European, North American, Australian and Asian germplasm. We identified the non-dormant TaMKK3-A allele at low frequencies within the A-genome diploid progenitor Triticum urartu genepool, and show an increase in the allele frequency in modern varieties. In United Kingdom varieties, the frequency of the dormant TaMKK3-A allele was significantly higher in bread-making quality varieties compared to feed and biscuit-making cultivars. Analysis of exome capture data from 58 diverse hexaploid wheat accessions identified fourteen haplotypes across the extended Phs-A1 locus and four haplotypes for TaMKK3-A . Analysis of these haplotypes in a collection of United Kingdom and Australian cultivars revealed distinct major dormant and non-dormant Phs-A1 haplotypes in each country, which were either rare or absent in the opposing germplasm set. The diagnostic markers and haplotype information reported in the study will

  11. Haplotype Analysis of the Pre-harvest Sprouting Resistance Locus Phs-A1 Reveals a Causal Role of TaMKK3-A in Global Germplasm

    Directory of Open Access Journals (Sweden)

    Oluwaseyi Shorinola

    2017-09-01

    Full Text Available Pre-harvest sprouting (PHS is an important cause of quality loss in many cereal crops and is particularly prevalent and damaging in wheat. Resistance to PHS is therefore a valuable target trait in many breeding programs. The Phs-A1 locus on wheat chromosome arm 4AL has been consistently shown to account for a significant proportion of natural variation to PHS in diverse mapping populations. However, the deployment of sprouting resistance is confounded by the fact that different candidate genes, including the tandem duplicated Plasma Membrane 19 (PM19 genes and the mitogen-activated protein kinase kinase 3 (TaMKK3-A gene, have been proposed to underlie Phs-A1. To further define the Phs-A1 locus, we constructed a physical map across this interval in hexaploid and tetraploid wheat. We established close proximity of the proposed candidate genes which are located within a 1.2 Mb interval. Genetic characterization of diverse germplasm used in previous genetic mapping studies suggests that TaMKK3-A, and not PM19, is the major gene underlying the Phs-A1 effect in European, North American, Australian and Asian germplasm. We identified the non-dormant TaMKK3-A allele at low frequencies within the A-genome diploid progenitor Triticum urartu genepool, and show an increase in the allele frequency in modern varieties. In United Kingdom varieties, the frequency of the dormant TaMKK3-A allele was significantly higher in bread-making quality varieties compared to feed and biscuit-making cultivars. Analysis of exome capture data from 58 diverse hexaploid wheat accessions identified fourteen haplotypes across the extended Phs-A1 locus and four haplotypes for TaMKK3-A. Analysis of these haplotypes in a collection of United Kingdom and Australian cultivars revealed distinct major dormant and non-dormant Phs-A1 haplotypes in each country, which were either rare or absent in the opposing germplasm set. The diagnostic markers and haplotype information reported in the

  12. Renormalization of Extended QCD2

    International Nuclear Information System (INIS)

    Fukaya, Hidenori; Yamamura, Ryo

    2015-01-01

    Extended QCD (XQCD), proposed by Kaplan [D. B. Kaplan, arXiv:1306.5818], is an interesting reformulation of QCD with additional bosonic auxiliary fields. While its partition function is kept exactly the same as that of original QCD, XQCD naturally contains properties of low-energy hadronic models. We analyze the renormalization group flow of 2D (X)QCD, which is solvable in the limit of a large number of colors N c , to understand what kind of roles the auxiliary degrees of freedom play and how the hadronic picture emerges in the low-energy region

  13. Extended spider cognition.

    Science.gov (United States)

    Japyassú, Hilton F; Laland, Kevin N

    2017-05-01

    There is a tension between the conception of cognition as a central nervous system (CNS) process and a view of cognition as extending towards the body or the contiguous environment. The centralised conception requires large or complex nervous systems to cope with complex environments. Conversely, the extended conception involves the outsourcing of information processing to the body or environment, thus making fewer demands on the processing power of the CNS. The evolution of extended cognition should be particularly favoured among small, generalist predators such as spiders, and here, we review the literature to evaluate the fit of empirical data with these contrasting models of cognition. Spiders do not seem to be cognitively limited, displaying a large diversity of learning processes, from habituation to contextual learning, including a sense of numerosity. To tease apart the central from the extended cognition, we apply the mutual manipulability criterion, testing the existence of reciprocal causal links between the putative elements of the system. We conclude that the web threads and configurations are integral parts of the cognitive systems. The extension of cognition to the web helps to explain some puzzling features of spider behaviour and seems to promote evolvability within the group, enhancing innovation through cognitive connectivity to variable habitat features. Graded changes in relative brain size could also be explained by outsourcing information processing to environmental features. More generally, niche-constructed structures emerge as prime candidates for extending animal cognition, generating the selective pressures that help to shape the evolving cognitive system.

  14. The goya mouse mutant reveals distinct newly identified roles for MAP3K1 in the development and survival of cochlear sensory hair cells.

    Science.gov (United States)

    Parker, Andrew; Cross, Sally H; Jackson, Ian J; Hardisty-Hughes, Rachel; Morse, Susan; Nicholson, George; Coghill, Emma; Bowl, Michael R; Brown, Steve D M

    2015-12-01

    Mitogen-activated protein kinase, MAP3K1, plays an important role in a number of cellular processes, including epithelial migration during eye organogenesis. In addition, studies in keratinocytes indicate that MAP3K1 signalling through JNK is important for actin stress fibre formation and cell migration. However, MAP3K1 can also act independently of JNK in the regulation of cell proliferation and apoptosis. We have identified a mouse mutant, goya, which exhibits the eyes-open-at-birth and microphthalmia phenotypes. In addition, these mice also have hearing loss. The goya mice carry a splice site mutation in the Map3k1 gene. We show that goya and kinase-deficient Map3k1 homozygotes initially develop supernumerary cochlear outer hair cells (OHCs) that subsequently degenerate, and a progressive profound hearing loss is observed by 9 weeks of age. Heterozygote mice also develop supernumerary OHCs, but no cellular degeneration or hearing loss is observed. MAP3K1 is expressed in a number of inner-ear cell types, including outer and inner hair cells, stria vascularis and spiral ganglion. Investigation of targets downstream of MAP3K1 identified an increase in p38 phosphorylation (Thr180/Tyr182) in multiple cochlear tissues. We also show that the extra OHCs do not arise from aberrant control of proliferation via p27KIP1. The identification of the goya mutant reveals a signalling molecule involved with hair-cell development and survival. Mammalian hair cells do not have the ability to regenerate after damage, which can lead to irreversible sensorineural hearing loss. Given the observed goya phenotype, and the many diverse cellular processes that MAP3K1 is known to act upon, further investigation of this model might help to elaborate upon the mechanisms underlying sensory hair cell specification, and pathways important for their survival. In addition, MAP3K1 is revealed as a new candidate gene for human sensorineural hearing loss. © 2015. Published by The Company of

  15. Attributing Increased River Flooding in the Future: Hydrodynamic Downscaling Reveals Role of Plant Physiological Responses to Increased CO2 is First Order

    Science.gov (United States)

    Fowler, M. D.; Kooperman, G. J.; Pritchard, M. S.; Randerson, J. T.

    2017-12-01

    River flooding events, which are the most frequently occurring natural disaster today, are expected to become more frequent and intense in response to climate change. However, the magnitude of these changes remains debated, in part due to uncertainty in our understanding of the physical processes that contribute to these events and their representation in global climate models. While the intensification of precipitation has been shown to be a primary driver of increased flooding, plant physiological responses to increasing CO2 may also play an important role. As the atmospheric concentration of CO2 increases, plants may respond by reducing the width of their stomata (i.e. stomatal conductance), which can decrease water lost through transpiration and in turn maintain higher soil moisture levels. On long timescales, reduced transpiration has been shown to increase average runoff, but on short timescales elevated soil moisture can also increase instantaneous runoff by limiting the rate at which water is able to infiltrate the soil surface. Here, through hydrodynamic downscaling, we isolate the portion of flooding amplification that can be attributed to the physiological response to increasing CO2. This builds on a new analysis that has revealed such physiological effects can rival changes caused by the atmospheric response alone in the tails of the runoff distribution. We use a set of four simulations run with the Community Earth System Model: one pre-industrial control simulation and three others that are forced with four times CO2. In the three climate change simulations, the increased CO2 is applied only to the land-surface, only to the atmosphere, and to both, respectively. Thirty years of daily runoff from these experiments are used as input for the hydrodynamic CaMa-Flood model. Our results reveal that both the radiative and physiological responses to climate change contribute significantly to future changes in flood return period and inundated area. This

  16. Extending quantum mechanics entails extending special relativity

    International Nuclear Information System (INIS)

    Aravinda, S; Srikanth, R

    2016-01-01

    The complementarity between signaling and randomness in any communicated resource that can simulate singlet statistics is generalized by relaxing the assumption of free will in the choice of measurement settings. We show how to construct an ontological extension for quantum mechanics (QMs) through the oblivious embedding of a sound simulation protocol in a Newtonian spacetime. Minkowski or other intermediate spacetimes are ruled out as the locus of the embedding by virtue of hidden influence inequalities. The complementarity transferred from a simulation to the extension unifies a number of results about quantum non-locality, and implies that special relativity has a different significance for the ontological model and for the operational theory it reproduces. Only the latter, being experimentally accessible, is required to be Lorentz covariant. There may be certain Lorentz non-covariant elements at the ontological level, but they will be inaccessible at the operational level in a valid extension. Certain arguments against the extendability of QM, due to Conway and Kochen (2009) and Colbeck and Renner (2012), are attributed to their assumption that the spacetime at the ontological level has Minkowski causal structure. (paper)

  17. Transcriptional Activities of the Microbial Consortium Living with the Marine Nitrogen-Fixing Cyanobacterium Trichodesmium Reveal Potential Roles in Community-Level Nitrogen Cycling.

    Science.gov (United States)

    Lee, Michael D; Webb, Eric A; Walworth, Nathan G; Fu, Fei-Xue; Held, Noelle A; Saito, Mak A; Hutchins, David A

    2018-01-01

    Trichodesmium is a globally distributed cyanobacterium whose nitrogen-fixing capability fuels primary production in warm oligotrophic oceans. Like many photoautotrophs, Trichodesmium serves as a host to various other microorganisms, yet little is known about how this associated community modulates fluxes of environmentally relevant chemical species into and out of the supraorganismal structure. Here, we utilized metatranscriptomics to examine gene expression activities of microbial communities associated with Trichodesmium erythraeum (strain IMS101) using laboratory-maintained enrichment cultures that have previously been shown to harbor microbial communities similar to those of natural populations. In enrichments maintained under two distinct CO 2 concentrations for ∼8 years, the community transcriptional profiles were found to be specific to the treatment, demonstrating a restructuring of overall gene expression had occurred. Some of this restructuring involved significant increases in community respiration-related transcripts under elevated CO 2 , potentially facilitating the corresponding measured increases in host nitrogen fixation rates. Particularly of note, in both treatments, community transcripts involved in the reduction of nitrate, nitrite, and nitrous oxide were detected, suggesting the associated organisms may play a role in colony-level nitrogen cycling. Lastly, a taxon-specific analysis revealed distinct ecological niches of consistently cooccurring major taxa that may enable, or even encourage, the stable cohabitation of a diverse community within Trichodesmium consortia. IMPORTANCE Trichodesmium is a genus of globally distributed, nitrogen-fixing marine cyanobacteria. As a source of new nitrogen in otherwise nitrogen-deficient systems, these organisms help fuel carbon fixation carried out by other more abundant photoautotrophs and thereby have significant roles in global nitrogen and carbon cycling. Members of the Trichodesmium genus tend to

  18. 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.

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

    International Nuclear Information System (INIS)

    Shin, Kwang-Soo; Kim, Young Hwan; Yu, Jae-Hyuk

    2015-01-01

    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

  20. Crystal Structure of the Extended-Spectrum β-Lactamase PER-2 and Insights into the Role of Specific Residues in the Interaction with β-Lactams and β-Lactamase Inhibitors

    Science.gov (United States)

    Ruggiero, Melina; Kerff, Frédéric; Herman, Raphaël; Sapunaric, Frédéric; Galleni, Moreno; Gutkind, Gabriel; Charlier, Paulette; Sauvage, Eric

    2014-01-01

    PER-2 belongs to a small (7 members to date) group of extended-spectrum β-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most β-lactams. In this study, we determined the X-ray structure of PER-2 at 2.20 Å and evaluated the possible role of several residues in the structure and activity toward β-lactams and mechanism-based inhibitors. PER-2 is defined by the presence of a singular trans bond between residues 166 to 167, which generates an inverted Ω loop, an expanded fold of this domain that results in a wide active site cavity that allows for efficient hydrolysis of antibiotics like the oxyimino-cephalosporins, and a series of exclusive interactions between residues not frequently involved in the stabilization of the active site in other class A β-lactamases. PER β-lactamases might be included within a cluster of evolutionarily related enzymes harboring the conserved residues Asp136 and Asn179. Other signature residues that define these enzymes seem to be Gln69, Arg220, Thr237, and probably Arg/Lys240A (“A” indicates an insertion according to Ambler's scheme for residue numbering in PER β-lactamases), with structurally important roles in the stabilization of the active site and proper orientation of catalytic water molecules, among others. We propose, supported by simulated models of PER-2 in combination with different β-lactams, the presence of a hydrogen-bond network connecting Ser70-Gln69-water-Thr237-Arg220 that might be important for the proper activity and inhibition of the enzyme. Therefore, we expect that mutations occurring in these positions will have impacts on the overall hydrolytic behavior. PMID:25070104

  1. Antiferromagnetism, charge density wave, and d-wave superconductivity in the extended t-J-U model: role of intersite Coulomb interaction and a critical overview of renormalized mean field theory.

    Science.gov (United States)

    Abram, M; Zegrodnik, M; Spałek, J

    2017-09-13

    In the first part of the paper, we study the stability of antiferromagnetic (AF), charge density wave (CDW), and superconducting (SC) states within the t-J-U-V model of strongly correlated electrons by using the statistically consistent Gutzwiller approximation (SGA). We concentrate on the role of the intersite Coulomb interaction term V in stabilizing the CDW phase. In particular, we show that the charge ordering appears only above a critical value of V in a limited hole-doping range δ. The effect of the V term on SC and AF phases is that a strong interaction suppresses SC, whereas the AF order is not significantly influenced by its presence. In the second part, separate calculations for the case of a pure SC phase have been carried out within an extended approach (the diagrammatic expansion for the Gutzwiller wave function, DE-GWF) in order to analyze the influence of the intersite Coulomb repulsion on the SC phase with the higher-order corrections included beyond the SGA method. The upper concentration for the SC disappearance decreases with increasing V, bringing the results closer to experiment. In appendices A and B we discuss the ambiguity connected with the choice of the Gutzwiller renormalization factors within the renormalized mean filed theory when either AF or CDW orders are considered. At the end, we overview briefly the possible extensions of the current models to put descriptions of the SC, AF, and CDW states on equal footing.

  2. Transcriptional regulation of the outer membrane porin gene ompW reveals its physiological role during the transition from the aerobic to the anaerobic lifestyle of Escherichia coli

    Directory of Open Access Journals (Sweden)

    Minfeng eXiao

    2016-05-01

    Full Text Available Understanding bacterial physiology relies on elucidating the regulatory mechanisms and cellular functions of those differentially expressed genes in response to environmental changes. A widespread Gram-negative bacterial outer membrane protein OmpW has been implicated in the adaptation to stresses in various species. It is recently found to be present in the regulon of the global anaerobic transcription factor FNR and ArcA in E. coli. However, little is known about the physiological implications of this regulatory disposition. In this study, we demonstrate that transcription of ompW is indeed mediated by a series of global regulators involved in the anaerobiosis of E. coli. We show that FNR can both activate and repress the expression of ompW through its direct binding to two distinctive sites, -81.5 and -126.5 bp respectively, on ompW promoter. ArcA also participates in repression of ompW under anaerobic condition, but in an FNR dependent manner. Additionally, ompW is also subject to the regulation by CRP and NarL which senses the availability and types of carbon sources and respiration electron acceptors in the environment respectively, implying a role of OmpW in the carbon and energy metabolism of E. coli during its anaerobic adaptation. Molecular docking reveals that OmpW can bind fumarate, an alternative electron acceptor in anaerobic respiration, with sufficient affinity. Moreover, supplement of fumarate or succinate which belongs to the C4-dicarboxylates family of metabolite, to E. coli culture rescues OmpW-mediated colicin S4 killing. Taken together, we propose that OmpW is involved in anaerobic carbon and energy metabolism to mediate the transition from aerobic to anaerobic lifestyle in E. coli.

  3. A crucial role of paralogous β-defensin genes in the Chinese alligator innate immune system revealed by the first determination of a Crocodilia defensin cluster.

    Science.gov (United States)

    Tang, Ke-Yi; Wang, Xin; Wan, Qiu-Hong; Fang, Sheng-Guo

    2018-04-01

    The β-defensin, one of the antimicrobial peptides (AMPs), is a significant component of the innate immune with a broad range of antimicrobial activities. Differing from the widely-studied mammals and birds, limited information about β-defensins has been reported in reptiles, especially in crocodilians. As a same ancient species as dinosaurs and the most endangered species of 23 crocodilians, the survival of Chinese alligator (Alligator sinensis) means a powerful immune system and possible involvement of AMPs in its immune resistance. In this study, we identified 20 novel Alligator sinensisβ-defensin genes (AsBDs) from a 390 kb region using bioinformatic and experimental approaches, and successfully distinguished six orthologous AsBDs to birds and nine paralogous AsBDs undergoing gene duplication events. The amino acid alignment shows that the AsBD paralogs, like α-defensins, encode a significantly longer pro-piece comparing with the orthologs. The calculation of non-synonymous (d N ) and synonymous (d S ) substitutions in the mature peptide reveals that the AsBD paralogs experience a significantly higher selective pressure (d N /d S ) than the orthologs, but a similar evolutionary force to α-defensins. The gene expression result indicates that the AsBD paralogs have a significantly higher expression level than the orthologos in gastrointestinal tract where the host is vulnerable to enteric pathogenic bacteria, as observed in α-defensins. These three pieces of evidence demonstrate that the AsBD paralogs do play an important role in maintaining long-term survival of this endangered reptile. Thus, this survey of AsBDs on the genomic structure, evolutionary characteristics, and expression pattern provides a genetic and immunological foundation for further investigating their antimicrobial function and alternative antibiotics potentiality. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  4. A synthetic eicosanoid LX-mimetic unravels host-donor interactions in allogeneic BMT-induced GvHD to reveal an early protective role for host neutrophils.

    Science.gov (United States)

    Devchand, Pallavi R; Schmidt, Birgitta A; Primo, Valeria C; Zhang, Qing-yin; Arnaout, M Amin; Serhan, Charles N; Nikolic, Boris

    2005-02-01

    Lipoxin A(4) (LXA(4)) and aspirin-triggered 15-epi-LXA(4) are potent endogenous lipid mediators thought to define the inflammatory set-point. We used single prophylactic administrations of a synthetic aspirin-triggered lipoxin A(4) signal mimetic, ATLa, to probe dynamics of early host-donor interactions in a mouse model for the inflammation-associated multifactorial disease of allogeneic bone marrow transplant (BMT) -induced graft-vs.-host disease (GvHD). We first demonstrated that both host and donor are responsive to the ATLa signals. The simple and restricted regimen of a single prophylactic administration of ATLa [100 ng/mL to donor cells or 1 microg (approximately 50 microg/kg) i.v. to host] was sufficient to delay death. Clinical indicators of weight, skin lesions, diarrhea and eye inflammation were monitored. Histological analyses on day 45 post-BMT showed that the degree of cellular trafficking, particularly neutrophil infiltrate, and protection of end-organ target pathology are different, depending on whether the host or donor was treated with ATLa. Taken together, these results chart some ATLa protective effects on GvHD cellular dynamics over time and identify a previously unrecognized effect of host neutrophils in the early phase post-BMT as important determinants in the dynamics of GvHD onset and progression.-Devchand, P. R., Schmidt, B. A., Primo, V. C., Zhang, Q.-y., Arnaout, M. A., Serhan, C. N., Nikolic, B. A synthetic eicosanoid LX-mimetic unravels host-donor interactions in allogeneic BMT-induced GvHD to reveal an early protective role for host neutrophils.

  5. Phenotypic characterization of Grm1crv4 mice reveals a functional role for the type 1 metabotropic glutamate receptor in bone mineralization.

    Science.gov (United States)

    Musante, Ilaria; Mattinzoli, Deborah; Otescu, Lavinia Alexandra; Bossi, Simone; Ikehata, Masami; Gentili, Chiara; Cangemi, Giuliana; Gatti, Cinzia; Emionite, Laura; Messa, Piergiorgio; Ravazzolo, Roberto; Rastaldi, Maria Pia; Riccardi, Daniela; Puliti, Aldamaria

    2017-01-01

    Recent increasing evidence supports a role for neuronal type signaling in bone. Specifically glutamate receptors have been found in cells responsible for bone remodeling, namely the osteoblasts and the osteoclasts. While most studies have focused on ionotropic glutamate receptors, the relevance of the metabotropic glutamate signaling in bone is poorly understood. Specifically type 1 metabotropic glutamate (mGlu1) receptors are expressed in bone, but the effect of its ablation on skeletal development has never been investigated. Here we report that Grm1 crv4/crv4 mice, homozygous for an inactivating mutation of the mGlu1 receptor, and mainly characterized by ataxia and renal dysfunction, exhibit decreased body weight, bone length and bone mineral density compared to wild type (WT) animals. Blood analyses of the affected mice demonstrate the absence of changes in circulating factors, such as vitamin D and PTH, suggesting renal damage is not the main culprit of the skeletal phenotype. Cultures of osteoblasts lacking functional mGlu1 receptors exhibit less homogeneous collagen deposition than WT cells, and present increased expression of osteocalcin, a marker of osteoblast maturation. These data suggest that the skeletal damage is directly linked to the absence of the receptor, which in turn leads to osteoblasts dysfunction and earlier maturation. Accordingly, skeletal histomorphology suggests that Grm1 crv4/crv4 mice exhibit enhanced bone maturation, resulting in premature fusion of the growth plate and shortened long bones, and further slowdown of bone apposition rate compared to the WT animals. In summary, this work reveals novel functions of mGlu1 receptors in the bone and indicates that in osteoblasts mGlu1 receptors are necessary for production of normal bone matrix, longitudinal bone growth, and normal skeletal development. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Yeast Interspecies Comparative Proteomics Reveals Divergence in Expression Profiles and Provides Insights into Proteome Resource Allocation and Evolutionary Roles of Gene Duplication*

    Science.gov (United States)

    Kito, Keiji; Ito, Haruka; Nohara, Takehiro; Ohnishi, Mihoko; Ishibashi, Yuko; Takeda, Daisuke

    2016-01-01

    Omics analysis is a versatile approach for understanding the conservation and diversity of molecular systems across multiple taxa. In this study, we compared the proteome expression profiles of four yeast species (Saccharomyces cerevisiae, Saccharomyces mikatae, Kluyveromyces waltii, and Kluyveromyces lactis) grown on glucose- or glycerol-containing media. Conserved expression changes across all species were observed only for a small proportion of all proteins differentially expressed between the two growth conditions. Two Kluyveromyces species, both of which exhibited a high growth rate on glycerol, a nonfermentative carbon source, showed distinct species-specific expression profiles. In K. waltii grown on glycerol, proteins involved in the glyoxylate cycle and gluconeogenesis were expressed in high abundance. In K. lactis grown on glycerol, the expression of glycolytic and ethanol metabolic enzymes was unexpectedly low, whereas proteins involved in cytoplasmic translation, including ribosomal proteins and elongation factors, were highly expressed. These marked differences in the types of predominantly expressed proteins suggest that K. lactis optimizes the balance of proteome resource allocation between metabolism and protein synthesis giving priority to cellular growth. In S. cerevisiae, about 450 duplicate gene pairs were retained after whole-genome duplication. Intriguingly, we found that in the case of duplicates with conserved sequences, the total abundance of proteins encoded by a duplicate pair in S. cerevisiae was similar to that of protein encoded by nonduplicated ortholog in Kluyveromyces yeast. Given the frequency of haploinsufficiency, this observation suggests that conserved duplicate genes, even though minor cases of retained duplicates, do not exhibit a dosage effect in yeast, except for ribosomal proteins. Thus, comparative proteomic analyses across multiple species may reveal not only species-specific characteristics of metabolic processes under

  7. Small interference RNA profiling reveals the essential role of human membrane trafficking genes in mediating the infectious entry of dengue virus

    Directory of Open Access Journals (Sweden)

    Chu Justin

    2010-02-01

    Full Text Available Abstract Background Dengue virus (DENV is the causative agent of Dengue fever and the life-threatening Dengue Haemorrhagic fever or Dengue shock syndrome. In the absence of anti-viral agents or vaccine, there is an urgent need to develop an effective anti-viral strategy against this medically important viral pathogen. The initial interplay between DENV and the host cells may represent one of the potential anti-viral targeting sites. Currently the involvements of human membrane trafficking host genes or factors that mediate the infectious cellular entry of dengue virus are not well defined. Results In this study, we have used a targeted small interfering RNA (siRNA library to identify and profile key cellular genes involved in processes of endocytosis, cytoskeletal dynamics and endosome trafficking that are important and essential for DENV infection. The infectious entry of DENV into Huh7 cells was shown to be potently inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis. The important role of clathrin-mediated endocytosis was confirmed by the expression of well-characterized dominant-negative mutants of genes in this pathway and by using the clathrin endocytosis inhibitor chlorpromazine. Furthermore, DENV infection was shown to be sensitive to the disruption of human genes in regulating the early to late endosomal trafficking as well as the endosomal acidic pH. The importance and involvement of both actin and microtubule dynamics in mediating the infectious entry of DENV was also revealed in this study. Conclusions Together, the findings from this study have provided a detail profiling of the human membrane trafficking cellular genes and the mechanistic insight into the interplay of these host genes with DENV to initiate an infection, hence broadening our understanding on the entry pathway of this medically important viral pathogen. These data may also provide a new potential avenue for development of anti

  8. Resistance to pyridine-based inhibitor KF116 reveals an unexpected role of integrase in HIV-1 Gag-Pol polyprotein proteolytic processing.

    Science.gov (United States)

    Hoyte, Ashley C; Jamin, Augusta V; Koneru, Pratibha C; Kobe, Matthew J; Larue, Ross C; Fuchs, James R; Engelman, Alan N; Kvaratskhelia, Mamuka

    2017-12-01

    The pyridine-based multimerization selective HIV-1 integrase (IN) inhibitors (MINIs) are a distinct subclass of allosteric IN inhibitors. MINIs potently inhibit HIV-1 replication during virion maturation by inducing hyper- or aberrant IN multimerization but are largely ineffective during the early steps of viral replication. Here, we investigated the mechanism for the evolution of a triple IN substitution (T124N/V165I/T174I) that emerges in cell culture with a representative MINI, KF116. We show that HIV-1 NL4-3(IN T124N/V165I/T174I) confers marked (>2000-fold) resistance to KF116. Two IN substitutions (T124N/T174I) directly weaken inhibitor binding at the dimer interface of the catalytic core domain but at the same time markedly impair HIV-1 replication capacity. Unexpectedly, T124N/T174I IN substitutions inhibited proteolytic processing of HIV-1 polyproteins Gag and Gag-Pol, resulting in immature virions. Strikingly, the addition of the third IN substitution (V165I) restored polyprotein processing, virus particle maturation, and significant levels of replication capacity. These results reveal an unanticipated role of IN for polyprotein proteolytic processing during virion morphogenesis. The complex evolutionary pathway for the emergence of resistant viruses, which includes the need for the compensatory V165I IN substitution, highlights a relatively high genetic barrier exerted by MINI KF116. Additionally, we have solved the X-ray structure of the drug-resistant catalytic core domain protein, which provides means for rational development of second-generation MINIs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Correlation-based network analysis of metabolite and enzyme profiles reveals a role of citrate biosynthesis in modulating N and C metabolism in Zea mays

    Directory of Open Access Journals (Sweden)

    David Toubiana

    2016-07-01

    Full Text Available To investigate the natural variability of leaf metabolism and enzymatic activity in a maize inbred population, statistical and network analyses were employed on metabolite and enzyme profiles. The test of coefficient of variation showed that sugars and amino acids displayed opposite trends in their variance within the population, consistently with their related enzymes. The overall higher CV values for metabolites as compared to the tested enzymes are indicative for their greater phenotypic plasticity. H2 tests revealed galactinol (1 and asparagine (0.91 as the highest scorers among metabolites and nitrate reductase (0.73, NAD-glutamate dehydrogenase (0.52, and phosphoglucomutase (0.51 among enzymes. The overall low H2 scores for metabolites and enzymes are suggestive for a great environmental impact or gene-environment interaction. Correlation-based network generation followed by community detection analysis, partitioned the network into three main communities and one dyad, (i reflecting the different levels of phenotypic plasticity of the two molecular classes as observed for the CV values and (ii highlighting the concerted changes between classes of chemically related metabolites. Community 1 is composed mainly of enzymes and specialized metabolites, community 2’ is enriched in N-containing compounds and phosphorylated-intermediates. The third community contains mainly organic acids and sugars. Cross-community linkages are supported by aspartate, by the photorespiration amino acids glycine and serine, by the metabolically related GABA and putrescine, and by citrate. The latter displayed the strongest node-betweenness value (185.25 of all nodes highlighting its fundamental structural role in the connectivity of the network by linking between different communities and to the also strongly connected enzyme aldolase.

  10. Role of DNA Repair Factor Xeroderma Pigmentosum Protein Group C in Response to Replication Stress As Revealed by DNA Fragile Site Affinity Chromatography and Quantitative Proteomics.

    Science.gov (United States)

    Beresova, Lucie; Vesela, Eva; Chamrad, Ivo; Voller, Jiri; Yamada, Masayuki; Furst, Tomas; Lenobel, Rene; Chroma, Katarina; Gursky, Jan; Krizova, Katerina; Mistrik, Martin; Bartek, Jiri

    2016-12-02

    Replication stress (RS) fuels genomic instability and cancer development and may contribute to aging, raising the need to identify factors involved in cellular responses to such stress. Here, we present a strategy for identification of factors affecting the maintenance of common fragile sites (CFSs), which are genomic loci that are particularly sensitive to RS and suffer from increased breakage and rearrangements in tumors. A DNA probe designed to match the high flexibility island sequence typical for the commonly expressed CFS (FRA16D) was used as specific DNA affinity bait. Proteins significantly enriched at the FRA16D fragment under normal and replication stress conditions were identified using stable isotope labeling of amino acids in cell culture-based quantitative mass spectrometry. The identified proteins interacting with the FRA16D fragment included some known CFS stabilizers, thereby validating this screening approach. Among the hits from our screen so far not implicated in CFS maintenance, we chose Xeroderma pigmentosum protein group C (XPC) for further characterization. XPC is a key factor in the DNA repair pathway known as global genomic nucleotide excision repair (GG-NER), a mechanism whose several components were enriched at the FRA16D fragment in our screen. Functional experiments revealed defective checkpoint signaling and escape of DNA replication intermediates into mitosis and the next generation of XPC-depleted cells exposed to RS. Overall, our results provide insights into an unexpected biological role of XPC in response to replication stress and document the power of proteomics-based screening strategies to elucidate mechanisms of pathophysiological significance.

  11. Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3′-5′)-cyclic-GMP in virulence

    Science.gov (United States)

    Kulesekara, Hemantha; Lee, Vincent; Brencic, Anja; Liberati, Nicole; Urbach, Jonathan; Miyata, Sachiko; Lee, Daniel G.; Neely, Alice N.; Hyodo, Mamoru; Hayakawa, Yoshihiro; Ausubel, Frederick M.; Lory, Stephen

    2006-01-01

    The opportunistic pathogen Pseudomonas aeruginosa is responsible for systemic infections in immunocompromised individuals and chronic respiratory disease in patients with cystic fibrosis. Cyclic nucleotides are known to play a variety of roles in the regulation of virulence-related factors in pathogenic bacteria. A set of P. aeruginosa genes, encoding proteins that contain putative domains characteristic of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) that are responsible for the maintenance of cellular levels of the second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) was identified in the annotated genomes of P. aeruginosa strains PAO1 and PA14. Although the majority of these genes are components of the P. aeruginosa core genome, several are located on presumptive horizontally acquired genomic islands. A comprehensive analysis of P. aeruginosa genes encoding the enzymes of c-di-GMP metabolism (DGC- and PDE-encoding genes) was carried out to analyze the function of c-di-GMP in two disease-related phenomena, cytotoxicity and biofilm formation. Analysis of the phenotypes of DGC and PDE mutants and overexpressing clones revealed that certain virulence-associated traits are controlled by multiple DGCs and PDEs through alterations in c-di-GMP levels. A set of mutants in selected DGC- and PDE-encoding genes exhibited attenuated virulence in a mouse infection model. Given that insertions in different DGC and PDE genes result in distinct phenotypes, it seems likely that the formation or degradation of c-di-GMP by these enzymes is in highly localized and intimately linked to particular targets of c-di-GMP action. PMID:16477007

  12. An Extended Duopoly Game.

    Science.gov (United States)

    Eckalbar, John C.

    2002-01-01

    Illustrates how principles and intermediate microeconomic students can gain an understanding for strategic price setting by playing a relatively large oligopoly game. Explains that the game extends to a continuous price space and outlines appropriate applications. Offers the Mathematica code to instructors so that the assumptions of the game can…

  13. Transversally extended string

    International Nuclear Information System (INIS)

    Akama, Keiichi

    1988-01-01

    Starting with the space-time action of the transversally extended string, we derive its world-sheet action, which is that of a gravitational and gauge theory with matter fields on the world-sheet, with additional effects of the second fundamental quantity. (author)

  14. Extended artistic appreciation.

    Science.gov (United States)

    Wilson, Robert A

    2013-04-01

    I propose that in at least some cases, objects of artistic appreciation are best thought of not simply as causes of artistic appreciation, but as parts of the cognitive machinery that drives aesthetic appreciation. In effect, this is to say that aesthetic appreciation operates via extended cognitive systems.

  15. Towards Extended Vantage Theory

    Science.gov (United States)

    Glaz, Adam

    2010-01-01

    The applicability of Vantage Theory (VT), a model of (colour) categorization, to linguistic data largely depends on the modifications and adaptations of the model for the purpose. An attempt to do so proposed here, called Extended Vantage Theory (EVT), slightly reformulates the VT conception of vantage by capitalizing on some of the entailments of…

  16. RNA-seq reveals the RNA binding proteins, Hfq and RsmA, play various roles in virulence, antibiotic production and genomic flux in Serratia sp. ATCC 39006.

    Science.gov (United States)

    Wilf, Nabil M; Reid, Adam J; Ramsay, Joshua P; Williamson, Neil R; Croucher, Nicholas J; Gatto, Laurent; Hester, Svenja S; Goulding, David; Barquist, Lars; Lilley, Kathryn S; Kingsley, Robert A; Dougan, Gordon; Salmond, George Pc

    2013-11-22

    Serratia sp. ATCC 39006 (S39006) is a Gram-negative enterobacterium that is virulent in plant and animal models. It produces a red-pigmented trypyrrole secondary metabolite, prodigiosin (Pig), and a carbapenem antibiotic (Car), as well as the exoenzymes, pectate lyase and cellulase. Secondary metabolite production in this strain is controlled by a complex regulatory network involving quorum sensing (QS). Hfq and RsmA (two RNA binding proteins and major post-transcriptional regulators of gene expression) play opposing roles in the regulation of several key phenotypes within S39006. Prodigiosin and carbapenem production was abolished, and virulence attenuated, in an S39006 ∆hfq mutant, while the converse was observed in an S39006 rsmA transposon insertion mutant. In order to define the complete regulon of Hfq and RsmA, deep sequencing of cDNA libraries (RNA-seq) was used to analyse the whole transcriptome of S39006 ∆hfq and rsmA::Tn mutants. Moreover, we investigated global changes in the proteome using an LC-MS/MS approach. Analysis of differential gene expression showed that Hfq and RsmA directly or indirectly regulate (at the level of RNA) 4% and 19% of the genome, respectively, with some correlation between RNA and protein expression. Pathways affected include those involved in antibiotic regulation, virulence, flagella synthesis, and surfactant production. Although Hfq and RsmA are reported to activate flagellum production in E. coli and an adherent-invasive E. coli hfq mutant was shown to have no flagella by electron microscopy, we found that flagellar production was increased in the S39006 rsmA and hfq mutants. Additionally, deletion of rsmA resulted in greater genomic flux with increased activity of two mobile genetic elements. This was confirmed by qPCR and analysis of rsmA culture supernatant revealed the presence of prophage DNA and phage particles. Finally, expression of a hypothetical protein containing DUF364 increased prodigiosin production and was

  17. Extended Irreversible Thermodynamics

    CERN Document Server

    Jou, David

    2010-01-01

    This is the 4th edition of the highly acclaimed monograph on Extended Irreversible Thermodynamics, a theory that goes beyond the classical theory of irreversible processes. In contrast to the classical approach, the basic variables describing the system are complemented by non-equilibrium quantities. The claims made for extended thermodynamics are confirmed by the kinetic theory of gases and statistical mechanics. The book covers a wide spectrum of applications, and also contains a thorough discussion of the foundations and the scope of the current theories on non-equilibrium thermodynamics. For this new edition, the authors critically revised existing material while taking into account the most recent developments in fast moving fields such as heat transport in micro- and nanosystems or fast solidification fronts in materials sciences. Several fundamental chapters have been revisited emphasizing physics and applications over mathematical derivations. Also, fundamental questions on the definition of non-equil...

  18. Propelling Extended Objects

    Science.gov (United States)

    Humbert, Richard

    2010-01-01

    A force acting on just part of an extended object (either a solid or a volume of a liquid) can cause all of it to move. That motion is due to the transmission of the force through the object by its material. This paper discusses how the force is distributed to all of the object by a gradient of stress or pressure in it, which creates the local…

  19. Extending Critical Performativity

    DEFF Research Database (Denmark)

    Spicer, André; Alvesson, Mats; Kärreman, Dan

    2016-01-01

    In this article we extend the debate about critical performativity. We begin by outlining the basic tenets of critical performativity and how this has been applied in the study of management and organization. We then address recent critiques of critical performance. We note these arguments suffer...... of public importance; engaging with non-academic groups using dialectical reasoning; scaling up insights through movement building; and propagating deliberation...

  20. Proteomic plasma membrane profiling reveals an essential role for gp96 in the cell surface expression of LDLR family members, including the LDL receptor and LRP6.

    Science.gov (United States)

    Weekes, Michael P; Antrobus, Robin; Talbot, Suzanne; Hör, Simon; Simecek, Nikol; Smith, Duncan L; Bloor, Stuart; Randow, Felix; Lehner, Paul J

    2012-03-02

    The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96.

  1. Revealing Hearts

    DEFF Research Database (Denmark)

    Saghaug, Kristin Falck; Pattison, George; Lindgren, Peter

    2014-01-01

    with reference to Tillich’s account of the meaning of revelation through culture and art, summed up in the statement that “(...) revelation is the manifestation of the ground of being for human knowledge” (Tillich, 1951, p.94), which, we argue, can be extended to everyday experiences, for example, in business......Some small business owners want to balance personal values as well as economic values. “I have to follow my heart” or “it must be meaningful” some of them say. But how might they be able to know what gives meaning to the heart? The philosophical theologian Paul Tillich finds that the problem...... life. In Tillich’s own terms, even preliminary concerns may point at an ultimate concern (Tillich, 1964), which can also be understood as ‘knowledge of the heart’. Our account is also connected to wider discussions concerning the nature of intuition....

  2. Extended suicide with a pet.

    Science.gov (United States)

    Cooke, Brian K

    2013-01-01

    The combination of the killing of a pet and a suicide is a perplexing scenario that is largely unexplored in the literature. Many forensic psychiatrists and psychologists may be unaccustomed to considering the significance of the killing of a pet. The subject is important, however, because many people regard their pets as members of their family. A case is presented of a woman who killed her pet dog and herself by carbon monoxide poisoning. The purpose of this article is to provide an initial exploration of the topic of extended suicide with a pet. Forensic mental health evaluations may have a role in understanding the etiology of this event and in opining as to the culpability of individuals who attempt to or successfully kill a pet and then commit suicide. Because the scientific literature is lacking, there is a need to understand this act from a variety of perspectives. First, a social and anthropological perspective will be presented that summarizes the history of the practice of killing of one's pet, with a focus on the ancient Egyptians. A clinical context will examine what relationship animals have to mental illness. A vast body of existing scientific data showing the relevance of human attachment to pets suggests that conclusions from the phenomena of homicide-suicide and filicide-suicide are applicable to extended suicide with a pet. Finally, recommendations will be proposed for both clinical and forensic psychiatrists faced with similar cases.

  3. Extended Testability Analysis Tool

    Science.gov (United States)

    Melcher, Kevin; Maul, William A.; Fulton, Christopher

    2012-01-01

    The Extended Testability Analysis (ETA) Tool is a software application that supports fault management (FM) by performing testability analyses on the fault propagation model of a given system. Fault management includes the prevention of faults through robust design margins and quality assurance methods, or the mitigation of system failures. Fault management requires an understanding of the system design and operation, potential failure mechanisms within the system, and the propagation of those potential failures through the system. The purpose of the ETA Tool software is to process the testability analysis results from a commercial software program called TEAMS Designer in order to provide a detailed set of diagnostic assessment reports. The ETA Tool is a command-line process with several user-selectable report output options. The ETA Tool also extends the COTS testability analysis and enables variation studies with sensor sensitivity impacts on system diagnostics and component isolation using a single testability output. The ETA Tool can also provide extended analyses from a single set of testability output files. The following analysis reports are available to the user: (1) the Detectability Report provides a breakdown of how each tested failure mode was detected, (2) the Test Utilization Report identifies all the failure modes that each test detects, (3) the Failure Mode Isolation Report demonstrates the system s ability to discriminate between failure modes, (4) the Component Isolation Report demonstrates the system s ability to discriminate between failure modes relative to the components containing the failure modes, (5) the Sensor Sensor Sensitivity Analysis Report shows the diagnostic impact due to loss of sensor information, and (6) the Effect Mapping Report identifies failure modes that result in specified system-level effects.

  4. Mathematics revealed

    CERN Document Server

    Berman, Elizabeth

    1979-01-01

    Mathematics Revealed focuses on the principles, processes, operations, and exercises in mathematics.The book first offers information on whole numbers, fractions, and decimals and percents. Discussions focus on measuring length, percent, decimals, numbers as products, addition and subtraction of fractions, mixed numbers and ratios, division of fractions, addition, subtraction, multiplication, and division. The text then examines positive and negative numbers and powers and computation. Topics include division and averages, multiplication, ratios, and measurements, scientific notation and estim

  5. Extended Wordsearches in Chemistry

    Science.gov (United States)

    Cotton, Simon

    1998-04-01

    Students can be encouraged to develop their factual knowledge by use of puzzles. One strategy described here is the extended wordsearch, where the wordsearch element generates a number of words or phrases from which the answers to a series of questions are selected. The wordsearch can be generated with the aid of computer programs, though in order to make them suitable for students with dyslexia or other learning difficulties, a simpler form is more appropriate. These problems can be employed in a variety of contexts, for example, as topic tests and classroom end-of-lesson fillers. An example is provided in the area of calcium chemistry. Sources of suitable software are listed.

  6. Classical extended superconformal symmetries

    International Nuclear Information System (INIS)

    Viswanathan, R.R.

    1990-10-01

    Super-covariant differential operators are defined in two dimensions which map supersymmetry doublets to other doublets. The possibility of constructing a closed algebra among the fields appearing in such operators is explored. Such an algebra exists for Grassmann-odd differential operators. A representation for these operators in terms of free-field doublets is constructed. An explicit closed algebra involving fields of spin 2 and 5/2, in addition to the stress tensor and the supersymmetry generator, is constructed from such a free-field representation as an example of a non-linear extended superconformal algebra. (author). 9 refs

  7. Hamiltonian dynamics of extended objects

    Science.gov (United States)

    Capovilla, R.; Guven, J.; Rojas, E.

    2004-12-01

    We consider relativistic extended objects described by a reparametrization-invariant local action that depends on the extrinsic curvature of the worldvolume swept out by the object as it evolves. We provide a Hamiltonian formulation of the dynamics of such higher derivative models which is motivated by the ADM formulation of general relativity. The canonical momenta are identified by looking at boundary behaviour under small deformations of the action; the relationship between the momentum conjugate to the embedding functions and the conserved momentum density is established. The canonical Hamiltonian is constructed explicitly; the constraints on the phase space, both primary and secondary, are identified and the role they play in the theory is described. The multipliers implementing the primary constraints are identified in terms of the ADM lapse and shift variables and Hamilton's equations are shown to be consistent with the Euler Lagrange equations.

  8. Hamiltonian dynamics of extended objects

    International Nuclear Information System (INIS)

    Capovilla, R; Guven, J; Rojas, E

    2004-01-01

    We consider relativistic extended objects described by a reparametrization-invariant local action that depends on the extrinsic curvature of the worldvolume swept out by the object as it evolves. We provide a Hamiltonian formulation of the dynamics of such higher derivative models which is motivated by the ADM formulation of general relativity. The canonical momenta are identified by looking at boundary behaviour under small deformations of the action; the relationship between the momentum conjugate to the embedding functions and the conserved momentum density is established. The canonical Hamiltonian is constructed explicitly; the constraints on the phase space, both primary and secondary, are identified and the role they play in the theory is described. The multipliers implementing the primary constraints are identified in terms of the ADM lapse and shift variables and Hamilton's equations are shown to be consistent with the Euler-Lagrange equations

  9. Hamiltonian dynamics of extended objects

    Energy Technology Data Exchange (ETDEWEB)

    Capovilla, R [Departamento de FIsica, Centro de Investigacion y de Estudios Avanzados del IPN, Apdo Postal 14-740, 07000 Mexico, DF (Mexico); Guven, J [School of Theoretical Physics, Dublin Institute for Advanced Studies, 10 Burlington Road, Dublin 4 (Ireland); Rojas, E [Instituto de Ciencias Nucleares, Universidad Nacional Autonoma de Mexico, Apdo Postal 70-543, 04510 Mexico, DF (Mexico)

    2004-12-07

    We consider relativistic extended objects described by a reparametrization-invariant local action that depends on the extrinsic curvature of the worldvolume swept out by the object as it evolves. We provide a Hamiltonian formulation of the dynamics of such higher derivative models which is motivated by the ADM formulation of general relativity. The canonical momenta are identified by looking at boundary behaviour under small deformations of the action; the relationship between the momentum conjugate to the embedding functions and the conserved momentum density is established. The canonical Hamiltonian is constructed explicitly; the constraints on the phase space, both primary and secondary, are identified and the role they play in the theory is described. The multipliers implementing the primary constraints are identified in terms of the ADM lapse and shift variables and Hamilton's equations are shown to be consistent with the Euler-Lagrange equations.

  10. Extended Interneuronal Network of the Dentate Gyrus

    Directory of Open Access Journals (Sweden)

    Gergely G. Szabo

    2017-08-01

    Full Text Available Local interneurons control principal cells within individual brain areas, but anecdotal observations indicate that interneuronal axons sometimes extend beyond strict anatomical boundaries. Here, we use the case of the dentate gyrus (DG to show that boundary-crossing interneurons with cell bodies in CA3 and CA1 constitute a numerically significant and diverse population that relays patterns of activity generated within the CA regions back to granule cells. These results reveal the existence of a sophisticated retrograde GABAergic circuit that fundamentally extends the canonical interneuronal network.

  11. Ant-infecting Ophiocordyceps genomes reveal a high diversity of potential behavioral manipulation genes and a possible major role for enterotoxins

    NARCIS (Netherlands)

    De Bekker, Charissa; Ohm, Robin A.; Evans, Harry C.; Brachmann, Andreas; Hughes, David P

    2017-01-01

    Much can be gained from revealing the mechanisms fungal entomopathogens employ. Especially intriguing are fungal parasites that manipulate insect behavior because, presumably, they secrete a wealth of bioactive compounds. To gain more insight into their strategies, we compared the genomes of five

  12. The Roles of Teachers' Science Talk in Revealing Language Demands within Diverse Elementary School Classrooms: A Study of Teaching Heat and Temperature in Singapore

    Science.gov (United States)

    Seah, Lay Hoon; Yore, Larry D.

    2017-01-01

    This study of three science teachers' lessons on heat and temperature seeks to characterise classroom talk that highlighted the ways language is used and to examine the nature of the language demands revealed in constructing, negotiating, arguing and communicating science ideas. The transcripts from the entire instructional units for these…

  13. Extending juvenility in grasses

    Energy Technology Data Exchange (ETDEWEB)

    Kaeppler, Shawn; de Leon Gatti, Natalia; Foerster, Jillian

    2017-04-11

    The present invention relates to compositions and methods for modulating the juvenile to adult developmental growth transition in plants, such as grasses (e.g. maize). In particular, the invention provides methods for enhancing agronomic properties in plants by modulating expression of GRMZM2G362718, GRMZM2G096016, or homologs thereof. Modulation of expression of one or more additional genes which affect juvenile to adult developmental growth transition such as Glossy15 or Cg1, in conjunction with such modulation of expression is also contemplated. Nucleic acid constructs for down-regulation of GRMZM2G362718 and/or GRMZM2G096016 are also contemplated, as are transgenic plants and products produced there from, that demonstrate altered, such as extended juvenile growth, and display associated phenotypes such as enhanced yield, improved digestibility, and increased disease resistance. Plants described herein may be used, for example, as improved forage or feed crops or in biofuel production.

  14. Extended biorthogonal matrix polynomials

    Directory of Open Access Journals (Sweden)

    Ayman Shehata

    2017-01-01

    Full Text Available The pair of biorthogonal matrix polynomials for commutative matrices were first introduced by Varma and Tasdelen in [22]. The main aim of this paper is to extend the properties of the pair of biorthogonal matrix polynomials of Varma and Tasdelen and certain generating matrix functions, finite series, some matrix recurrence relations, several important properties of matrix differential recurrence relations, biorthogonality relations and matrix differential equation for the pair of biorthogonal matrix polynomials J(A,B n (x, k and K(A,B n (x, k are discussed. For the matrix polynomials J(A,B n (x, k, various families of bilinear and bilateral generating matrix functions are constructed in the sequel.

  15. Extended conformal algebras

    International Nuclear Information System (INIS)

    Goddard, Peter

    1990-01-01

    The algebra of the group of conformal transformations in two dimensions consists of two commuting copies of the Virasoro algebra. In many mathematical and physical contexts, the representations of ν which are relevant satisfy two conditions: they are unitary and they have the ''positive energy'' property that L o is bounded below. In an irreducible unitary representation the central element c takes a fixed real value. In physical contexts, the value of c is a characteristic of a theory. If c < 1, it turns out that the conformal algebra is sufficient to ''solve'' the theory, in the sense of relating the calculation of the infinite set of physically interesting quantities to a finite subset which can be handled in principle. For c ≥ 1, this is no longer the case for the algebra alone and one needs some sort of extended conformal algebra, such as the superconformal algebra. It is these algebras that this paper aims at addressing. (author)

  16. Extended Poisson Exponential Distribution

    Directory of Open Access Journals (Sweden)

    Anum Fatima

    2015-09-01

    Full Text Available A new mixture of Modified Exponential (ME and Poisson distribution has been introduced in this paper. Taking the Maximum of Modified Exponential random variable when the sample size follows a zero truncated Poisson distribution we have derived the new distribution, named as Extended Poisson Exponential distribution. This distribution possesses increasing and decreasing failure rates. The Poisson-Exponential, Modified Exponential and Exponential distributions are special cases of this distribution. We have also investigated some mathematical properties of the distribution along with Information entropies and Order statistics of the distribution. The estimation of parameters has been obtained using the Maximum Likelihood Estimation procedure. Finally we have illustrated a real data application of our distribution.

  17. Extended fuel cycle length

    International Nuclear Information System (INIS)

    Bruyere, M.; Vallee, A.; Collette, C.

    1986-09-01

    Extended fuel cycle length and burnup are currently offered by Framatome and Fragema in order to satisfy the needs of the utilities in terms of fuel cycle cost and of overall systems cost optimization. We intend to point out the consequences of an increased fuel cycle length and burnup on reactor safety, in order to determine whether the bounding safety analyses presented in the Safety Analysis Report are applicable and to evaluate the effect on plant licensing. This paper presents the results of this examination. The first part indicates the consequences of increased fuel cycle length and burnup on the nuclear data used in the bounding accident analyses. In the second part of this paper, the required safety reanalyses are presented and the impact on the safety margins of different fuel management strategies is examined. In addition, systems modifications which can be required are indicated

  18. Extending Cultural Literacy.

    Science.gov (United States)

    Riecken, Ted J.; Court, Deborah

    1992-01-01

    Advocates defining cultural literacy to recognize the mass media's role in transmitting and maintaining cultural stereotypes and shaping values and beliefs. Distinguishes between ideational and material aspects of culture. Advocates teaching critical thinking and respect for persons in light of questionable moral perspectives in certain media…

  19. Nonstructural leaf carbohydrates dynamics of Pinus edulis during drought-induced tree mortality reveal role for carbon metabolism in mortality mechanism

    Science.gov (United States)

    Adams, Henry D.; Germino, Matthew J.; Breshears, David D.; Barron-Gafford, Greg A.; Guardiola-Claramonte, Maite; Zou, Chris B.; Huxman, Travis E.

    2013-01-01

    * Vegetation change is expected with global climate change, potentially altering ecosystem function and climate feedbacks. However, causes of plant mortality, which are central to vegetation change, are understudied, and physiological mechanisms remain unclear, particularly the roles of carbon metabolism and xylem function.

  20. What is said or how it is said makes a difference : role of the right fronto-parietal operculum in emotional prosody as revealed by repetitive TMS

    NARCIS (Netherlands)

    van Rijn, S; Aleman, A; van Diessen, E; Berckmoes, C; Vingerhoets, G; Kahn, RS

    Emotional signals in spoken language can be conveyed by semantic as well as prosodic cues. We investigated the role of the fronto-parietal operculum, a somatosensory area where the lips, tongue and jaw are represented, in the right hemisphere to detection of emotion in prosody vs. semantics. A total

  1. Effective connectivity reveals important roles for both the hyperdirect (fronto-subthalamic) and indirect (fronto-striatal-pallidal) fronto-basal ganglia pathways during response inhibition

    NARCIS (Netherlands)

    Jahfari, S.; Waldorp, L.; van den Wildenberg, W.P.M.; Scholte, H.S.; Ridderinkhof, K.R.; Forstmann, B.U.

    2011-01-01

    Fronto-basal ganglia pathways play a crucial role in voluntary action control, including the ability to inhibit motor responses. Response inhibition might be mediated via a fast hyperdirect pathway connecting the right inferior frontal gyrus (rIFG) and the presupplementary motor area (preSMA) with

  2. Recombinant Adeno-Associated Virus-Mediated microRNA Delivery into the Postnatal Mouse Brain Reveals a Role for miR-134 in Dendritogenesis in Vivo

    DEFF Research Database (Denmark)

    Christensen, Mette; Larsen, Lars A; Kauppinen, Sakari

    2010-01-01

    delivery of microRNAs in vivo by use of recombinant adeno-associated virus (rAAV). rAAV-mediated overexpression of miR-134 in neurons of the postnatal mouse brain provided evidence for a negative role of miR-134 in dendritic arborization of cortical layer V pyramidal neurons in vivo, thereby confirming...

  3. The central role of mosquito cytochrome P450 CYP6Zs in insecticide detoxification revealed by functional expression and structural modelling.

    Science.gov (United States)

    Chandor-Proust, Alexia; Bibby, Jaclyn; Régent-Kloeckner, Myriam; Roux, Jessica; Guittard-Crilat, Emilie; Poupardin, Rodolphe; Riaz, Muhammad Asam; Paine, Mark; Dauphin-Villemant, Chantal; Reynaud, Stéphane; David, Jean-Philippe

    2013-10-01

    The resistance of mosquitoes to chemical insecticides is threatening vector control programmes worldwide. Cytochrome P450 monooxygenases (CYPs) are known to play a major role in insecticide resistance, allowing resistant insects to metabolize insecticides at a higher rate. Among them, members of the mosquito CYP6Z subfamily, like Aedes aegypti CYP6Z8 and its Anopheles gambiae orthologue CYP6Z2, have been frequently associated with pyrethroid resistance. However, their role in the pyrethroid degradation pathway remains unclear. In the present study, we created a genetically modified yeast strain overexpressing Ae. aegypti cytochrome P450 reductase and CYP6Z8, thereby producing the first mosquito P450-CPR (NADPH-cytochrome P450-reductase) complex in a yeast recombinant system. The results of the present study show that: (i) CYP6Z8 metabolizes PBAlc (3-phenoxybenzoic alcohol) and PBAld (3-phenoxybenzaldehyde), common pyrethroid metabolites produced by carboxylesterases, producing PBA (3-phenoxybenzoic acid); (ii) CYP6Z8 transcription is induced by PBAlc, PBAld and PBA; (iii) An. gambiae CYP6Z2 metabolizes PBAlc and PBAld in the same way; (iv) PBA is the major metabolite produced in vivo and is excreted without further modification; and (v) in silico modelling of substrate-enzyme interactions supports a similar role of other mosquito CYP6Zs in pyrethroid degradation. By playing a pivotal role in the degradation of pyrethroid insecticides, mosquito CYP6Zs thus represent good targets for mosquito-resistance management strategies.

  4. Partial loss-of-function alleles reveal a role for GNOM in auxin transport-related, post-embryonic development of Arabidopsis

    DEFF Research Database (Denmark)

    Geldner, Niko; Richter, Sandra; Vieten, Anne

    2004-01-01

    The Arabidopsis GNOM gene encodes an ARF GDP/GTP exchange factor involved in embryonic axis formation and polar localisation of the auxin efflux regulator PIN1. To examine whether GNOM also plays a role in post-embryonic development and to clarify its involvement in auxin transport, we have...

  5. Heterotypic contact reveals a COX-2-mediated suppression of osteoblast differentiation by endothelial cells: A negative modulatory role for prostanoids in VEGF-mediated cell: cell communication?

    International Nuclear Information System (INIS)

    Clarkin, Claire E.; Garonna, Elena; Pitsillides, Andrew A.; Wheeler-Jones, Caroline P.D.

    2008-01-01

    In bone, angiogenesis must be initiated appropriately, but limited once remodelling or repair is complete. Our recent findings have supported a role for prostaglandins (PG), known modulators of osteoblast (OB) and endothelial cell (EC) behaviour, in facilitating VEGF-mediated paracrine communication from OBs to 'remotely located' ECs, but the mechanism(s) regulating OB:EC crosstalk when these cells are closely opposed are undefined. In this study we have examined: (i) the effects of exogenous PGE 2 on VEGF-driven events in ECs, and (ii) the role of endogenous COX-2-derived prostanoids in mediating communication between intimately opposed OBs and ECs in direct contact. Exposure of ECs to PGE 2 increased ERK1/2 phosphorylation, COX-2 induction, 6-keto-PGF 1α release and EC proliferation. In contrast, PGE 2 attenuated VEGF 165 -induced VEGFR2/Flk1 phosphorylation, ERK1/2 activation and proliferation of ECs, suggesting that exogenous PGE 2 restricts the actions of VEGF. However, the COX-2-selective inhibitor, NS398, also attenuated VEGF-induced proliferation, implying a distinct role for endogenous COX-2 activity in regulating EC behaviour. To examine the effect of OB:EC proximity and the role of COX-2 products further, we used a confrontational co-culture model. These studies showed that COX-2 blockade with NS398 enhanced EC-dependent increases in OB differentiation, that this effect was reversed by exogenous PGH 2 (immediate COX-2 product), and that exogenous VEGF did not influence EC-dependent OB differentiation under these conditions. Our findings indicate that locally produced prostanoids may serve distinct roles depending on OB:EC proximity and negatively modulate VEGF-mediated changes in EC behaviour when these cells are closely opposed to control angiogenesis during bone (re)modelling

  6. The roles of teachers' science talk in revealing language demands within diverse elementary school classrooms: a study of teaching heat and temperature in Singapore

    Science.gov (United States)

    Seah, Lay Hoon; Yore, Larry D.

    2017-01-01

    This study of three science teachers' lessons on heat and temperature seeks to characterise classroom talk that highlighted the ways language is used and to examine the nature of the language demands revealed in constructing, negotiating, arguing and communicating science ideas. The transcripts from the entire instructional units for these teachers' four culturally and linguistically diverse Grade 4 classes (10 years old) with English as the language of instruction constitute the data for this investigation. Analysis of these transcripts focused on teachers' talk that made explicit reference to the form or function of the language of science and led to the inductive development of the 'Attending to Language Demands in Science' analytical framework. This framework in turn revealed that the major foregrounding purposes of teachers' talk include labelling, explaining, differentiating, selecting and constructing. Further classification of the instances within these categories revealed the extensive and contextualised nature of the language demands. The results challenge the conventional assumption that basic literacy skills dominate over disciplinary literacy skills in primary school science. Potential uses of the analytical framework that could further expand our understanding of the forms, functions and demands of language used in elementary school science are also discussed.

  7. Extending the amygdala in theories of threat processing

    Science.gov (United States)

    Fox, Andrew S.; Oler, Jonathan A.; Tromp, Do P.M.; Fudge, Julie L.; Kalin, Ned H.

    2015-01-01

    The central extended amygdala is an evolutionarily conserved set of interconnected brain regions that play an important role in threat processing to promote survival. Two core components of the central extended amygdala, the central nucleus of the amygdala (Ce) and the lateral bed nucleus of the stria terminalis (BST) are highly similar regions that serve complimentary roles by integrating fear- and anxiety-relevant information. Survival depends on the central extended amygdala's ability to rapidly integrate and respond to threats that vary in their immediacy, proximity, and characteristics. Future studies will benefit from understanding alterations in central extended amygdala function in relation to stress-related psychopathology. PMID:25851307

  8. Revealing Rembrandt

    Directory of Open Access Journals (Sweden)

    Andrew J Parker

    2014-04-01

    Full Text Available The power and significance of artwork in shaping human cognition is self-evident. The starting point for our empirical investigations is the view that the task of neuroscience is to integrate itself with other forms of knowledge, rather than to seek to supplant them. In our recent work, we examined a particular aspect of the appreciation of artwork using present-day functional magnetic resonance imaging (fMRI. Our results emphasised the continuity between viewing artwork and other human cognitive activities. We also showed that appreciation of a particular aspect of artwork, namely authenticity, depends upon the co-ordinated activity between the brain regions involved in multiple decision making and those responsible for processing visual information. The findings about brain function probably have no specific consequences for understanding how people respond to the art of Rembrandt in comparison with their response to other artworks. However, the use of images of Rembrandt’s portraits, his most intimate and personal works, clearly had a significant impact upon our viewers, even though they have been spatially confined to the interior of an MRI scanner at the time of viewing. Neuroscientific studies of humans viewing artwork have the capacity to reveal the diversity of human cognitive responses that may be induced by external advice or context as people view artwork in a variety of frameworks and settings.

  9. Quantitative measures of sexual selection reveal no evidence for sex-role reversal in a sea spider with prolonged paternal care

    OpenAIRE

    Barreto, Felipe S.; Avise, John C.

    2010-01-01

    Taxa in which males alone invest in postzygotic care of offspring are often considered good models for investigating the proffered relationships between sexual selection and mating systems. In the pycnogonid sea spider Pycnogonum stearnsi, males carry large egg masses on their bodies for several weeks, so this species is a plausible candidate for sex-role reversal (greater intensity of sexual selection on females than on males). Here, we couple a microsatellite-based assessment of the mating ...

  10. Survey of High Throughput RNA-Seq Data Reveals Potential Roles for lncRNAs during Development and Stress Response in Bread Wheat

    Directory of Open Access Journals (Sweden)

    Shumayla

    2017-06-01

    Full Text Available Long non-coding RNAs (lncRNAs are a family of regulatory RNAs that play essential role in the various developmental processes and stress responses. Recent advances in sequencing technology and computational methods enabled identification and characterization of lncRNAs in certain plant species, but they are less known in Triticum aestivum (bread wheat. Herein, we analyzed 52 RNA seq data (>30 billion reads and identified 44,698 lncRNAs in T. aestivum genome, which were characterized in comparison to the coding sequences (mRNAs. Similar to the mRNAs, lncRNAs were also derived from each sub-genome and chromosome, and showed tissue developmental stage specific and differential expression, as well. The modulated expression of lncRNAs during abiotic stresses like heat, drought, and salt indicated their putative role in stress response. The co-expression of lncRNAs with vital mRNAs including various transcription factors and enzymes involved in Abscisic acid (ABA biosynthesis, and gene ontology mapping inferred their regulatory roles in numerous biological processes. A few lncRNAs were predicted as precursor (19 lncRNAs, while some as target mimics (1,047 lncRNAs of known miRNAs involved in various regulatory functions. The results suggested numerous functions of lncRNAs in T. aestivum, and unfolded the opportunities for functional characterization of individual lncRNA in future studies.

  11. Identification of factors required for m6 A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI.

    Science.gov (United States)

    Růžička, Kamil; Zhang, Mi; Campilho, Ana; Bodi, Zsuzsanna; Kashif, Muhammad; Saleh, Mária; Eeckhout, Dominique; El-Showk, Sedeer; Li, Hongying; Zhong, Silin; De Jaeger, Geert; Mongan, Nigel P; Hejátko, Jan; Helariutta, Ykä; Fray, Rupert G

    2017-07-01

    N6-adenosine methylation (m 6 A) of mRNA is an essential process in most eukaryotes, but its role and the status of factors accompanying this modification are still poorly understood. Using combined methods of genetics, proteomics and RNA biochemistry, we identified a core set of mRNA m 6 A writer proteins in Arabidopsis thaliana. The components required for m 6 A in Arabidopsis included MTA, MTB, FIP37, VIRILIZER and the E3 ubiquitin ligase HAKAI. Downregulation of these proteins led to reduced relative m 6 A levels and shared pleiotropic phenotypes, which included aberrant vascular formation in the root, indicating that correct m 6 A methylation plays a role in developmental decisions during pattern formation. The conservation of these proteins amongst eukaryotes and the demonstration of a role in writing m 6 A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  12. Genome-wide identification of microRNA targets in human ES cells reveals a role for miR-302 in modulating BMP response

    Science.gov (United States)

    Lipchina, Inna; Elkabetz, Yechiel; Hafner, Markus; Sheridan, Robert; Mihailovic, Aleksandra; Tuschl, Thomas; Sander, Chris; Studer, Lorenz; Betel, Doron

    2011-01-01

    MicroRNAs are important regulators in many cellular processes, including stem cell self-renewal. Recent studies demonstrated their function as pluripotency factors with the capacity for somatic cell reprogramming. However, their role in human embryonic stem (ES) cells (hESCs) remains poorly understood, partially due to the lack of genome-wide strategies to identify their targets. Here, we performed comprehensive microRNA profiling in hESCs and in purified neural and mesenchymal derivatives. Using a combination of AGO cross-linking and microRNA perturbation experiments, together with computational prediction, we identified the targets of the miR-302/367 cluster, the most abundant microRNAs in hESCs. Functional studies identified novel roles of miR-302/367 in maintaining pluripotency and regulating hESC differentiation. We show that in addition to its role in TGF-β signaling, miR-302/367 promotes bone morphogenetic protein (BMP) signaling by targeting BMP inhibitors TOB2, DAZAP2, and SLAIN1. This study broadens our understanding of microRNA function in hESCs and is a valuable resource for future studies in this area. PMID:22012620

  13. Classical extended conformal symmetries

    International Nuclear Information System (INIS)

    Viswanathan, R.

    1990-02-01

    Extensions of the Virasoro algebra are constructed as Poisson brackets of higher spin fields which appear as coefficient fields in certain covariant derivative operators of order N. These differential operators are constructed so as to be covariant under reparametrizations on fields of definite conformal dimension. Factorization of such an N-th order operator in terms of first order operators, together with the inclusion of a spin one U(1) current, is shown to lead to a two-parameter W-algebra. One of these parameters plays the role of interpolating between W-algebras based on different Lie algebras of the same rank. (author). 11 refs

  14. Proteomic characterization of EL4 lymphoma-derived tumors upon chemotherapy treatment reveals potential roles for lysosomes and caspase-6 during tumor cell death in vivo.

    Science.gov (United States)

    Kramer, David A; Eldeeb, Mohamed A; Wuest, Melinda; Mercer, John; Fahlman, Richard P

    2017-06-01

    The murine mouse lymphoblastic lymphoma cell line (EL4) tumor model is an established in vivo apoptosis model for the investigation of novel cancer imaging agents and immunological treatments due to the rapid and significant response of the EL4 tumors to cyclophosphamide and etoposide combination chemotherapy. Despite the utility of this model system in cancer research, little is known regarding the molecular details of in vivo tumor cell death. Here, we report the first in-depth quantitative proteomic analysis of the changes that occur in these tumors upon cyclophosphamide and etoposide treatment in vivo. Using a label-free quantitative proteomic approach a total of 5838 proteins were identified in the treated and untreated tumors, of which 875 were determined to change in abundance with statistical significance. Initial analysis of the data reveals changes that may have been predicted, such as the downregulation of ribosomes, but demonstrates the robustness of the dataset. Analysis of the dataset also reveals the unexpected downregulation of caspase-3 and an upregulation of caspase-6 in addition to a global upregulation of lysosomal proteins in the bulk of the tumor. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. TATN-1 mutations reveal a novel role for tyrosine as a metabolic signal that influences developmental decisions and longevity in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Annabel A Ferguson

    Full Text Available Recent work has identified changes in the metabolism of the aromatic amino acid tyrosine as a risk factor for diabetes and a contributor to the development of liver cancer. While these findings could suggest a role for tyrosine as a direct regulator of the behavior of cells and tissues, evidence for this model is currently lacking. Through the use of RNAi and genetic mutants, we identify tatn-1, which is the worm ortholog of tyrosine aminotransferase and catalyzes the first step of the conserved tyrosine degradation pathway, as a novel regulator of the dauer decision and modulator of the daf-2 insulin/IGF-1-like (IGFR signaling pathway in Caenorhabditis elegans. Mutations affecting tatn-1 elevate tyrosine levels in the animal, and enhance the effects of mutations in genes that lie within the daf-2/insulin signaling pathway or are otherwise upstream of daf-16/FOXO on both dauer formation and worm longevity. These effects are mediated by elevated tyrosine levels as supplemental dietary tyrosine mimics the phenotypes produced by a tatn-1 mutation, and the effects still occur when the enzymes needed to convert tyrosine into catecholamine neurotransmitters are missing. The effects on dauer formation and lifespan require the aak-2/AMPK gene, and tatn-1 mutations increase phospho-AAK-2 levels. In contrast, the daf-16/FOXO transcription factor is only partially required for the effects on dauer formation and not required for increased longevity. We also find that the controlled metabolism of tyrosine by tatn-1 may function normally in dauer formation because the expression of the TATN-1 protein is regulated both by daf-2/IGFR signaling and also by the same dietary and environmental cues which influence dauer formation. Our findings point to a novel role for tyrosine as a developmental regulator and modulator of longevity, and support a model where elevated tyrosine levels play a causal role in the development of diabetes and cancer in people.

  16. Bone marrow chimeric mice reveal a role for CX₃CR1 in maintenance of the monocyte-derived cell population in the olfactory neuroepithelium.

    Science.gov (United States)

    Vukovic, Jana; Blomster, Linda V; Chinnery, Holly R; Weninger, Wolfgang; Jung, Steffen; McMenamin, Paul G; Ruitenberg, Marc J

    2010-10-01

    Macrophages in the olfactory neuroepithelium are thought to play major roles in tissue homeostasis and repair. However, little information is available at present about possible heterogeneity of these monocyte-derived cells, their turnover rates, and the role of chemokine receptors in this process. To start addressing these issues, this study used Cx₃cr1(gfp) mice, in which the gene sequence for eGFP was knocked into the CX₃CR1 gene locus in the mutant allele. Using neuroepithelial whole-mounts from Cx₃cr1(gfp/+) mice, we show that eGFP(+) cells of monocytic origin are distributed in a loose network throughout this tissue and can be subdivided further into two immunophenotypically distinct subsets based on MHC-II glycoprotein expression. BM chimeric mice were created using Cx₃cr1(gfp/+) donors to investigate turnover of macrophages (and other monocyte-derived cells) in the olfactory neuroepithelium. Our data indicate that the monocyte-derived cell population in the olfactory neuroepithelium is actively replenished by circulating monocytes and under the experimental conditions, completely turned over within 6 months. Transplantation of Cx₃cr1(gfp/gfp) (i.e., CX₃CR1-deficient) BM partially impaired the replenishment process and resulted in an overall decline of the total monocyte-derived cell number in the olfactory epithelium. Interestingly, replenishment of the CD68(low)MHC-II(+) subset appeared minimally affected by CX₃CR1 deficiency. Taken together, the established baseline data about heterogeneity of monocyte-derived cells, their replenishment rates, and the role of CX₃CR1 provide a solid basis to further examine the importance of different monocyte subsets for neuroregeneration at this unique frontier with the external environment.

  17. TATN-1 Mutations Reveal a Novel Role for Tyrosine as a Metabolic Signal That Influences Developmental Decisions and Longevity in Caenorhabditis elegans

    Science.gov (United States)

    Ferguson, Annabel A.; Dumas, Kathleen J.; Ritov, Vladimir B.; Matern, Dietrich; Hu, Patrick J.; Fisher, Alfred L.

    2013-01-01

    Recent work has identified changes in the metabolism of the aromatic amino acid tyrosine as a risk factor for diabetes and a contributor to the development of liver cancer. While these findings could suggest a role for tyrosine as a direct regulator of the behavior of cells and tissues, evidence for this model is currently lacking. Through the use of RNAi and genetic mutants, we identify tatn-1, which is the worm ortholog of tyrosine aminotransferase and catalyzes the first step of the conserved tyrosine degradation pathway, as a novel regulator of the dauer decision and modulator of the daf-2 insulin/IGF-1-like (IGFR) signaling pathway in Caenorhabditis elegans. Mutations affecting tatn-1 elevate tyrosine levels in the animal, and enhance the effects of mutations in genes that lie within the daf-2/insulin signaling pathway or are otherwise upstream of daf-16/FOXO on both dauer formation and worm longevity. These effects are mediated by elevated tyrosine levels as supplemental dietary tyrosine mimics the phenotypes produced by a tatn-1 mutation, and the effects still occur when the enzymes needed to convert tyrosine into catecholamine neurotransmitters are missing. The effects on dauer formation and lifespan require the aak-2/AMPK gene, and tatn-1 mutations increase phospho-AAK-2 levels. In contrast, the daf-16/FOXO transcription factor is only partially required for the effects on dauer formation and not required for increased longevity. We also find that the controlled metabolism of tyrosine by tatn-1 may function normally in dauer formation because the expression of the TATN-1 protein is regulated both by daf-2/IGFR signaling and also by the same dietary and environmental cues which influence dauer formation. Our findings point to a novel role for tyrosine as a developmental regulator and modulator of longevity, and support a model where elevated tyrosine levels play a causal role in the development of diabetes and cancer in people. PMID:24385923

  18. The Arabidopsis szl1 Mutant Reveals a Critical Role of β-Carotene in Photosystem I Photoprotection1[C][W

    Science.gov (United States)

    Cazzaniga, Stefano; Li, Zhirong; Niyogi, Krishna K.; Bassi, Roberto; Dall’Osto, Luca

    2012-01-01

    Carotenes and their oxygenated derivatives, the xanthophylls, are structural determinants in both photosystems (PS) I and II. They bind and stabilize photosynthetic complexes, increase the light-harvesting capacity of chlorophyll-binding proteins, and have a major role in chloroplast photoprotection. Localization of carotenoid species within each PS is highly conserved: Core complexes bind carotenes, whereas peripheral light-harvesting systems bind xanthophylls. The specific functional role of each xanthophyll species has been recently described by genetic dissection, however the in vivo role of carotenes has not been similarly defined. Here, we have analyzed the function of carotenes in photosynthesis and photoprotection, distinct from that of xanthophylls, by characterizing the suppressor of zeaxanthin-less (szl) mutant of Arabidopsis (Arabidopsis thaliana) which, due to the decreased activity of the lycopene-β-cyclase, shows a lower carotene content than wild-type plants. When grown at room temperature, mutant plants showed a lower content in PSI light-harvesting complex I complex than the wild type, and a reduced capacity for chlorophyll fluorescence quenching, the rapidly reversible component of nonphotochemical quenching. When exposed to high light at chilling temperature, szl1 plants showed stronger photoxidation than wild-type plants. Both PSI and PSII from szl1 were similarly depleted in carotenes and yet PSI activity was more sensitive to light stress than PSII as shown by the stronger photoinhibition of PSI and increased rate of singlet oxygen release from isolated PSI light-harvesting complex I complexes of szl1 compared with the wild type. We conclude that carotene depletion in the core complexes impairs photoprotection of both PS under high light at chilling temperature, with PSI being far more affected than PSII. PMID:23029671

  19. Proteome analysis reveals phosphorylation of ATP synthase beta -subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes

    DEFF Research Database (Denmark)

    Højlund, Kurt; Wrzesinski, Krzysztof; Larsen, Peter Mose

    2003-01-01

    quantitate a large number of proteins and their post-translational modifications simultaneously and is a powerful tool to study polygenic diseases like type 2 diabetes. Using this approach on human skeletal muscle biopsies, we have identified eight potential protein markers for type 2 diabetes in the fasting...... synthase beta-subunit phosphoisoform in diabetic muscle correlated inversely with fasting plasma glucose levels. These data suggest a role for phosphorylation of ATP synthase beta-subunit in the regulation of ATP synthesis and that alterations in the regulation of ATP synthesis and cellular stress proteins...

  20. Genetic Disruption of the Sh3pxd2a Gene Reveals an Essential Role in Mouse Development and the Existence of a Novel Isoform of Tks5

    OpenAIRE

    Cejudo-Martin, Pilar; Yuen, Angela; Vlahovich, Nicole; Lock, Peter; Courtneidge, Sara A.; Díaz, Begoña

    2014-01-01

    Tks5 is a scaffold protein and Src substrate involved in cell migration and matrix degradation through its essential role in invadosome formation and function. We have previously described that Tks5 is fundamental for zebrafish neural crest cell migration in vivo. In the present study, we sought to investigate the function of Tks5 in mammalian development by analyzing mice mutant for sh3pxd2a, the gene encoding Tks5. Homozygous disruption of the sh3pxd2a gene by gene-trapping in mouse resulte...

  1. Selective abrogation of the uPA-uPAR interaction in vivo reveals a novel role in suppression of fibrin-associated inflammation

    DEFF Research Database (Denmark)

    Connolly, Brian M; Choi, Eun Young; Gårdsvoll, Henrik

    2010-01-01

    the interaction between endogenous uPA and uPAR is selectively abrogated, whereas other functions of both the protease and its receptor are retained. Specifically, we introduced 4 amino acid substitutions into the growth factor domain (GFD) of uPA that abrogate uPAR binding while preserving the overall structure...... a principal in vivo role of the uPA-uPAR interaction in cell-associated fibrinolysis critical for suppression of fibrin accumulation and fibrin-associated inflammation and provides a valuable model for further exploration of this multifunctional receptor....

  2. A Role of Medial Olivocochlear Reflex as a Protection Mechanism from Noise-Induced Hearing Loss Revealed in Short-Practicing Violinists.

    Directory of Open Access Journals (Sweden)

    Sho Otsuka

    Full Text Available Previous studies have indicated that extended exposure to a high level of sound might increase the risk of hearing loss among professional symphony orchestra musicians. One of the major problems associated with musicians' hearing loss is difficulty in estimating its risk simply on the basis of the physical amount of exposure, i.e. the exposure level and duration. The aim of this study was to examine whether the measurement of the medial olivocochlear reflex (MOCR, which is assumed to protect the cochlear from acoustic damage, could enable us to assess the risk of hearing loss among musicians. To test this, we compared the MOCR strength and the hearing deterioration caused by one-hour instrument practice. The participants in the study were music university students who are majoring in the violin, whose left ear is exposed to intense violin sounds (broadband sounds containing a significant number of high-frequency components during their regular instrument practice. Audiogram and click-evoked otoacoustic emissions (CEOAEs were measured before and after a one-hour violin practice. There was a larger exposure to the left ear than to the right ear, and we observed a left-ear specific temporary threshold shift (TTS after the violin practice. Left-ear CEOAEs decreased proportionally to the TTS. The exposure level, however, could not entirely explain the inter-individual variation in the TTS and the decrease in CEOAE. On the other hand, the MOCR strength could predict the size of the TTS and CEOAE decrease. Our findings imply that, among other factors, the MOCR is a promising measure for assessing the risk of hearing loss among musicians.

  3. Gene knockout and overexpression analysis revealed the role of N-acetylmuramidase in autolysis of Lactobacillus delbrueckii subsp. bulgaricus ljj-6.

    Directory of Open Access Journals (Sweden)

    Xiao-Yang Pang

    Full Text Available Autolysis of lactic acid bacteria (LAB plays a vital role in dairy processing. During cheese making, autolysis of LAB affects cheese flavor development through release of intracellular enzymes and restricts the proliferation of cells in yogurt fermentation and probiotics production. In order to explore the mechanism of autolysis, the gene for the autolytic enzymes of L. bulgaricus, N-acetylmuramidase (mur, was cloned and sequenced (GenBank accession number: KF157911. Mur gene overexpression and gene knockout vectors were constructed based on pMG76e and pUC19 vectors. Recombinant plasmids were transformed into L. bulgaricus ljj-6 by electroporation, then three engineered strains with pMG76e-mur vector and fifteen engineered strains with pUC19-mur::EryBII were screened. The autolysis of the mur knockout strain was significantly lower and autolysis of the mur overexpressed strain was significantly higher compared with that of the wild type strain ljj-6. This result suggested that the mur gene played an important role in autolysis of L. bulgaricus. On the other hand, autolytic activity in a low degree was still observed in the mur knockout strain, which implied that other enzymes but autolysin encoded by mur were also involved in autolysis of L. bulgaricus.

  4. Subtype-Selective Small Molecule Inhibitors Reveal a Fundamental Role for Nav1.7 in Nociceptor Electrogenesis, Axonal Conduction and Presynaptic Release

    Science.gov (United States)

    Estacion, Mark; Turner, Jamie; Mis, Malgorzata A.; Wilbrey, Anna; Payne, Elizabeth C.; Gutteridge, Alex; Cox, Peter J.; Doyle, Rachel; Printzenhoff, David; Lin, Zhixin; Marron, Brian E.; West, Christopher; Swain, Nigel A.; Storer, R. Ian; Stupple, Paul A.; Castle, Neil A.; Hounshell, James A.; Rivara, Mirko; Randall, Andrew; Dib-Hajj, Sulayman D.; Krafte, Douglas; Waxman, Stephen G.; Patel, Manoj K.; Butt, Richard P.; Stevens, Edward B.

    2016-01-01

    Human genetic studies show that the voltage gated sodium channel 1.7 (Nav1.7) is a key molecular determinant of pain sensation. However, defining the Nav1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Nav1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Nav1.7’s role in nociceptor physiology. We report that Nav1.7 is the predominant functional TTX-sensitive Nav in mouse and human nociceptors and contributes to the initiation and the upstroke phase of the nociceptor action potential. Moreover, we confirm a role for Nav1.7 in influencing synaptic transmission in the dorsal horn of the spinal cord as well as peripheral neuropeptide release in the skin. These findings demonstrate multiple contributions of Nav1.7 to nociceptor signalling and shed new light on the relative functional contribution of this channel to peripheral and central noxious signal transmission. PMID:27050761

  5. A genome-wide scan reveals important roles of DNA methylation in human longevity by regulating age-related disease genes.

    Directory of Open Access Journals (Sweden)

    Fu-Hui Xiao

    Full Text Available It is recognized that genetic factors contribute to human longevity. Besides the hypothesis of existence of longevity genes, another suggests that a lower frequency of risk alleles decreases the incidence of age-related diseases in the long-lived people. However, the latter finds no support from recent genetic studies. Considering the crucial role of epigenetic modification in gene regulation, we then hypothesize that suppressing disease-related genes in longevity individuals is likely achieved by epigenetic modification, e.g. DNA methylation. To test this hypothesis, we investigated the genome-wide methylation profile in 4 Chinese female centenarians and 4 middle-aged controls using methyl-DNA immunoprecipitation sequencing. 626 differentially methylated regions (DMRs were observed between both groups. Interestingly, genes with these DMRs were enriched in age-related diseases, including type-2 diabetes, cardiovascular disease, stroke and Alzheimer's disease. This pattern remains rather stable after including methylomes of two white individuals. Further analyses suggest that the observed DMRs likely have functional roles in regulating disease-associated gene expressions, with some genes [e.g. caspase 3 (CASP3] being down-regulated whereas the others [i.e. interleukin 1 receptor, type 2 (IL1R2] up-regulated. Therefore, our study suggests that suppressing the disease-related genes via epigenetic modification is an important contributor to human longevity.

  6. Integrative RNA- and miRNA-Profile Analysis Reveals a Likely Role of BR and Auxin Signaling in Branch Angle Regulation of B. napus

    Directory of Open Access Journals (Sweden)

    Hongtao Cheng

    2017-05-01

    Full Text Available Oilseed rape (Brassica napus L. is the second largest oilseed crop worldwide and one of the most important oil crops in China. As a component of plant architecture, branch angle plays an important role in yield performance, especially under high-density planting conditions. However, the mechanisms underlying the regulation of branch angle are still largely not understood. Two oilseed rape lines with significantly different branch angles were used to conduct RNA- and miRNA-profiling at two developmental stages, identifying differential expression of a large number of genes involved in auxin- and brassinosteroid (BR-related pathways. Many auxin response genes, including AUX1, IAA, GH3, and ARF, were enriched in the compact line. However, a number of genes involved in BR signaling transduction and biosynthesis were down-regulated. Differentially expressed miRNAs included those involved in auxin signaling transduction. Expression patterns of most target genes were fine-tuned by related miRNAs, such as miR156, miR172, and miR319. Some miRNAs were found to be differentially expressed at both developmental stages, including three miR827 members. Our results provide insight that auxin- and BR-signaling may play a pivotal role in branch angle regulation.

  7. A transgenic model for conditional induction and rescue of portal hypertension reveals a role of VEGF-mediated regulation of sinusoidal fenestrations.

    Directory of Open Access Journals (Sweden)

    Dalit May

    Full Text Available Portal hypertension (PH is a common complication and a leading cause of death in patients with chronic liver diseases. PH is underlined by structural and functional derangement of liver sinusoid vessels and its fenestrated endothelium. Because in most clinical settings PH is accompanied by parenchymal injury, it has been difficult to determine the precise role of microvascular perturbations in causing PH. Reasoning that Vascular Endothelial Growth Factor (VEGF is required to maintain functional integrity of the hepatic microcirculation, we developed a transgenic mouse system for a liver-specific-, reversible VEGF inhibition. The system is based on conditional induction and de-induction of a VEGF decoy receptor that sequesters VEGF and preclude signaling. VEGF blockade results in sinusoidal endothelial cells (SECs fenestrations closure and in accumulation and transformation of the normally quiescent hepatic stellate cells, i.e. provoking the two processes underlying sinusoidal capillarization. Importantly, sinusoidal capillarization was sufficient to cause PH and its typical sequela, ascites, splenomegaly and venous collateralization without inflicting parenchymal damage or fibrosis. Remarkably, these dramatic phenotypes were fully reversed within few days from lifting-off VEGF blockade and resultant re-opening of SECs' fenestrations. This study not only uncovered an indispensible role for VEGF in maintaining structure and function of mature SECs, but also highlights the vasculo-centric nature of PH pathogenesis. Unprecedented ability to rescue PH and its secondary manifestations via manipulating a single vascular factor may also be harnessed for examining the potential utility of de-capillarization treatment modalities.

  8. Apoptosis modulation in the immune system reveals a role of neutrophils in tissue damage in a murine model of chlamydial genital infection.

    Science.gov (United States)

    Zortel, Tom; Schmitt-Graeff, Annette; Kirschnek, Susanne; Häcker, Georg

    2018-03-07

    Chlamydial infection frequently causes damage to the female genital tract. The precise mechanisms of chlamydial clearance and tissue damage are unknown but studies suggest immunopathology with a particular role of neutrophils. The goal of this study was to understand the contribution of the immune system, in particular neutrophils. Using Chlamydia muridarum, we infected mice with a prolonged immune response due to expression of Bcl-2 in haematopoietic cells (Bcl-2-mice), and mice where mature neutrophils are lacking due to the deletion of Mcl-1 in myeloid cells (LysM-cre-mcl-1-flox-mice; Mcl-1-mice). We monitored bacterial clearance, cellular infiltrate and long-term tissue damage. Both mutant strains showed slightly delayed clearance of the acute infection. Bcl-2-mice had a strongly increased inflammatory infiltrate concerning almost all cell lineages. The infection of Bcl-2-mice caused increased tissue damage. The loss of neutrophils in Mcl-1-mice was associated with substantial quantitative and qualitative alterations of the inflammatory infiltrate. Mcl-1-mice had higher chlamydial burden and reduced tissue damage, including lower incidence of hydrosalpinx and less uterine dilation. Inhibition of apoptosis in the haematopoietic system increases inflammation and tissue damage. Neutrophils have broad functions, including a role in chlamydial clearance and in tissue destruction.

  9. Nonstructural leaf carbohydrate dynamics of Pinus edulis during drought-induced tree mortality reveal role for carbon metabolism in mortality mechanism.

    Science.gov (United States)

    Adams, Henry D; Germino, Matthew J; Breshears, David D; Barron-Gafford, Greg A; Guardiola-Claramonte, Maite; Zou, Chris B; Huxman, Travis E

    2013-03-01

    Vegetation change is expected with global climate change, potentially altering ecosystem function and climate feedbacks. However, causes of plant mortality, which are central to vegetation change, are understudied, and physiological mechanisms remain unclear, particularly the roles of carbon metabolism and xylem function. We report analysis of foliar nonstructural carbohydrates (NSCs) and associated physiology from a previous experiment where earlier drought-induced mortality of Pinus edulis at elevated temperatures was associated with greater cumulative respiration. Here, we predicted faster NSC decline for warmed trees than for ambient-temperature trees. Foliar NSC in droughted trees declined by 30% through mortality and was lower than in watered controls. NSC decline resulted primarily from decreased sugar concentrations. Starch initially declined, and then increased above pre-drought concentrations before mortality. Although temperature did not affect NSC and sugar, starch concentrations ceased declining and increased earlier with higher temperatures. Reduced foliar NSC during lethal drought indicates a carbon metabolism role in mortality mechanism. Although carbohydrates were not completely exhausted at mortality, temperature differences in starch accumulation timing suggest that carbon metabolism changes are associated with time to death. Drought mortality appears to be related to temperature-dependent carbon dynamics concurrent with increasing hydraulic stress in P. edulis and potentially other similar species. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  10. Quantitative analysis of male germline stem cell differentiation reveals a role for the p53-mTORC1 pathway in spermatogonial maintenance.

    Science.gov (United States)

    Xiong, Mulin; Ferder, Ianina C; Ohguchi, Yasuyo; Wang, Ning

    2015-01-01

    p53 protects cells from DNA damage by inducing cell-cycle arrest upon encountering genomic stress. Among other pathways, p53 elicits such an effect by inhibiting mammalian target of rapamycin complex 1 (mTORC1), the master regulator of cell proliferation and growth. Although recent studies have indicated roles for both p53 and mTORC1 in stem cell maintenance, it remains unclear whether the p53-mTORC1 pathway is conserved to mediate this process under normal physiological conditions. Spermatogenesis is a classic stem cell-dependent process in which undifferentiated spermatogonia undergo self-renewal and differentiation to maintain the lifelong production of spermatozoa. To better understand this process, we have developed a novel flow cytometry (FACS)-based approach that isolates spermatogonia at consecutive differentiation stages. By using this as a tool, we show that genetic loss of p53 augments mTORC1 activity during early spermatogonial differentiation. Functionally, loss of p53 drives spermatogonia out of the undifferentiated state and causes a consistent expansion of early differentiating spermatogonia until the stage of preleptotene (premeiotic) spermatocyte. The frequency of early meiotic spermatocytes is, however, dramatically decreased. Thus, these data suggest that p53-mTORC1 pathway plays a critical role in maintaining the homeostasis of early spermatogonial differentiation. Moreover, our FACS approach could be a valuable tool in understanding spermatogonial differentiation.

  11. Transcriptome analysis of an mvp mutant reveals important changes in global gene expression and a role for methyl jasmonate in vernalization and flowering in wheat.

    Science.gov (United States)

    Diallo, Amadou Oury; Agharbaoui, Zahra; Badawi, Mohamed A; Ali-Benali, Mohamed Ali; Moheb, Amira; Houde, Mario; Sarhan, Fathey

    2014-06-01

    The einkorn wheat mutant mvp-1 (maintained vegetative phase 1) has a non-flowering phenotype caused by deletions including, but not limited to, the genes CYS, PHYC, and VRN1. However, the impact of these deletions on global gene expression is still unknown. Transcriptome analysis showed that these deletions caused the upregulation of several pathogenesis-related (PR) and jasmonate-responsive genes. These results suggest that jasmonates may be involved in flowering and vernalization in wheat. To test this hypothesis, jasmonic acid (JA) and methyl jasmonate (MeJA) content in mvp and wild-type plants was measured. The content of JA was comparable in all plants, whereas the content of MeJA was higher by more than 6-fold in mvp plants. The accumulation of MeJA was also observed in vernalization-sensitive hexaploid winter wheat during cold exposure. This accumulation declined rapidly once plants were deacclimated under floral-inductive growth conditions. This suggests that MeJA may have a role in floral transition. To confirm this result, we treated vernalization-insensitive spring wheat with MeJA. The treatment delayed flowering with significant downregulation of both TaVRN1 and TaFT1 genes. These data suggest a role for MeJA in modulating vernalization and flowering time in wheat. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  12. Quantitative measures of sexual selection reveal no evidence for sex-role reversal in a sea spider with prolonged paternal care.

    Science.gov (United States)

    Barreto, Felipe S; Avise, John C

    2010-10-07

    Taxa in which males alone invest in postzygotic care of offspring are often considered good models for investigating the proffered relationships between sexual selection and mating systems. In the pycnogonid sea spider Pycnogonum stearnsi, males carry large egg masses on their bodies for several weeks, so this species is a plausible candidate for sex-role reversal (greater intensity of sexual selection on females than on males). Here, we couple a microsatellite-based assessment of the mating system in a natural population with formal quantitative measures of genetic fitness to investigate the direction of sexual selection in P. stearnsi. Both sexes proved to be highly polygamous and showed similar standardized variances in reproductive and mating successes. Moreover, the fertility (number of progeny) of males and females appeared to be equally and highly dependent on mate access, as shown by similar Bateman gradients for the two sexes. The absence of sex-role reversal in this population of P. stearnsi is probably attributable to the fact that males are not limited by brooding space but have evolved an ability to carry large numbers of progeny. Body length was not a good predictor of male mating or reproductive success, so the aim of future studies should be to determine what traits are the targets of sexual selection in this species.

  13. Genome-Wide Identification, Characterization, and Expression Analysis of Small RNA Biogenesis Purveyors Reveal Their Role in Regulation of Biotic Stress Responses in Three Legume Crops

    Directory of Open Access Journals (Sweden)

    Rajeev K. Varshney

    2017-04-01

    Full Text Available Biotic stress in legume crops is one of the major threats to crop yield and productivity. Being sessile organisms, plants have evolved a myriad of mechanisms to combat different stresses imposed on them. One such mechanism, deciphered in the last decade, is small RNA (sRNA mediated defense in plants. Small RNAs (sRNAs have emerged as one of the major players in gene expression regulation in plants during developmental stages and under stress conditions. They are known to act both at transcriptional and post-transcriptional levels. Dicer-like (DCL, Argonaute (AGO, and RNA dependent RNA polymerase (RDR constitute the major components of sRNA biogenesis machinery and are known to play a significant role in combating biotic and abiotic stresses. This study is, therefore, focused on identification and characterization of sRNA biogenesis proteins in three important legume crops, namely chickpea, pigeonpea, and groundnut. Phylogenetic analysis of these proteins between legume species classified them into distinct clades and suggests the evolutionary conservation of these genes across the members of Papillionidoids subfamily. Variable expression of sRNA biogenesis genes in response to the biotic stresses among the three legumes indicate the possible existence of specialized regulatory mechanisms in different legumes. This is the first ever study to understand the role of sRNA biogenesis genes in response to pathogen attacks in the studied legumes.

  14. Targeted inactivation of integrin-linked kinase in hair follicle stem cells reveals an important modulatory role in skin repair after injury.

    Science.gov (United States)

    Nakrieko, Kerry-Ann; Rudkouskaya, Alena; Irvine, Timothy S; D'Souza, Sudhir J A; Dagnino, Lina

    2011-07-15

    Integrin-linked kinase (ILK) is key for normal epidermal morphogenesis, but little is known about its role in hair follicle stem cells and epidermal regeneration. Hair follicle stem cells are important contributors to newly formed epidermis following injury. We inactivated the Ilk gene in the keratin 15--expressing stem cell population of the mouse hair follicle bulge. Loss of ILK expression in these cells resulted in impaired cutaneous wound healing, with substantially decreased wound closure rates. ILK-deficient stem cells produced very few descendants that moved toward the epidermal surface and into the advancing epithelium that covers the wound. Furthermore, those few mutant cells that homed in the regenerated epidermis exhibited a reduced residence time. Paradoxically, ILK-deficient bulge stem cells responded to anagen growth signals and contributed to newly regenerated hair follicles during this phase of hair follicle growth. Thus ILK plays an important modulatory role in the normal contribution of hair follicle stem cell progeny to the regenerating epidermis following injury.

  15. LCM-seq reveals the crucial role of LsSOC1 in heat-promoted bolting of lettuce (Lactuca sativa L.).

    Science.gov (United States)

    Chen, Zijing; Zhao, Wensheng; Ge, Danfeng; Han, Yingyan; Ning, Kang; Luo, Chen; Wang, Shenglin; Liu, Renyi; Zhang, Xiaolan; Wang, Qian

    2018-05-17

    Lettuce (Lactuca sativa L.) is one of the most economically important vegetables. The floral transition in lettuce is accelerated under high temperatures, which can significantly decrease yields. However, the molecular mechanism underlying the floral tranition in lettuce is poorly known. Using laser capture microdissection coupled with RNA sequencing, we isolated shoot apical meristem cells from the bolting-sensitive lettuce line S39 at four critical stages of development. Subsequently, we screened specifically for the flowering-related gene LsSOC1 during the floral transition through comparative transcriptomic analysis. Molecular biology, developmental biology, and biochemical tools were combined to investigate the biological function of LsSOC1 in lettuce. LsSOC1 knockdown by RNA interference resulted in a significant delay in the timing of bolting and insensitivity to high temperature, which indicated that LsSOC1 functions as an activator during heat-promoted bolting in lettuce. We determined that two heat-shock transcription factors, HsfA1e and HsfA4c, bound to the promoter of LsSOC1 to confirm that LsSOC1 played an important role in heat-promoted bolting. This study indicates that LsSOC1 plays a crucial role in the heat-promoted bolting process in lettuce. Further investigation of LsSOC1 may be useful for clarification of the bolting mechanism in lettuce. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  16. Gene knockout and overexpression analysis revealed the role of N-acetylmuramidase in autolysis of Lactobacillus delbrueckii subsp. bulgaricus ljj-6.

    Science.gov (United States)

    Pang, Xiao-Yang; Cui, Wen-Ming; Liu, Lu; Zhang, Shu-Wen; Lv, Jia-Ping

    2014-01-01

    Autolysis of lactic acid bacteria (LAB) plays a vital role in dairy processing. During cheese making, autolysis of LAB affects cheese flavor development through release of intracellular enzymes and restricts the proliferation of cells in yogurt fermentation and probiotics production. In order to explore the mechanism of autolysis, the gene for the autolytic enzymes of L. bulgaricus, N-acetylmuramidase (mur), was cloned and sequenced (GenBank accession number: KF157911). Mur gene overexpression and gene knockout vectors were constructed based on pMG76e and pUC19 vectors. Recombinant plasmids were transformed into L. bulgaricus ljj-6 by electroporation, then three engineered strains with pMG76e-mur vector and fifteen engineered strains with pUC19-mur::EryBII were screened. The autolysis of the mur knockout strain was significantly lower and autolysis of the mur overexpressed strain was significantly higher compared with that of the wild type strain ljj-6. This result suggested that the mur gene played an important role in autolysis of L. bulgaricus. On the other hand, autolytic activity in a low degree was still observed in the mur knockout strain, which implied that other enzymes but autolysin encoded by mur were also involved in autolysis of L. bulgaricus.

  17. Subtype-Selective Small Molecule Inhibitors Reveal a Fundamental Role for Nav1.7 in Nociceptor Electrogenesis, Axonal Conduction and Presynaptic Release.

    Directory of Open Access Journals (Sweden)

    Aristos J Alexandrou

    Full Text Available Human genetic studies show that the voltage gated sodium channel 1.7 (Nav1.7 is a key molecular determinant of pain sensation. However, defining the Nav1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Nav1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Nav1.7's role in nociceptor physiology. We report that Nav1.7 is the predominant functional TTX-sensitive Nav in mouse and human nociceptors and contributes to the initiation and the upstroke phase of the nociceptor action potential. Moreover, we confirm a role for Nav1.7 in influencing synaptic transmission in the dorsal horn of the spinal cord as well as peripheral neuropeptide release in the skin. These findings demonstrate multiple contributions of Nav1.7 to nociceptor signalling and shed new light on the relative functional contribution of this channel to peripheral and central noxious signal transmission.

  18. Structure of the first representative of Pfam family PF09410 (DUF2006) reveals a structural signature of the calycin superfamily that suggests a role in lipid metabolism

    International Nuclear Information System (INIS)

    Chiu, Hsiu-Ju; Bakolitsa, Constantina; Skerra, Arne; Lomize, Andrei; Carlton, Dennis; Miller, Mitchell D.; Krishna, S. Sri; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Slawomir K.; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Kumar, Abhinav; Marciano, David; McMullan, Daniel; Morse, Andrew T.; Nigoghossian, Edward; Okach, Linda; Paulsen, Jessica; Reyes, Ron; Rife, Christopher L.; Bedem, Henry van den; Weekes, Dana; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2009-01-01

    NE1406, the first structural representative of PF09410, reveals a lipocalin-like fold with features that suggest involvement in lipid metabolism. In addition, NE1406 provides potential structural templates for two other protein families (PF07143 and PF08622). The first structural representative of the domain of unknown function DUF2006 family, also known as Pfam family PF09410, comprises a lipocalin-like fold with domain duplication. The finding of the calycin signature in the N-terminal domain, combined with remote sequence similarity to two other protein families (PF07143 and PF08622) implicated in isoprenoid metabolism and the oxidative stress response, support an involvement in lipid metabolism. Clusters of conserved residues that interact with ligand mimetics suggest that the binding and regulation sites map to the N-terminal domain and to the interdomain interface, respectively

  19. Knockout of the PKN Family of Rho Effector Kinases Reveals a Non-redundant Role for PKN2 in Developmental Mesoderm Expansion

    Directory of Open Access Journals (Sweden)

    Ivan Quétier

    2016-01-01

    Full Text Available In animals, the protein kinase C (PKC family has expanded into diversely regulated subgroups, including the Rho family-responsive PKN kinases. Here, we describe knockouts of all three mouse PKN isoforms and reveal that PKN2 loss results in lethality at embryonic day 10 (E10, with associated cardiovascular and morphogenetic defects. The cardiovascular phenotype was not recapitulated by conditional deletion of PKN2 in endothelial cells or the developing heart. In contrast, inducible systemic deletion of PKN2 after E7 provoked collapse of the embryonic mesoderm. Furthermore, mouse embryonic fibroblasts, which arise from the embryonic mesoderm, depend on PKN2 for proliferation and motility. These cellular defects are reflected in vivo as dependence on PKN2 for mesoderm proliferation and neural crest migration. We conclude that failure of the mesoderm to expand in the absence of PKN2 compromises cardiovascular integrity and development, resulting in lethality.

  20. Electron microprobe analysis (WDS EPMA) of Zhamanshin glass reveals the impactor and a common role of accretion in the origin of splash-form impact glass

    International Nuclear Information System (INIS)

    Vetvicka, I; Frank, J; Drtina, J

    2010-01-01

    Impact glass samples collected during expeditions to the Zhamashin and Lonar craters were subjected to a morphology survey and compared to Wabar, Henbury and Darwin impact glasses to reveal that the accretion of fibres and spherules is not exclusive for irghizites but occurs in other splash form glasses over the world. WDS EPMA and LA-ICP-MS assays of Zhamanshin and Lonar glasses enabled the definition of akmurynites as Zhamanshin glass of specific morphology, chemistry and absence of extraterrestrial contamination. However, extraterrestrial contamination in irghizites was verified and further WDS EPMA analyses led to the conclusion that the Zhamanshin crater had been formed by the impact of a primitive achondrite of Lodran chemistry.

  1. Proteomic analysis of HIV-1 Nef cellular binding partners reveals a role for exocyst complex proteins in mediating enhancement of intercellular nanotube formation

    Directory of Open Access Journals (Sweden)

    Mukerji Joya

    2012-06-01

    Full Text Available Abstract Background HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunneling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. Results To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3. We report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6, an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. Conclusions Exocyst complex proteins are likely a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Linkages revealed between Nef and the exocyst complex suggest a new paradigm of

  2. Structural and functional studies of the biotin protein ligase from Aquifex aeolicus reveal a critical role for a conserved residue in target specificity.

    Science.gov (United States)

    Tron, Cecile M; McNae, Iain W; Nutley, Margaret; Clarke, David J; Cooper, Alan; Walkinshaw, Malcolm D; Baxter, Robert L; Campopiano, Dominic J

    2009-03-20

    Biotin protein ligase (BPL; EC 6.3.4.15) catalyses the formation of biotinyl-5'-AMP from biotin and ATP, and the succeeding biotinylation of the biotin carboxyl carrier protein. We describe the crystal structures, at 2.4 A resolution, of the class I BPL from the hyperthermophilic bacteria Aquifex aeolicus (AaBPL) in its ligand-free form and in complex with biotin and ATP. The solvent-exposed beta- and gamma-phosphates of ATP are located in the inter-subunit cavity formed by the N- and C-terminal domains. The Arg40 residue from the conserved GXGRXG motif is shown to interact with the carboxyl group of biotin and to stabilise the alpha- and beta-phosphates of the nucleotide. The structure of the mutant AaBPL R40G in both the ligand-free and biotin-bound forms reveals that the mutated loop has collapsed, thus hindering ATP binding. Isothermal titration calorimetry indicated that the presence of biotin is not required for ATP binding to wild-type AaBPL in the absence of Mg(2+), and the binding of biotin and ATP has been determined to occur via a random but cooperative process. The affinity for biotin is relatively unaffected by the R40G mutation. In contrast, the thermodynamic data indicate that binding of ATP to AaBPL R40G is very weak in the absence or in the presence of biotin. The AaBPL R40G mutant remains catalytically active but shows poor substrate specificity; mass spectrometry and Western blot studies revealed that the mutant biotinylates both the target A. aeolicus BCCPDelta67 fragment and BSA, and is subject to self-biotinylation.

  3. Dynamic genome wide expression profiling of Drosophila head development reveals a novel role of Hunchback in retinal glia cell development and blood-brain barrier integrity.

    Directory of Open Access Journals (Sweden)

    Montserrat Torres-Oliva

    2018-01-01

    Full Text Available Drosophila melanogaster head development represents a valuable process to study the developmental control of various organs, such as the antennae, the dorsal ocelli and the compound eyes from a common precursor, the eye-antennal imaginal disc. While the gene regulatory network underlying compound eye development has been extensively studied, the key transcription factors regulating the formation of other head structures from the same imaginal disc are largely unknown. We obtained the developmental transcriptome of the eye-antennal discs covering late patterning processes at the late 2nd larval instar stage to the onset and progression of differentiation at the end of larval development. We revealed the expression profiles of all genes expressed during eye-antennal disc development and we determined temporally co-expressed genes by hierarchical clustering. Since co-expressed genes may be regulated by common transcriptional regulators, we combined our transcriptome dataset with publicly available ChIP-seq data to identify central transcription factors that co-regulate genes during head development. Besides the identification of already known and well-described transcription factors, we show that the transcription factor Hunchback (Hb regulates a significant number of genes that are expressed during late differentiation stages. We confirm that hb is expressed in two polyploid subperineurial glia cells (carpet cells and a thorough functional analysis shows that loss of Hb function results in a loss of carpet cells in the eye-antennal disc. Additionally, we provide for the first time functional data indicating that carpet cells are an integral part of the blood-brain barrier. Eventually, we combined our expression data with a de novo Hb motif search to reveal stage specific putative target genes of which we find a significant number indeed expressed in carpet cells.

  4. Dynamic genome wide expression profiling of Drosophila head development reveals a novel role of Hunchback in retinal glia cell development and blood-brain barrier integrity

    Science.gov (United States)

    Torres-Oliva, Montserrat; Schneider, Julia; Wiegleb, Gordon

    2018-01-01

    Drosophila melanogaster head development represents a valuable process to study the developmental control of various organs, such as the antennae, the dorsal ocelli and the compound eyes from a common precursor, the eye-antennal imaginal disc. While the gene regulatory network underlying compound eye development has been extensively studied, the key transcription factors regulating the formation of other head structures from the same imaginal disc are largely unknown. We obtained the developmental transcriptome of the eye-antennal discs covering late patterning processes at the late 2nd larval instar stage to the onset and progression of differentiation at the end of larval development. We revealed the expression profiles of all genes expressed during eye-antennal disc development and we determined temporally co-expressed genes by hierarchical clustering. Since co-expressed genes may be regulated by common transcriptional regulators, we combined our transcriptome dataset with publicly available ChIP-seq data to identify central transcription factors that co-regulate genes during head development. Besides the identification of already known and well-described transcription factors, we show that the transcription factor Hunchback (Hb) regulates a significant number of genes that are expressed during late differentiation stages. We confirm that hb is expressed in two polyploid subperineurial glia cells (carpet cells) and a thorough functional analysis shows that loss of Hb function results in a loss of carpet cells in the eye-antennal disc. Additionally, we provide for the first time functional data indicating that carpet cells are an integral part of the blood-brain barrier. Eventually, we combined our expression data with a de novo Hb motif search to reveal stage specific putative target genes of which we find a significant number indeed expressed in carpet cells. PMID:29360820

  5. Genome-Resolved Metagenomic Analysis Reveals Roles for Candidate Phyla and Other Microbial Community Members in Biogeochemical Transformations in Oil Reservoirs.

    Science.gov (United States)

    Hu, Ping; Tom, Lauren; Singh, Andrea; Thomas, Brian C; Baker, Brett J; Piceno, Yvette M; Andersen, Gary L; Banfield, Jillian F

    2016-01-19

    Oil reservoirs are major sites of methane production and carbon turnover, processes with significant impacts on energy resources and global biogeochemical cycles. We applied a cultivation-independent genomic approach to define microbial community membership and predict roles for specific organisms in biogeochemical transformations in Alaska North Slope oil fields. Produced water samples were collected from six locations between 1,128 m (24 to 27°C) and 2,743 m (80 to 83°C) below the surface. Microbial community complexity decreased with increasing temperature, and the potential to degrade hydrocarbon compounds was most prevalent in the lower-temperature reservoirs. Sulfate availability, rather than sulfate reduction potential, seems to be the limiting factor for sulfide production in some of the reservoirs under investigation. Most microorganisms in the intermediate- and higher-temperature samples were related to previously studied methanogenic and nonmethanogenic archaea and thermophilic bacteria, but one candidate phylum bacterium, a member of the Acetothermia (OP1), was present in Kuparuk sample K3. The greatest numbers of candidate phyla were recovered from the mesothermic reservoir samples SB1 and SB2. We reconstructed a nearly complete genome for an organism from the candidate phylum Parcubacteria (OD1) that was abundant in sample SB1. Consistent with prior findings for members of this lineage, the OD1 genome is small, and metabolic predictions support an obligately anaerobic, fermentation-based lifestyle. At moderate abundance in samples SB1 and SB2 were members of bacteria from other candidate phyla, including Microgenomates (OP11), Atribacteria (OP9), candidate phyla TA06 and WS6, and Marinimicrobia (SAR406). The results presented here elucidate potential roles of organisms in oil reservoir biological processes. The activities of microorganisms in oil reservoirs impact petroleum resource quality and the global carbon cycle. We show that bacteria

  6. Characterization of the rubber tree metallothionein family reveals a role in mitigating the effects of reactive oxygen species associated with physiological stress.

    Science.gov (United States)

    Huang, Yacheng; Fang, Yongjun; Long, Xiangyu; Liu, Linya; Wang, Jia; Zhu, Jinheng; Ma, Yanyan; Qin, Yunxia; Qi, Jiyan; Hu, Xinwen; Tang, Chaorong

    2018-02-07

    Metallothioneins (MTs) as reactive oxygen species (ROS) scavengers play important roles in stress response and heavy metal homeostasis. In Hevea brasiliensis (the para rubber tree that is the source of commercial natural rubber) and in other trees, the functions of MTs are not well understood. Latex exudes when the rubber tree is tapped. The flow of latex and its regeneration can be enhanced by tapping, wounding and ethylene treatment, all of which produce ROS as a by-product. Here, we show the presence of four MT genes in H. brasiliensis, comprising three Type 2 (HbMT2, -2a and -2b) and one Type 3 (HbMT3L) isoforms, representing one of the smallest MT gene families among angiosperms. The four HbMTs exhibited distinct tissue expression patterns: HbMT2 and HbMT3L mainly in leaves, HbMT2a specifically in flowers and HbMT2b in diverse tissues. The expression of HbMT2b, an isoform present in latex, decreased significantly in the latex following the stress-inducing treatments of tapping, wounding and ethephon (an ethylene generator). The expressions of the leaf-abundant isoforms, HbMT2 and -3L were up-regulated following pathogenic fungus infection and high-temperature stress, but down-regulated by low-temperature stress. These reactions were consistent with multiple defense- and hormone-responsive cis-acting elements in the HbMT promoters. Nine transcription factors were shown to implicate in the high-temperature responsiveness of HbMT2 and -3L in leaves. Overexpression of HbMT2 in Escherichia coli enhanced the bacterium's tolerance to heavy metals and ROS, consistent with its predicted role as an ROS scavenger. Taken together, our results, along with other relevant studies, suggest an important role of HbMTs in latex regeneration as well as species adaptation via the regulation of ROS homeostasis. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  7. Detoxification of ammonia in mouse cortical GABAergic cell cultures increases neuronal oxidative metabolism and reveals an emerging role for release of glucose-derived alanine

    DEFF Research Database (Denmark)

    Leke, Renata; Bak, Lasse Kristoffer; Anker, Malene

    2011-01-01

    Cerebral hyperammonemia is believed to play a pivotal role in the development of hepatic encephalopathy (HE), a debilitating condition arising due to acute or chronic liver disease. In the brain, ammonia is thought to be detoxified via the activity of glutamine synthetase, an astrocytic enzyme....... Moreover, it has been suggested that cerebral tricarboxylic acid (TCA) cycle metabolism is inhibited and glycolysis enhanced during hyperammonemia. The aim of this study was to characterize the ammonia-detoxifying mechanisms as well as the effects of ammonia on energy-generating metabolic pathways...... in a mouse neuronal-astrocytic co-culture model of the GABAergic system. We found that 5 mM ammonium chloride affected energy metabolism by increasing the neuronal TCA cycle activity and switching the astrocytic TCA cycle toward synthesis of substrate for glutamine synthesis. Furthermore, ammonia exposure...

  8. What is said or how it is said makes a difference: role of the right fronto-parietal operculum in emotional prosody as revealed by repetitive TMS.

    Science.gov (United States)

    van Rijn, Sophie; Aleman, André; van Diessen, Eric; Berckmoes, Celine; Vingerhoets, Guy; Kahn, René S

    2005-06-01

    Emotional signals in spoken language can be conveyed by semantic as well as prosodic cues. We investigated the role of the fronto-parietal operculum, a somatosensory area where the lips, tongue and jaw are represented, in the right hemisphere to detection of emotion in prosody vs. semantics. A total of 14 healthy volunteers participated in the present experiment, which involved transcranial magnetic stimulation (TMS) in combination with frameless stereotaxy. As predicted, compared with sham stimulation, TMS over the right fronto-parietal operculum differentially affected the reaction times for detection of emotional prosody vs. emotional semantics, showing that there is a dissociation at a neuroanatomical level. Detection of withdrawal emotions (fear and sadness) in prosody was delayed significantly by TMS. No effects of TMS were observed for approach emotions (happiness and anger). We propose that the right fronto-parietal operculum is not globally involved in emotion evaluation, but sensitive to specific forms of emotional discrimination and emotion types.

  9. In vivo FRET imaging revealed a regulatory role of RanGTP in kinetochore-microtubule attachments via Aurora B kinase.

    Directory of Open Access Journals (Sweden)

    Yoke-Peng Lee

    Full Text Available Under the fluctuating circumstances provided by the innate dynamics of microtubules and opposing tensions resulted from microtubule-associated motors, it is vital to ensure stable kinetochore-microtubule attachments for accurate segregation. However, a comprehensive understanding of how this regulation is mechanistically achieved remains elusive. Using our newly designed live cell FRET time-lapse imaging, we found that post-metaphase RanGTP is crucial in the maintenance of stable kinetochore-microtubule attachments by regulating Aurora B kinase via the NES-bearing Mst1. More importantly, our study demonstrates that by ensuring stable alignment of metaphase chromosomes prior to segregation, RanGTP is indispensible in governing the genomic integrity and the fidelity of cell cycle progression. Our findings suggest an additional role of RanGTP beyond its known function in mitotic spindle assembly during the prometaphase-metaphase transition.

  10. Possible roles of vacuolar H+-ATPase and mitochondrial function in tolerance to air-drying stress revealed by genome-wide screening of Saccharomyces cerevisiae deletion strains.

    Science.gov (United States)

    Shima, Jun; Ando, Akira; Takagi, Hiroshi

    2008-03-01

    Yeasts used in bread making are exposed to air-drying stress during dried yeast production processes. To clarify the genes required for air-drying tolerance, we performed genome-wide screening using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 278 gene deletions responsible for air-drying sensitivity. These genes were classified based on their cellular function and on the localization of their gene products. The results showed that the genes required for air-drying tolerance were frequently involved in mitochondrial functions and in connection with vacuolar H(+)-ATPase, which plays a role in vacuolar acidification. To determine the role of vacuolar acidification in air-drying stress tolerance, we monitored intracellular pH. The results showed that intracellular acidification was induced during air-drying and that this acidification was amplified in a deletion mutant of the VMA2 gene encoding a component of vacuolar H(+)-ATPase, suggesting that vacuolar H(+)-ATPase helps maintain intracellular pH homeostasis, which is affected by air-drying stress. To determine the effects of air-drying stress on mitochondria, we analysed the mitochondrial membrane potential under air-drying stress conditions using MitoTracker. The results showed that mitochondria were extremely sensitive to air-drying stress, suggesting that a mitochondrial function is required for tolerance to air-drying stress. We also analysed the correlation between oxidative-stress sensitivity and air-drying-stress sensitivity. The results suggested that oxidative stress is a critical determinant of sensitivity to air-drying stress, although ROS-scavenging systems are not necessary for air-drying stress tolerance. (c) 2008 John Wiley & Sons, Ltd.

  11. Genotypic variation in growth and physiological response to drought stress and re-watering reveals the critical role of recovery in drought adaptation in maize seedlings

    Directory of Open Access Journals (Sweden)

    Daoqian eChen

    2016-01-01

    Full Text Available Non-irrigated crops in temperate climates and irrigated crops in arid climates are subjected to continuous cycles of water stress and re-watering. Thus, fast and efficient recovery from water stress may be among the key determinants of plant drought adaptation. The present study was designed to comparatively analyze the roles of drought resistance and drought recovery in drought adaptation and to investigate the physiological basis of genotypic variation in drought adaptation in maize (Zea mays seedlings. As the seedlings behavior in growth associate with yield under drought, it could partly reflect the potential of drought adaptability. Growth and physiological responses to progressive drought stress and recovery were observed in seedlings of ten maize lines. The results showed that drought adaptability is closely related to drought recovery (r = 0.714**, but not to drought resistance (r = 0.332. Drought induced decreases in leaf water content, water potential, osmotic potential, gas exchange parameters, chlorophyll content, Fv/Fm and nitrogen content, and increased H2O2 accumulation and lipid peroxidation. After recovery, most of these physiological parameters rapidly returned to normal levels. The physiological responses varied between lines. Further correlation analysis indicated that the physiological bases of drought resistance and drought recovery are definitely different, and that maintaining higher chlorophyll content (r = 0.874*** and Fv/Fm (r = 0.626* under drought stress contributes to drought recovery. Our results suggest that both drought resistance and recovery are key determinants of plant drought adaptation, and that drought recovery may play a more important role than previously thought. In addition, leaf water potential, chlorophyll content and Fv/Fm could be used as efficient reference indicators in the selection of drought-adaptive genotypes.

  12. Genome-Resolved Metagenomic Analysis Reveals Roles for Candidate Phyla and Other Microbial Community Members in Biogeochemical Transformations in Oil Reservoirs

    Directory of Open Access Journals (Sweden)

    Ping Hu

    2016-01-01

    Full Text Available Oil reservoirs are major sites of methane production and carbon turnover, processes with significant impacts on energy resources and global biogeochemical cycles. We applied a cultivation-independent genomic approach to define microbial community membership and predict roles for specific organisms in biogeochemical transformations in Alaska North Slope oil fields. Produced water samples were collected from six locations between 1,128 m (24 to 27°C and 2,743 m (80 to 83°C below the surface. Microbial community complexity decreased with increasing temperature, and the potential to degrade hydrocarbon compounds was most prevalent in the lower-temperature reservoirs. Sulfate availability, rather than sulfate reduction potential, seems to be the limiting factor for sulfide production in some of the reservoirs under investigation. Most microorganisms in the intermediate- and higher-temperature samples were related to previously studied methanogenic and nonmethanogenic archaea and thermophilic bacteria, but one candidate phylum bacterium, a member of the Acetothermia (OP1, was present in Kuparuk sample K3. The greatest numbers of candidate phyla were recovered from the mesothermic reservoir samples SB1 and SB2. We reconstructed a nearly complete genome for an organism from the candidate phylum Parcubacteria (OD1 that was abundant in sample SB1. Consistent with prior findings for members of this lineage, the OD1 genome is small, and metabolic predictions support an obligately anaerobic, fermentation-based lifestyle. At moderate abundance in samples SB1 and SB2 were members of bacteria from other candidate phyla, including Microgenomates (OP11, Atribacteria (OP9, candidate phyla TA06 and WS6, and Marinimicrobia (SAR406. The results presented here elucidate potential roles of organisms in oil reservoir biological processes.

  13. Reexamination of the Physiological Role of PykA in Escherichia coli Revealed that It Negatively Regulates the Intracellular ATP Levels under Anaerobic Conditions.

    Science.gov (United States)

    Zhao, Chunhua; Lin, Zhao; Dong, Hongjun; Zhang, Yanping; Li, Yin

    2017-06-01

    Pyruvate kinase is one of the three rate-limiting glycolytic enzymes that catalyze the last step of glycolysis, conversion of phosphoenolpyruvate (PEP) into pyruvate, which is associated with ATP generation. Two isozymes of pyruvate kinase, PykF and PykA, are identified in Escherichia coli PykF is considered important, whereas PykA has a less-defined role. Prior studies inactivated the pykA gene to increase the level of its substrate, PEP, and thereby increased the yield of end products derived from PEP. We were surprised when we found a pykA ::Tn 5 mutant in a screen for increased yield of an end product derived from pyruvate ( n -butanol), suggesting that the role of PykA needs to be reexamined. We show that the pykA mutant exhibited elevated intracellular ATP levels, biomass concentrations, glucose consumption, and n -butanol production. We also discovered that the pykA mutant expresses higher levels of a presumed pyruvate transporter, YhjX, permitting the mutant to recapture and metabolize excreted pyruvate. Furthermore, we demonstrated that the nucleotide diphosphate kinase activity of PykA leads to negative regulation of the intracellular ATP levels. Taking the data together, we propose that inactivation of pykA can be considered a general strategy to enhance the production of pyruvate-derived metabolites under anaerobic conditions. IMPORTANCE This study showed that knocking out pykA significantly increased the intracellular ATP level and thus significantly increased the levels of glucose consumption, biomass formation, and pyruvate-derived product formation under anaerobic conditions. pykA was considered to be encoding a dispensable pyruvate kinase; here we show that pykA negatively regulates the anaerobic glycolysis rate through regulating the energy distribution. Thus, knocking out pykA can be used as a general strategy to increase the level of pyruvate-derived fermentative products. Copyright © 2017 American Society for Microbiology.

  14. Novel ethyl methanesulfonate (EMS-induced null alleles of the Drosophila homolog of LRRK2 reveal a crucial role in endolysosomal functions and autophagy in vivo

    Directory of Open Access Journals (Sweden)

    Mark W. Dodson

    2014-12-01

    Full Text Available Mutations in LRRK2 cause a dominantly inherited form of Parkinson’s disease (PD and are the most common known genetic determinant of PD. Inhibitor-based therapies targeting LRRK2 have emerged as a key therapeutic strategy in PD; thus, understanding the consequences of inhibiting the normal cellular functions of this protein is vital. Despite much interest, the physiological functions of LRRK2 remain unclear. Several recent studies have linked the toxicity caused by overexpression of pathogenic mutant forms of LRRK2 to defects in the endolysosomal and autophagy pathways, raising the question of whether endogenous LRRK2 might play a role in these processes. Here, we report the characterization of multiple novel ethyl methanesulfonate (EMS-induced nonsense alleles in the Drosophila LRRK2 homolog, lrrk. Using these alleles, we show that lrrk loss-of-function causes striking defects in the endolysosomal and autophagy pathways, including the accumulation of markedly enlarged lysosomes that are laden with undigested contents, consistent with a defect in lysosomal degradation. lrrk loss-of-function also results in the accumulation of autophagosomes, as well as the presence of enlarged early endosomes laden with mono-ubiquitylated cargo proteins, suggesting an additional defect in lysosomal substrate delivery. Interestingly, the lysosomal abnormalities in these lrrk mutants can be suppressed by a constitutively active form of the small GTPase rab9, which promotes retromer-dependent recycling from late endosomes to the Golgi. Collectively, our data provides compelling evidence of a vital role for lrrk in lysosomal function and endolysosomal membrane transport in vivo, and suggests a link between lrrk and retromer-mediated endosomal recycling.

  15. Novel ethyl methanesulfonate (EMS)-induced null alleles of the Drosophila homolog of LRRK2 reveal a crucial role in endolysosomal functions and autophagy in vivo.

    Science.gov (United States)

    Dodson, Mark W; Leung, Lok K; Lone, Mohiddin; Lizzio, Michael A; Guo, Ming

    2014-12-01

    Mutations in LRRK2 cause a dominantly inherited form of Parkinson's disease (PD) and are the most common known genetic determinant of PD. Inhibitor-based therapies targeting LRRK2 have emerged as a key therapeutic strategy in PD; thus, understanding the consequences of inhibiting the normal cellular functions of this protein is vital. Despite much interest, the physiological functions of LRRK2 remain unclear. Several recent studies have linked the toxicity caused by overexpression of pathogenic mutant forms of LRRK2 to defects in the endolysosomal and autophagy pathways, raising the question of whether endogenous LRRK2 might play a role in these processes. Here, we report the characterization of multiple novel ethyl methanesulfonate (EMS)-induced nonsense alleles in the Drosophila LRRK2 homolog, lrrk. Using these alleles, we show that lrrk loss-of-function causes striking defects in the endolysosomal and autophagy pathways, including the accumulation of markedly enlarged lysosomes that are laden with undigested contents, consistent with a defect in lysosomal degradation. lrrk loss-of-function also results in the accumulation of autophagosomes, as well as the presence of enlarged early endosomes laden with mono-ubiquitylated cargo proteins, suggesting an additional defect in lysosomal substrate delivery. Interestingly, the lysosomal abnormalities in these lrrk mutants can be suppressed by a constitutively active form of the small GTPase rab9, which promotes retromer-dependent recycling from late endosomes to the Golgi. Collectively, our data provides compelling evidence of a vital role for lrrk in lysosomal function and endolysosomal membrane transport in vivo, and suggests a link between lrrk and retromer-mediated endosomal recycling. © 2014. Published by The Company of Biologists Ltd.

  16. Distinct roles of autophagy-dependent and -independent functions of FIP200 revealed by generation and analysis of a mutant knock-in mouse model

    Science.gov (United States)

    Chen, Song; Wang, Chenran; Yeo, Syn; Liang, Chun-Chi; Okamoto, Takako; Sun, Shaogang; Wen, Jian; Guan, Jun-Lin

    2016-01-01

    Autophagy is an evolutionarily conserved cellular process controlled through a set of essential autophagy genes (Atgs). However, there is increasing evidence that most, if not all, Atgs also possess functions independent of their requirement in canonical autophagy, making it difficult to distinguish the contributions of autophagy-dependent or -independent functions of a particular Atg to various biological processes. To distinguish these functions for FIP200 (FAK family-interacting protein of 200 kDa), an Atg in autophagy induction, we examined FIP200 interaction with its autophagy partner, Atg13. We found that residues 582–585 (LQFL) in FIP200 are required for interaction with Atg13, and mutation of these residues to AAAA (designated the FIP200-4A mutant) abolished its canonical autophagy function in vitro. Furthermore, we created a FIP200-4A mutant knock-in mouse model and found that specifically blocking FIP200 interaction with Atg13 abolishes autophagy in vivo, providing direct support for the essential role of the ULK1/Atg13/FIP200/Atg101 complex in the process beyond previous studies relying on the complete knockout of individual components. Analysis of the new mouse model showed that nonautophagic functions of FIP200 are sufficient to fully support embryogenesis by maintaining a protective role in TNFα-induced apoptosis. However, FIP200-mediated canonical autophagy is required to support neonatal survival and tumor cell growth. These studies provide the first genetic evidence linking an Atg's autophagy and nonautophagic functions to different biological processes in vivo. PMID:27013233

  17. Roles of the 15-kDa Selenoprotein (Sep15) in Redox Homeostasis and Cataract Development Revealed by the Analysis of Sep 15 Knockout Mice*

    Science.gov (United States)

    Kasaikina, Marina V.; Fomenko, Dmitri E.; Labunskyy, Vyacheslav M.; Lachke, Salil A.; Qiu, Wenya; Moncaster, Juliet A.; Zhang, Jie; Wojnarowicz, Mark W.; Natarajan, Sathish Kumar; Malinouski, Mikalai; Schweizer, Ulrich; Tsuji, Petra A.; Carlson, Bradley A.; Maas, Richard L.; Lou, Marjorie F.; Goldstein, Lee E.; Hatfield, Dolph L.; Gladyshev, Vadim N.

    2011-01-01

    The 15-kDa selenoprotein (Sep15) is a thioredoxin-like, endoplasmic reticulum-resident protein involved in the quality control of glycoprotein folding through its interaction with UDP-glucose:glycoprotein glucosyltransferase. Expression of Sep15 is regulated by dietary selenium and the unfolded protein response, but its specific function is not known. In this study, we developed and characterized Sep15 KO mice by targeted removal of exon 2 of the Sep15 gene coding for the cysteine-rich UDP-glucose:glycoprotein glucosyltransferase-binding domain. These KO mice synthesized a mutant mRNA, but the shortened protein product could be detected neither in tissues nor in Sep15 KO embryonic fibroblasts. Sep15 KO mice were viable and fertile, showed normal brain morphology, and did not activate endoplasmic reticulum stress pathways. However, parameters of oxidative stress were elevated in the livers of these mice. We found that Sep15 mRNA was enriched during lens development. Further phenotypic characterization of Sep15 KO mice revealed a prominent nuclear cataract that developed at an early age. These cataracts did not appear to be associated with severe oxidative stress or glucose dysregulation. We suggest that the cataracts resulted from an improper folding status of lens proteins caused by Sep15 deficiency. PMID:21768092

  18. Genetic mosaic analysis reveals a major role for frizzled 4 and frizzled 8 in controlling ureteric growth in the developing kidney.

    Science.gov (United States)

    Ye, Xin; Wang, Yanshu; Rattner, Amir; Nathans, Jeremy

    2011-03-01

    The developing mammalian kidney is an attractive system in which to study the control of organ growth. Targeted mutations in the Wnt receptors frizzled (Fz) 4 and Fz8 lead to reduced ureteric bud growth and a reduction in kidney size, a phenotype previously reported for loss of Wnt11. In cell culture, Fz4 and Fz8 can mediate noncanonical signaling stimulated by Wnt11, but only Fz4 mediates Wnt11-stimulated canonical signaling. In genetically mosaic mouse ureteric buds, competition between phenotypically mutant Fz4(-/-) or Fz4(-/-);Fz8(-/-) cells and adjacent phenotypically wild-type Fz4(+/-) or Fz4(+/-);Fz8(-/-) cells results in under-representation of the mutant cells to an extent far greater than would be predicted from the size reduction of homogeneously mutant kidneys. This discrepancy presumably reflects the compensatory action of a network of growth regulatory systems that minimize developmental perturbations. The present work represents the first description of a kidney phenotype referable to one or more Wnt receptors and demonstrates a general strategy for revealing the contribution of an individual growth regulatory pathway when it is part of a larger homeostatic network.

  19. Proteomics of Fusarium oxysporum race 1 and race 4 reveals enzymes involved in carbohydrate metabolism and ion transport that might play important roles in banana Fusarium wilt.

    Science.gov (United States)

    Sun, Yong; Yi, Xiaoping; Peng, Ming; Zeng, Huicai; Wang, Dan; Li, Bo; Tong, Zheng; Chang, Lili; Jin, Xiang; Wang, Xuchu

    2014-01-01

    Banana Fusarium wilt is a soil-spread fungal disease caused by Fusarium oxysporum. In China, the main virulence fungi in banana are F. oxysporum race 1 (F1, weak virulence) and race 4 (F4, strong virulence). To date, no proteomic analyses have compared the two races, but the difference in virulence between F1 and F4 might result from their differentially expressed proteins. Here we report the first comparative proteomics of F1 and F4 cultured under various conditions, and finally identify 99 protein species, which represent 59 unique proteins. These proteins are mainly involved in carbohydrate metabolism, post-translational modification, energy production, and inorganic ion transport. Bioinformatics analysis indicated that among the 46 proteins identified from F4 were several enzymes that might be important for virulence. Reverse transcription PCR analysis of the genes for 15 of the 56 proteins revealed that their transcriptional patterns were similar to their protein expression patterns. Taken together, these data suggest that proteins involved in carbohydrate metabolism and ion transport may be important in the pathogenesis of banana Fusarium wilt. Some enzymes such as catalase-peroxidase, galactosidase and chitinase might contribute to the strong virulence of F4. Overexpression or knockout of the genes for the F4-specific proteins will help us to further understand the molecular mechanism of Fusarium-induced banana wilt.

  20. Separating foliar physiology from morphology reveals the relative roles of vertically structured transpiration factors within red maple crowns and limitations of larger scale models

    Science.gov (United States)

    Bauerle, William L.; Bowden, Joseph D.

    2011-01-01

    A spatially explicit mechanistic model, MAESTRA, was used to separate key parameters affecting transpiration to provide insights into the most influential parameters for accurate predictions of within-crown and within-canopy transpiration. Once validated among Acer rubrum L. genotypes, model responses to different parameterization scenarios were scaled up to stand transpiration (expressed per unit leaf area) to assess how transpiration might be affected by the spatial distribution of foliage properties. For example, when physiological differences were accounted for, differences in leaf width among A. rubrum L. genotypes resulted in a 25% difference in transpiration. An in silico within-canopy sensitivity analysis was conducted over the range of genotype parameter variation observed and under different climate forcing conditions. The analysis revealed that seven of 16 leaf traits had a ≥5% impact on transpiration predictions. Under sparse foliage conditions, comparisons of the present findings with previous studies were in agreement that parameters such as the maximum Rubisco-limited rate of photosynthesis can explain ∼20% of the variability in predicted transpiration. However, the spatial analysis shows how such parameters can decrease or change in importance below the uppermost canopy layer. Alternatively, model sensitivity to leaf width and minimum stomatal conductance was continuous along a vertical canopy depth profile. Foremost, transpiration sensitivity to an observed range of morphological and physiological parameters is examined and the spatial sensitivity of transpiration model predictions to vertical variations in microclimate and foliage density is identified to reduce the uncertainty of current transpiration predictions. PMID:21617246

  1. Roles for SH2 and SH3 domains in Lyn kinase association with activated FcepsilonRI in RBL mast cells revealed by patterned surface analysis.

    Science.gov (United States)

    Hammond, Stephanie; Wagenknecht-Wiesner, Alice; Veatch, Sarah L; Holowka, David; Baird, Barbara

    2009-10-01

    In mast cells, antigen-mediated cross-linking of IgE bound to its high-affinity surface receptor, FcepsilonRI, initiates a signaling cascade that culminates in degranulation and release of allergic mediators. Antigen-patterned surfaces, in which the antigen is deposited in micron-sized features on a silicon substrate, were used to examine the spatial relationship between clustered IgE-FcepsilonRI complexes and Lyn, the signal-initiating tyrosine kinase. RBL mast cells expressing wild-type Lyn-EGFP showed co-redistribution of this protein with clustered IgE receptors on antigen-patterned surfaces, whereas Lyn-EGFP containing an inhibitory point mutation in its SH2 domain did not significantly accumulate with the patterned antigen, and Lyn-EGFP with an inhibitory point mutation in its SH3 domain exhibited reduced interactions. Our results using antigen-patterned surfaces and quantitative cross-correlation image analysis reveal that both the SH2 and SH3 domains contribute to interactions between Lyn kinase and cross-linked IgE receptors in stimulated mast cells.

  2. Sequence and Expression Analysis of Interferon Regulatory Factor 10 (IRF10 in Three Diverse Teleost Fish Reveals Its Role in Antiviral Defense.

    Directory of Open Access Journals (Sweden)

    Qiaoqing Xu

    Full Text Available Interferon regulatory factor (IRF 10 was first found in birds and is present in the genome of other tetrapods (but not humans and mice, as well as in teleost fish. The functional role of IRF10 in vertebrate immunity is relatively unknown compared to IRF1-9. The target of this research was to clone and characterize the IRF10 genes in three economically important fish species that will facilitate future evaluation of this molecule in fish innate and adaptive immunity.In the present study, a single IRF10 gene was cloned in grass carp Ctenopharyngodon idella and Asian swamp eel Monopterus albus, and two, named IRF10a and IRF10b, in rainbow trout Oncorhynchus mykiss. The fish IRF10 molecules share highest identities to other vertebrate IRF10s, and have a well conserved DNA binding domain, IRF-associated domain, and an 8 exon/7 intron structure with conserved intron phase. The presence of an upstream ATG or open reading frame (ORF in the 5'-untranslated region of different fish IRF10 cDNA sequences suggests potential regulation at the translational level, and this has been verified by in vitro transcription/translation experiments of the trout IRF10a cDNA, but would still need to be validated in fish cells.Both trout IRF10 paralogues are highly expressed in thymus, blood and spleen but are relatively low in head kidney and caudal kidney. Trout IRF10b expression is significantly higher than IRF10a in integumentary tissues i.e. gills, scales, skin, intestine, adipose fin and tail fins, suggesting that IRF10b may be more important in mucosal immunity. The expression of both trout IRF10 paralogues is up-regulated by recombinant IFN-γ. The expression of the IRF10 genes is highly induced by Poly I:C in vitro and in vivo, and by viral infection, but is less responsive to peptidoglycan and bacterial infection, suggesting an important role of fish IRF10 in antiviral defense.

  3. Chemical lesioning and glutamate administration reveal a major role for the nucleus tractus solitarius in the cardiac-somatic reflex in rats.

    Science.gov (United States)

    Liu, X-H; Sun, N; Du, J-Q; Tang, J-S; Han, M; Zhu, J-X; Huo, F-Q

    2012-04-05

    Many patients suffer from secondary muscle hyperalgesia after experiencing angina pectoris. In this study, we examined the role of the nucleus tractus solitarius (NTS) and glutamate receptors in modulating cardiac-evoked muscle hyperalgesia induced by pericardial capsaicin, which was monitored by recording electromyogram (EMG) activity from the spinotrapezius muscle in the anesthetized rat. Unilateral chemical lesioning of the commissural NTS with the neurotoxin ibotenic acid significantly depressed the cardiac-somatic reflex; the EMG responses decreased to 56.4 ± 6.9% of that of the controls (5 of 5). Microinjection of the excitatory amino acid glutamate, at 10, 20, and 50 nmol, into the commissural NTS increased the EMG response, in a dose-dependent manner, to 116.9 ± 4.9%, 143.9 ± 10.2%, and 214.2 ± 15.8% (n=8), respectively, of that of the controls. In contrast, microinjection of the N-methyl-D-aspartate (NMDA) receptor antagonist (+)-5-methyl-10, 11-dihydro-5H-dibenzo [a, d]-cyclohepten-5,10-imine maleate (MK-801) at 4 and 6 nmol, decreased the EMG response to 45.2 ± 10.6% and 36.8 ± 14.3%, respectively, of that of the controls (n=8 for each dose). Similarly, the metabotropic glutamate receptor (mGluR) antagonist (RS)-a-methyl-4-carboxyphenylglycine (MCPG), at 2.5 and 5 nmol, decreased the EMG response to 65.2 ± 16.3% and 57.0 ± 4.2%, respectively, of that of the controls. When a combination of MK-801 and MCPG was administrated, the EMG response further decreased to 22.5 ± 13.2% (n=6) of that of the controls. However, administration of a non-NMDA receptor antagonist 6, 7-dinitroquinoxaline-2, 3-dione (DNQX), at 2 and 5 nmol, had no effect on the EMG response. These results suggest that the NTS is involved in the facilitation of the cardiac-somatic reflex, and that the NMDA receptor and mGluRs play an important role in mediating this effect. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

  4. Optogenetic Manipulation of Cyclic Di-GMP (c-di-GMP) Levels Reveals the Role of c-di-GMP in Regulating Aerotaxis Receptor Activity in Azospirillum brasilense.

    Science.gov (United States)

    O'Neal, Lindsey; Ryu, Min-Hyung; Gomelsky, Mark; Alexandre, Gladys

    2017-09-15

    Bacterial chemotaxis receptors provide the sensory inputs that inform the direction of navigation in changing environments. Recently, we described the bacterial second messenger cyclic di-GMP (c-di-GMP) as a novel regulator of a subclass of chemotaxis receptors. In Azospirillum brasilense , c-di-GMP binds to a chemotaxis receptor, Tlp1, and modulates its signaling function during aerotaxis. Here, we further characterize the role of c-di-GMP in aerotaxis using a novel dichromatic optogenetic system engineered for manipulating intracellular c-di-GMP levels in real time. This system comprises a red/near-infrared-light-regulated diguanylate cyclase and a blue-light-regulated c-di-GMP phosphodiesterase. It allows the generation of transient changes in intracellular c-di-GMP concentrations within seconds of irradiation with appropriate light, which is compatible with the time scale of chemotaxis signaling. We provide experimental evidence that binding of c-di-GMP to the Tlp1 receptor activates its signaling function during aerotaxis, which supports the role of transient changes in c-di-GMP levels as a means of adjusting the response of A. brasilense to oxygen gradients. We also show that intracellular c-di-GMP levels in A. brasilense change with carbon metabolism. Our data support a model whereby c-di-GMP functions to imprint chemotaxis receptors with a record of recent metabolic experience, to adjust their contribution to the signaling output, thus allowing the cells to continually fine-tune chemotaxis sensory perception to their metabolic state. IMPORTANCE Motile bacteria use chemotaxis to change swimming direction in response to changes in environmental conditions. Chemotaxis receptors sense environmental signals and relay sensory information to the chemotaxis machinery, which ultimately controls the swimming pattern of cells. In bacteria studied to date, differential methylation has been known as a mechanism to control the activity of chemotaxis receptors and

  5. The extended arm of health professionals?

    DEFF Research Database (Denmark)

    Norlyk, Annelise; Martinsen, Bente

    2013-01-01

    responsibility for both the patient's well-being and for the patient's compliance with the daily regimen. Relatives were caught in a conflicting double role. They were the extended arm of the health professionals but also the caring, supporting partner. A tension arose between relatives' desire to help....... Due to the early discharge and the increasing demands on patients for self-care, patients' relatives seem to play a pivotal role in fast-track programmes. However, research is limited into how patients' close relatives are affected by and involved in the postoperative recovery process. Design...

  6. Irreducibility conditions for extended superfields

    International Nuclear Information System (INIS)

    Sokatchev, E.

    1981-05-01

    The irreducible supermultiplets contained in an extended superfield are presented as sets of covariant derivatives of the superfield. Differential irreducibility constraints are easily obtained from this decomposition. (author)

  7. Detoxification of ammonia in mouse cortical GABAergic cell cultures increases neuronal oxidative metabolism and reveals an emerging role for release of glucose-derived alanine.

    Science.gov (United States)

    Leke, Renata; Bak, Lasse K; Anker, Malene; Melø, Torun M; Sørensen, Michael; Keiding, Susanne; Vilstrup, Hendrik; Ott, Peter; Portela, Luis V; Sonnewald, Ursula; Schousboe, Arne; Waagepetersen, Helle S

    2011-04-01

    Cerebral hyperammonemia is believed to play a pivotal role in the development of hepatic encephalopathy (HE), a debilitating condition arising due to acute or chronic liver disease. In the brain, ammonia is thought to be detoxified via the activity of glutamine synthetase, an astrocytic enzyme. Moreover, it has been suggested that cerebral tricarboxylic acid (TCA) cycle metabolism is inhibited and glycolysis enhanced during hyperammonemia. The aim of this study was to characterize the ammonia-detoxifying mechanisms as well as the effects of ammonia on energy-generating metabolic pathways in a mouse neuronal-astrocytic co-culture model of the GABAergic system. We found that 5 mM ammonium chloride affected energy metabolism by increasing the neuronal TCA cycle activity and switching the astrocytic TCA cycle toward synthesis of substrate for glutamine synthesis. Furthermore, ammonia exposure enhanced the synthesis and release of alanine. Collectively, our results demonstrate that (1) formation of glutamine is seminal for detoxification of ammonia; (2) neuronal oxidative metabolism is increased in the presence of ammonia; and (3) synthesis and release of alanine is likely to be important for ammonia detoxification as a supplement to formation of glutamine.

  8. Role of DNA conformation & energetic insights in Msx-1-DNA recognition as revealed by molecular dynamics studies on specific and nonspecific complexes.

    Science.gov (United States)

    Kachhap, Sangita; Singh, Balvinder

    2015-01-01

    In most of homeodomain-DNA complexes, glutamine or lysine is present at 50th position and interacts with 5th and 6th nucleotide of core recognition region. Molecular dynamics simulations of Msx-1-DNA complex (Q50-TG) and its variant complexes, that is specific (Q50K-CC), nonspecific (Q50-CC) having mutation in DNA and (Q50K-TG) in protein, have been carried out. Analysis of protein-DNA interactions and structure of DNA in specific and nonspecific complexes show that amino acid residues use sequence-dependent shape of DNA to interact. The binding free energies of all four complexes were analysed to define role of amino acid residue at 50th position in terms of binding strength considering the variation in DNA on stability of protein-DNA complexes. The order of stability of protein-DNA complexes shows that specific complexes are more stable than nonspecific ones. Decomposition analysis shows that N-terminal amino acid residues have been found to contribute maximally in binding free energy of protein-DNA complexes. Among specific protein-DNA complexes, K50 contributes more as compared to Q50 towards binding free energy in respective complexes. The sequence dependence of local conformation of DNA enables Q50/Q50K to make hydrogen bond with nucleotide(s) of DNA. The changes in amino acid sequence of protein are accommodated and stabilized around TAAT core region of DNA having variation in nucleotides.

  9. Resveratrol treatment reveals a novel role for HMGB1 in regulation of the type 1 interferon response in dengue virus infection.

    Science.gov (United States)

    Zainal, Nurhafiza; Chang, Chih-Peng; Cheng, Yi-Lin; Wu, Yan-Wei; Anderson, Robert; Wan, Shu-Wen; Chen, Chia-Ling; Ho, Tzong-Shiann; AbuBakar, Sazaly; Lin, Yee-Shin

    2017-02-20

    Dengue is one of the most significant mosquito-borne virus diseases worldwide, particularly in tropical and subtropical regions. This study sought to examine the antiviral activity of resveratrol (RESV), a phytoalexin secreted naturally by plants, against dengue virus (DENV) infection. Our data showed that RESV inhibits the translocation of high mobility group box 1 (HMGB1), a DNA binding protein that normally resides in the nucleus, into the cytoplasm and extracellular milieu. HMGB1 migrates out of the nucleus during DENV infection. This migration is inhibited by RESV treatment and is mediated by induction of Sirt1 which leads to the retention of HMGB1 in the nucleus and consequently helps in the increased production of interferon-stimulated genes (ISGs). Nuclear HMGB1 was found to bind to the promoter region of the ISG and positively regulated the expression of ISG. The enhanced transcription of ISGs by nuclear HMGB1 thus contributes to the antiviral activity of RESV against DENV. To the best of our knowledge, this is the first report to demonstrate that RESV antagonizes DENV replication and that nuclear HMGB1 plays a role in regulating ISG production.

  10. Epigenomic analysis in a cell-based model reveals the roles of H3K9me3 in breast cancer transformation.

    Science.gov (United States)

    Li, Qing-Lan; Lei, Pin-Ji; Zhao, Quan-Yi; Li, Lianyun; Wei, Gang; Wu, Min

    2017-08-01

    Epigenetic marks are critical regulators of chromatin and gene activity. Their roles in normal physiology and disease states, including cancer development, still remain elusive. Herein, the epigenomic change of H3K9me3, as well as its potential impacts on gene activity and genome stability, was investigated in an in vitro breast cancer transformation model. The global H3K9me3 level was studied with western blotting. The distribution of H3K9me3 on chromatin and gene expression was studied with ChIP-Seq and RNA-Seq, respectively. The global H3K9me3 level decreases during transformation and its distribution on chromatin is reprogrammed. By combining with TCGA data, we identified 67 candidate oncogenes, among which five genes are totally novel. Our analysis further links H3K9me3 with transposon activity, and suggests H3K9me3 reduction increases the cell's sensitivity to DNA damage reagents. H3K9me3 reduction is possibly related with breast cancer transformation by regulating gene expression and chromatin stability during transformation.

  11. The Roles of Sphagnum and Cyperaceae in the Methane Cycle of an Ombrotrophic Bog Revealed by the Carbon Isotope Ratios of Leaf Waxes

    Science.gov (United States)

    Isles, P. D.; Nichols, J. E.; Peteet, D. M.; Kenna, T. C.

    2011-12-01

    Methane is a strong greenhouse gas, and the role of the terrestrial carbon cycle in the concentrations of atmospheric methane is poorly understood. What is clear, is that northern peatlands are a significant source of methane to the atmosphere. A recent discovery, and a topic of much scrutiny, has been the relationship between Sphagnum in peatlands and symbiotic methanotrophic bacteria. These bacteria oxidize methane produced at depth in peatlands before it is released to the atmosphere, contributing 13C-depleted CO2 to Sphagnum photosynthate. We seek to better understand the fate of methane produced in peatlands at depth, and the relationship between methane release from peatland surfaces and parameters such as temperature, moisture, and vegetation type. We compare carbon isotope ratios of leaf wax n-alkanes from sphagnum and vascular plants and major element chemistry at three different microhabitats, hummock, hollow, and sedge tussock, in Mer Bleue an ombrotrophic peatland near Ottowa, Ontario, Canada. We use these compound-specific carbon isotope measurements to constrain the amount of methane-derived CO2 incorporated by Sphagnum. We also compare our multiannually resolved down-core measurements to data from long-term monitoring of climate parameters and methane flux from the same microhabitats to ground-truth our sedimentary signature of methane with instrumental measurements.

  12. The Role of Water Distribution Controlled by Transmembrane Potentials in the Cytochrome c-Cardiolipin Interaction: Revealing from Surface-Enhanced Infrared Absorption Spectroscopy.

    Science.gov (United States)

    Zeng, Li; Wu, Lie; Liu, Li; Jiang, Xiue

    2017-11-02

    The interaction of cytochrome c (cyt c) with cardiolipin (CL) plays a crucial role in apoptotic functions, however, the changes of the transmembrane potential in governing the protein behavior at the membrane-water interface have not been studied due to the difficulties in simultaneously monitoring the interaction and regulating the electric field. Herein, surface-enhanced infrared absorption (SEIRA) spectroelectrochemistry is employed to study the mechanism of how the transmembrane potentials control the interaction of cyt c with CL membranes by regulating the electrode potentials of an Au film. When the transmembrane potential decreases, the water content at the interface of the membranes can be increased to slow down protein adsorption through decreasing the hydrogen-bond and hydrophobic interactions, but regulates the redox behavior of CL-bound cyt c through a possible water-facilitated proton-coupled electron transfer process. Our results suggest that the potential drop-induced restructure of the CL conformation and the hydration state could modify the structure and function of CL-bound cyt c on the lipid membrane. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. 2D IR spectroscopy reveals the role of water in the binding of channel-blocking drugs to the influenza M2 channel

    International Nuclear Information System (INIS)

    Ghosh, Ayanjeet; Gai, Feng; Hochstrasser, Robin M.; Wang, Jun; DeGrado, William F.; Moroz, Yurii S.; Korendovych, Ivan V.; Zanni, Martin

    2014-01-01

    Water is an integral part of the homotetrameric M2 proton channel of the influenza A virus, which not only assists proton conduction but could also play an important role in stabilizing channel-blocking drugs. Herein, we employ two dimensional infrared (2D IR) spectroscopy and site-specific IR probes, i.e., the amide I bands arising from isotopically labeled Ala30 and Gly34 residues, to probe how binding of either rimantadine or 7,7-spiran amine affects the water dynamics inside the M2 channel. Our results show, at neutral pH where the channel is non-conducting, that drug binding leads to a significant increase in the mobility of the channel water. A similar trend is also observed at pH 5.0 although the difference becomes smaller. Taken together, these results indicate that the channel water facilitates drug binding by increasing its entropy. Furthermore, the 2D IR spectral signatures obtained for both probes under different conditions collectively support a binding mechanism whereby amantadine-like drugs dock in the channel with their ammonium moiety pointing toward the histidine residues and interacting with a nearby water cluster, as predicted by molecular dynamics simulations. We believe these findings have important implications for designing new anti-influenza drugs

  14. Crucial role of dynamic linker histone binding and divalent ions for DNA accessibility and gene regulation revealed by mesoscale modeling of oligonucleosomes

    Science.gov (United States)

    Collepardo-Guevara, Rosana; Schlick, Tamar

    2012-01-01

    Monte Carlo simulations of a mesoscale model of oligonucleosomes are analyzed to examine the role of dynamic-linker histone (LH) binding/unbinding in high monovalent salt with divalent ions, and to further interpret noted chromatin fiber softening by dynamic LH in monovalent salt conditions. We find that divalent ions produce a fiber stiffening effect that competes with, but does not overshadow, the dramatic softening triggered by dynamic-LH behavior. Indeed, we find that in typical in vivo conditions, dynamic-LH binding/unbinding reduces fiber stiffening dramatically (by a factor of almost 5, as measured by the elasticity modulus) compared with rigidly fixed LH, and also the force needed to initiate chromatin unfolding, making it consistent with those of molecular motors. Our data also show that, during unfolding, divalent ions together with LHs induce linker-DNA bending and DNA–DNA repulsion screening, which guarantee formation of heteromorphic superbeads-on-a-string structures that combine regions of loose and compact fiber independently of the characteristics of the LH–core bond. These structures might be important for gene regulation as they expose regions of the DNA selectively. Dynamic control of LH binding/unbinding, either globally or locally, in the presence of divalent ions, might constitute a mechanism for regulation of gene expression. PMID:22790986

  15. Cross-species functional analyses reveal shared and separate roles for Sox11 in frog primary neurogenesis and mouse cortical neuronal differentiation

    Directory of Open Access Journals (Sweden)

    Chao Chen

    2016-04-01

    Full Text Available A well-functioning brain requires production of the correct number and types of cells during development; cascades of transcription factors are essential for cellular coordination. Sox proteins are transcription factors that affect various processes in the development of the nervous system. Sox11, a member of the SoxC family, is expressed in differentiated neurons and supports neuronal differentiation in several systems. To understand how generalizable the actions of Sox11 are across phylogeny, its function in the development of the frog nervous system and the mouse cerebral cortex were compared. Expression of Sox11 is largely conserved between these species; in the developing frog, Sox11 is expressed in the neural plate, neural tube and throughout the segmented brain, while in the mouse cerebral cortex, Sox11 is expressed in differentiated zones, including the preplate, subplate, marginal zone and cortical plate. In both frog and mouse, data demonstrate that Sox11 supports a role in promoting neuronal differentiation, with Sox11-positive cells expressing pan-neural markers and becoming morphologically complex. However, frog and mouse Sox11 cannot substitute for one another; a functional difference likely reflected in sequence divergence. Thus, Sox11 appears to act similarly in subserving neuronal differentiation but is species-specific in frog neural development and mouse corticogenesis.

  16. Structure–function analysis of mouse Sry reveals dual essential roles of the C-terminal polyglutamine tract in sex determination

    Science.gov (United States)

    Zhao, Liang; Ng, Ee Ting; Davidson, Tara-Lynne; Longmuss, Enya; Urschitz, Johann; Elston, Marlee; Moisyadi, Stefan; Bowles, Josephine; Koopman, Peter

    2014-01-01

    The mammalian sex-determining factor SRY comprises a conserved high-mobility group (HMG) box DNA-binding domain and poorly conserved regions outside the HMG box. Mouse Sry is unusual in that it includes a C-terminal polyglutamine (polyQ) tract that is absent in nonrodent SRY proteins, and yet, paradoxically, is essential for male sex determination. To dissect the molecular functions of this domain, we generated a series of Sry mutants, and studied their biochemical properties in cell lines and transgenic mouse embryos. Sry protein lacking the polyQ domain was unstable, due to proteasomal degradation. Replacing this domain with irrelevant sequences stabilized the protein but failed to restore Sry’s ability to up-regulate its key target gene SRY-box 9 (Sox9) and its sex-determining function in vivo. These functions were restored only when a VP16 transactivation domain was substituted. We conclude that the polyQ domain has important roles in protein stabilization and transcriptional activation, both of which are essential for male sex determination in mice. Our data disprove the hypothesis that the conserved HMG box domain is the only functional domain of Sry, and highlight an evolutionary paradox whereby mouse Sry has evolved a novel bifunctional module to activate Sox9 directly, whereas SRY proteins in other taxa, including humans, seem to lack this ability, presumably making them dependent on partner proteins(s) to provide this function. PMID:25074915

  17. Structure-function analysis of mouse Sry reveals dual essential roles of the C-terminal polyglutamine tract in sex determination.

    Science.gov (United States)

    Zhao, Liang; Ng, Ee Ting; Davidson, Tara-Lynne; Longmuss, Enya; Urschitz, Johann; Elston, Marlee; Moisyadi, Stefan; Bowles, Josephine; Koopman, Peter

    2014-08-12

    The mammalian sex-determining factor SRY comprises a conserved high-mobility group (HMG) box DNA-binding domain and poorly conserved regions outside the HMG box. Mouse Sry is unusual in that it includes a C-terminal polyglutamine (polyQ) tract that is absent in nonrodent SRY proteins, and yet, paradoxically, is essential for male sex determination. To dissect the molecular functions of this domain, we generated a series of Sry mutants, and studied their biochemical properties in cell lines and transgenic mouse embryos. Sry protein lacking the polyQ domain was unstable, due to proteasomal degradation. Replacing this domain with irrelevant sequences stabilized the protein but failed to restore Sry's ability to up-regulate its key target gene SRY-box 9 (Sox9) and its sex-determining function in vivo. These functions were restored only when a VP16 transactivation domain was substituted. We conclude that the polyQ domain has important roles in protein stabilization and transcriptional activation, both of which are essential for male sex determination in mice. Our data disprove the hypothesis that the conserved HMG box domain is the only functional domain of Sry, and highlight an evolutionary paradox whereby mouse Sry has evolved a novel bifunctional module to activate Sox9 directly, whereas SRY proteins in other taxa, including humans, seem to lack this ability, presumably making them dependent on partner proteins(s) to provide this function.

  18. Metabolic analysis of the increased adventitious rooting mutant of Artemisia annua reveals a role for the plant monoterpene borneol in adventitious root formation.

    Science.gov (United States)

    Tian, Na; Liu, Shuoqian; Li, Juan; Xu, Wenwen; Yuan, Lin; Huang, Jianan; Liu, Zhonghua

    2014-08-01

    Adventitious root (AR) formation is a critical process for plant clonal propagation. The role of plant secondary metabolites in AR formation is still poorly understood. Chemical and physical mutagenesis in combination with somatic variation were performed on Artemisia annua in order to obtain a mutant with changes in adventitious rooting and composition of plant secondary metabolites. Metabolic and morphological analyses of the iar (increased adventitious rooting) mutant coupled with in vitro assays were used to elucidate the relationship between plant secondary metabolites and AR formation. The only detected differences between the iar mutant and wild-type were rooting capacity and borneol/camphor content. Consistent with this, treatment with borneol in vitro promoted adventitious rooting in wild-type. The enhanced rooting did not continue upon removal of borneol. The iar mutant displayed no significant differences in AR formation upon treatment with camphor. Together, our results suggest that borneol promotes adventitious rooting whereas camphor has no effect on AR formation. © 2013 Scandinavian Plant Physiology Society.

  19. Role of alpha2C-adrenoceptor subtype in spatial working memory as revealed by mice with targeted disruption of the alpha2C-adrenoceptor gene.

    Science.gov (United States)

    Tanila, H; Mustonen, K; Sallinen, J; Scheinin, M; Riekkinen, P

    1999-02-01

    The role of the alpha2C-adrenoceptor subtype in mediating the beneficial effect of alpha2-adrenoceptor agonists on spatial working memory was studied in adult mice with targeted inactivation of the alpha2C-receptor gene (KO) and their wild-type controls (WT). A delayed alternation task was run in a T-maze with mixed delays varying from 20 s to 120 s. Dexmedetomidine, a specific but subtype nonselective alpha2-adrenoceptor agonist, dose-dependently decreased the total number of errors. The effect was strongest at the dose of 5 microg/kg (s.c.), and was observed similarly in KO and WT mice. KO mice performed inferior to WT mice due to a higher number of perseverative errors. Dexmedetomidine slowed initiation of the motor response in the start phase at lower doses in WT mice than in KO mice but no such difference was observed in the return phase of the task, suggesting involvement of alpha2C-adrenoceptors in the cognitive aspect of response preparation or in response sequence initiation. According to these findings, enhancement of spatial working memory is best achieved with alpha2-adrenoceptor agonists which have neither agonistic nor antagonistic effects at the alpha2C-adrenoceptor subtype.

  20. Intermittently-visual Tracking Experiments Reveal the Roles of Error-correction and Predictive Mechanisms in the Human Visual-motor Control System

    Science.gov (United States)

    Hayashi, Yoshikatsu; Tamura, Yurie; Sase, Kazuya; Sugawara, Ken; Sawada, Yasuji

    Prediction mechanism is necessary for human visual motion to compensate a delay of sensory-motor system. In a previous study, “proactive control” was discussed as one example of predictive function of human beings, in which motion of hands preceded the virtual moving target in visual tracking experiments. To study the roles of the positional-error correction mechanism and the prediction mechanism, we carried out an intermittently-visual tracking experiment where a circular orbit is segmented into the target-visible regions and the target-invisible regions. Main results found in this research were following. A rhythmic component appeared in the tracer velocity when the target velocity was relatively high. The period of the rhythm in the brain obtained from environmental stimuli is shortened more than 10%. The shortening of the period of rhythm in the brain accelerates the hand motion as soon as the visual information is cut-off, and causes the precedence of hand motion to the target motion. Although the precedence of the hand in the blind region is reset by the environmental information when the target enters the visible region, the hand motion precedes the target in average when the predictive mechanism dominates the error-corrective mechanism.

  1. Acute Smc5/6 depletion reveals its primary role in rDNA replication by restraining recombination at fork pausing sites.

    Directory of Open Access Journals (Sweden)

    Xiao P Peng

    2018-01-01

    Full Text Available Smc5/6, a member of the conserved SMC family of complexes, is essential for growth in most organisms. Its exact functions in a mitotic cell cycle are controversial, as chronic Smc5/6 loss-of-function alleles produce varying phenotypes. To circumvent this issue, we acutely depleted Smc5/6 in budding yeast and determined the first cell cycle consequences of Smc5/6 removal. We found a striking primary defect in replication of the ribosomal DNA (rDNA array. Each rDNA repeat contains a programmed replication fork barrier (RFB established by the Fob1 protein. Fob1 removal improves rDNA replication in Smc5/6 depleted cells, implicating Smc5/6 in the management of programmed fork pausing. A similar improvement is achieved by removing the DNA helicase Mph1 whose recombinogenic activity can be inhibited by Smc5/6 under DNA damage conditions. DNA 2D gel analyses further show that Smc5/6 loss increases recombination structures at RFB regions; moreover, mph1∆ and fob1∆ similarly reduce this accumulation. These findings point to an important mitotic role for Smc5/6 in restraining recombination events when protein barriers in rDNA stall replication forks. As rDNA maintenance influences multiple essential cellular processes, Smc5/6 likely links rDNA stability to overall mitotic growth.

  2. 2D IR spectroscopy reveals the role of water in the binding of channel-blocking drugs to the influenza M2 channel

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, Ayanjeet, E-mail: ayanjeet@sas.upenn.edu, E-mail: gai@sas.upenn.edu; Gai, Feng, E-mail: ayanjeet@sas.upenn.edu, E-mail: gai@sas.upenn.edu; Hochstrasser, Robin M. [Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104 (United States); Wang, Jun; DeGrado, William F. [Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California 94143 (United States); Moroz, Yurii S.; Korendovych, Ivan V. [Department of Chemistry, Syracuse University, Syracuse, New York 13244 (United States); Zanni, Martin [Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706 (United States)

    2014-06-21

    Water is an integral part of the homotetrameric M2 proton channel of the influenza A virus, which not only assists proton conduction but could also play an important role in stabilizing channel-blocking drugs. Herein, we employ two dimensional infrared (2D IR) spectroscopy and site-specific IR probes, i.e., the amide I bands arising from isotopically labeled Ala30 and Gly34 residues, to probe how binding of either rimantadine or 7,7-spiran amine affects the water dynamics inside the M2 channel. Our results show, at neutral pH where the channel is non-conducting, that drug binding leads to a significant increase in the mobility of the channel water. A similar trend is also observed at pH 5.0 although the difference becomes smaller. Taken together, these results indicate that the channel water facilitates drug binding by increasing its entropy. Furthermore, the 2D IR spectral signatures obtained for both probes under different conditions collectively support a binding mechanism whereby amantadine-like drugs dock in the channel with their ammonium moiety pointing toward the histidine residues and interacting with a nearby water cluster, as predicted by molecular dynamics simulations. We believe these findings have important implications for designing new anti-influenza drugs.

  3. Quantitative Phospho-proteomic Analysis of TNFα/NFκB Signaling Reveals a Role for RIPK1 Phosphorylation in Suppressing Necrotic Cell Death.

    Science.gov (United States)

    Mohideen, Firaz; Paulo, Joao A; Ordureau, Alban; Gygi, Steve P; Harper, J Wade

    2017-07-01

    TNFα is a potent inducer of inflammation due to its ability to promote gene expression, in part via the NFκB pathway. Moreover, in some contexts, TNFα promotes Caspase-dependent apoptosis or RIPK1/RIPK3/MLKL-dependent necrosis. Engagement of the TNF Receptor Signaling Complex (TNF-RSC), which contains multiple kinase activities, promotes phosphorylation of several downstream components, including TAK1, IKKα/IKKβ, IκBα, and NFκB. However, immediate downstream phosphorylation events occurring in response to TNFα signaling are poorly understood at a proteome-wide level. Here we use Tandem Mass Tagging-based proteomics to quantitatively characterize acute TNFα-mediated alterations in the proteome and phosphoproteome with or without inhibition of the cIAP-dependent survival arm of the pathway with a SMAC mimetic. We identify and quantify over 8,000 phosphorylated peptides, among which are numerous known sites in the TNF-RSC, NFκB, and MAP kinase signaling systems, as well as numerous previously unrecognized phosphorylation events. Functional analysis of S320 phosphorylation in RIPK1 demonstrates a role for this event in suppressing its kinase activity, association with CASPASE-8 and FADD proteins, and subsequent necrotic cell death during inflammatory TNFα stimulation. This study provides a resource for further elucidation of TNFα-dependent signaling pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. A Key Role of Xanthophylls That Are Not Embedded in Proteins in Regulation of the Photosynthetic Antenna Function in Plants, Revealed by Monomolecular Layer Studies.

    Science.gov (United States)

    Welc, Renata; Luchowski, Rafal; Grudzinski, Wojciech; Puzio, Michal; Sowinski, Karol; Gruszecki, Wieslaw I

    2016-12-29

    The main physiological function of LHCII (light-harvesting pigment-protein complex of photosystem II), the largest photosynthetic antenna complex of plants, is absorption of light quanta and transfer of excitation energy toward the reaction centers, to drive photosynthesis. However, under strong illumination, the photosynthetic apparatus faces the danger of photodegradation and therefore excitations in LHCII have to be down-regulated, e.g., via thermal energy dissipation. One of the elements of the regulatory system, operating in the photosynthetic apparatus under light stress conditions, is a conversion of violaxanthin, the xanthophyll present under low light, to zeaxanthin, accumulated under strong light. In the present study, an effect of violaxanthin and zeaxanthin on the molecular organization and the photophysical properties of LHCII was studied in a monomolecular layer system with application of molecular imaging (atomic force microscopy, fluorescence lifetime imaging microscopy) and spectroscopy (UV-Vis absorption, FTIR, fluorescence spectroscopy) techniques. The results of the experiments show that violaxanthin promotes the formation of supramolecular LHCII structures preventing dissipative excitation quenching while zeaxanthin is involved in the formation of excitonic energy states able to quench chlorophyll excitations in both the higher (B states) and lower (Q states) energy levels. The results point to a strategic role of xanthophylls that are not embedded in a protein environment, in regulation of the photosynthetic light harvesting activity in plants.

  5. A comparative study of ethylene growth response kinetics in eudicots and monocots reveals a role for gibberellin in growth inhibition and recovery.

    Science.gov (United States)

    Kim, Joonyup; Wilson, Rebecca L; Case, J Brett; Binder, Brad M

    2012-11-01

    Time-lapse imaging of dark-grown Arabidopsis (Arabidopsis thaliana) hypocotyls has revealed new aspects about ethylene signaling. This study expands upon these results by examining ethylene growth response kinetics of seedlings of several plant species. Although the response kinetics varied between the eudicots studied, all had prolonged growth inhibition for as long as ethylene was present. In contrast, with continued application of ethylene, white millet (Panicum miliaceum) seedlings had a rapid and transient growth inhibition response, rice (Oryza sativa 'Nipponbare') seedlings had a slow onset of growth stimulation, and barley (Hordeum vulgare) had a transient growth inhibition response followed, after a delay, by a prolonged inhibition response. Growth stimulation in rice correlated with a decrease in the levels of rice ETHYLENE INSENSTIVE3-LIKE2 (OsEIL2) and an increase in rice F-BOX DOMAIN AND LRR CONTAINING PROTEIN7 transcripts. The gibberellin (GA) biosynthesis inhibitor paclobutrazol caused millet seedlings to have a prolonged growth inhibition response when ethylene was applied. A transient ethylene growth inhibition response has previously been reported for Arabidopsis ethylene insensitive3-1 (ein3-1) eil1-1 double mutants. Paclobutrazol caused these mutants to have a prolonged response to ethylene, whereas constitutive GA signaling in this background eliminated ethylene responses. Sensitivity to paclobutrazol inversely correlated with the levels of EIN3 in Arabidopsis. Wild-type Arabidopsis seedlings treated with paclobutrazol and mutants deficient in GA levels or signaling had a delayed growth recovery after ethylene removal. It is interesting to note that ethylene caused alterations in gene expression that are predicted to increase GA levels in the ein3-1 eil1-1 seedlings. These results indicate that ethylene affects GA levels leading to modulation of ethylene growth inhibition kinetics.

  6. Crystal Structure of Acivicin-Inhibited [gamma]-Glutamyltranspeptidase Reveals Critical Roles for Its C-Terminus in Autoprocessing and Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Williams, Kristin; Cullati, Sierra; Sand, Aaron; Biterova, Ekaterina I.; Barycki, Joseph J.; (UNL)

    2009-03-27

    Helicobacter pylori {gamma}-glutamyltranspeptidase (HpGT) is a general {gamma}-glutamyl hydrolase and a demonstrated virulence factor. The enzyme confers a growth advantage to the bacterium, providing essential amino acid precursors by initiating the degradation of extracellular glutathione and glutamine. HpGT is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily and undergoes autoprocessing to generate the active form of the enzyme. Acivicin is a widely used {gamma}-glutamyltranspeptidase inhibitor that covalently modifies the enzyme, but its precise mechanism of action remains unclear. The time-dependent inactivation of HpGT exhibits a hyperbolic dependence on acivicin concentration with k{sub max} = 0.033 {+-} 0.006 s{sup -1} and K{sub I} = 19.7 {+-} 7.2 {micro}M. Structure determination of acivicin-modified HpGT (1.7 {angstrom}; R{sub factor} = 17.9%; R{sub free} = 20.8%) demonstrates that acivicin is accommodated within the {gamma}-glutamyl binding pocket of the enzyme. The hydroxyl group of Thr 380, the catalytic nucleophile in the autoprocessing and enzymatic reactions, displaces chloride from the acivicin ring to form the covalently linked complex. Within the acivicin-modified HpGT structure, the C-terminus of the protein becomes ordered with Phe 567 positioned over the active site. Substitution or deletion of Phe 567 leads to a >10-fold reduction in enzymatic activity, underscoring its importance in catalysis. The mobile C-terminus is positioned by several electrostatic interactions within the C-terminal region, most notably a salt bridge between Arg 475 and Glu 566. Mutational analysis reveals that Arg 475 is critical for the proper placement of the C-terminal region, the Tyr 433 containing loop, and the proposed oxyanion hole.

  7. Comparative Phosphoproteomics Reveals the Role of AmpC β-lactamase Phosphorylation in the Clinical Imipenem-resistant Strain Acinetobacter baumannii SK17.

    Science.gov (United States)

    Lai, Juo-Hsin; Yang, Jhih-Tian; Chern, Jeffy; Chen, Te-Li; Wu, Wan-Ling; Liao, Jiahn-Haur; Tsai, Shih-Feng; Liang, Suh-Yuen; Chou, Chi-Chi; Wu, Shih-Hsiung

    2016-01-01

    Nosocomial infectious outbreaks caused by multidrug-resistant Acinetobacter baumannii have emerged as a serious threat to human health. Phosphoproteomics of pathogenic bacteria has been used to identify the mechanisms of bacterial virulence and antimicrobial resistance. In this study, we used a shotgun strategy combined with high-accuracy mass spectrometry to analyze the phosphoproteomics of the imipenem-susceptible strain SK17-S and -resistant strain SK17-R. We identified 410 phosphosites on 248 unique phosphoproteins in SK17-S and 285 phosphosites on 211 unique phosphoproteins in SK17-R. The distributions of the Ser/Thr/Tyr/Asp/His phosphosites in SK17-S and SK17-R were 47.0%/27.6%/12.4%/8.0%/4.9% versus 41.4%/29.5%/17.5%/6.7%/4.9%, respectively. The Ser-90 phosphosite, located on the catalytic motif S(88)VS(90)K of the AmpC β-lactamase, was first identified in SK17-S. Based on site-directed mutagenesis, the nonphosphorylatable mutant S90A was found to be more resistant to imipenem, whereas the phosphorylation-simulated mutant S90D was sensitive to imipenem. Additionally, the S90A mutant protein exhibited higher β-lactamase activity and conferred greater bacterial protection against imipenem in SK17-S compared with the wild-type. In sum, our results revealed that in A. baumannii, Ser-90 phosphorylation of AmpC negatively regulates both β-lactamase activity and the ability to counteract the antibiotic effects of imipenem. These findings highlight the impact of phosphorylation-mediated regulation in antibiotic-resistant bacteria on future drug design and new therapies. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Comparative Phosphoproteomics Reveals the Role of AmpC β-lactamase Phosphorylation in the Clinical Imipenem-resistant Strain Acinetobacter baumannii SK17*

    Science.gov (United States)

    Lai, Juo-Hsin; Yang, Jhih-Tian; Chern, Jeffy; Chen, Te-Li; Wu, Wan-Ling; Liao, Jiahn-Haur; Tsai, Shih-Feng; Liang, Suh-Yuen; Chou, Chi-Chi

    2016-01-01

    Nosocomial infectious outbreaks caused by multidrug-resistant Acinetobacter baumannii have emerged as a serious threat to human health. Phosphoproteomics of pathogenic bacteria has been used to identify the mechanisms of bacterial virulence and antimicrobial resistance. In this study, we used a shotgun strategy combined with high-accuracy mass spectrometry to analyze the phosphoproteomics of the imipenem-susceptible strain SK17-S and -resistant strain SK17-R. We identified 410 phosphosites on 248 unique phosphoproteins in SK17-S and 285 phosphosites on 211 unique phosphoproteins in SK17-R. The distributions of the Ser/Thr/Tyr/Asp/His phosphosites in SK17-S and SK17-R were 47.0%/27.6%/12.4%/8.0%/4.9% versus 41.4%/29.5%/17.5%/6.7%/4.9%, respectively. The Ser-90 phosphosite, located on the catalytic motif S88VS90K of the AmpC β-lactamase, was first identified in SK17-S. Based on site-directed mutagenesis, the nonphosphorylatable mutant S90A was found to be more resistant to imipenem, whereas the phosphorylation-simulated mutant S90D was sensitive to imipenem. Additionally, the S90A mutant protein exhibited higher β-lactamase activity and conferred greater bacterial protection against imipenem in SK17-S compared with the wild-type. In sum, our results revealed that in A. baumannii, Ser-90 phosphorylation of AmpC negatively regulates both β-lactamase activity and the ability to counteract the antibiotic effects of imipenem. These findings highlight the impact of phosphorylation-mediated regulation in antibiotic-resistant bacteria on future drug design and new therapies. PMID:26499836

  9. A Comparative Study of Ethylene Growth Response Kinetics in Eudicots and Monocots Reveals a Role for Gibberellin in Growth Inhibition and Recovery1[W][OA

    Science.gov (United States)

    Kim, Joonyup; Wilson, Rebecca L.; Case, J. Brett; Binder, Brad M.

    2012-01-01

    Time-lapse imaging of dark-grown Arabidopsis (Arabidopsis thaliana) hypocotyls has revealed new aspects about ethylene signaling. This study expands upon these results by examining ethylene growth response kinetics of seedlings of several plant species. Although the response kinetics varied between the eudicots studied, all had prolonged growth inhibition for as long as ethylene was present. In contrast, with continued application of ethylene, white millet (Panicum miliaceum) seedlings had a rapid and transient growth inhibition response, rice (Oryza sativa ‘Nipponbare’) seedlings had a slow onset of growth stimulation, and barley (Hordeum vulgare) had a transient growth inhibition response followed, after a delay, by a prolonged inhibition response. Growth stimulation in rice correlated with a decrease in the levels of rice ETHYLENE INSENSTIVE3-LIKE2 (OsEIL2) and an increase in rice F-BOX DOMAIN AND LRR CONTAINING PROTEIN7 transcripts. The gibberellin (GA) biosynthesis inhibitor paclobutrazol caused millet seedlings to have a prolonged growth inhibition response when ethylene was applied. A transient ethylene growth inhibition response has previously been reported for Arabidopsis ethylene insensitive3-1 (ein3-1) eil1-1 double mutants. Paclobutrazol caused these mutants to have a prolonged response to ethylene, whereas constitutive GA signaling in this background eliminated ethylene responses. Sensitivity to paclobutrazol inversely correlated with the levels of EIN3 in Arabidopsis. Wild-type Arabidopsis seedlings treated with paclobutrazol and mutants deficient in GA levels or signaling had a delayed growth recovery after ethylene removal. It is interesting to note that ethylene caused alterations in gene expression that are predicted to increase GA levels in the ein3-1 eil1-1 seedlings. These results indicate that ethylene affects GA levels leading to modulation of ethylene growth inhibition kinetics. PMID:22977279

  10. Integrative Approach with Electrophysiological and Theoretical Methods Reveals a New Role of S4 Positively Charged Residues in PKD2L1 Channel Voltage-Sensing.

    Science.gov (United States)

    Numata, Tomohiro; Tsumoto, Kunichika; Yamada, Kazunori; Kurokawa, Tatsuki; Hirose, Shinichi; Nomura, Hideki; Kawano, Mitsuhiro; Kurachi, Yoshihisa; Inoue, Ryuji; Mori, Yasuo

    2017-08-29

    Numerical model-based simulations provide important insights into ion channel gating when experimental limitations exist. Here, a novel strategy combining numerical simulations with patch clamp experiments was used to investigate the net positive charges in the putative transmembrane segment 4 (S4) of the atypical, positively-shifted voltage-dependence of polycystic kidney disease 2-like 1 (PKD2L1) channel. Charge-neutralising mutations (K452Q, K455Q and K461Q) in S4 reduced gating charges, positively shifted the Boltzmann-type activation curve [i.e., open probability (P open )-V curve] and altered the time-courses of activation/deactivation of PKD2L1, indicating that this region constitutes part of a voltage sensor. Numerical reconstruction of wild-type (WT) and mutant PKD2L1-mediated currents necessitated, besides their voltage-dependent gating parameters, a scaling factor that describes the voltage-dependence of maximal conductance, G max . Subsequent single-channel conductance (γ) measurements revealed that voltage-dependence of G max in WT can be explained by the inward-rectifying property of γ, which is greatly changed in PKD2L1 mutants. Homology modelling based on PKD2 and Na V Ab structures suggest that such voltage dependence of P open and γ in PKD2L1 could both reflect the charged state of the S4 domain. The present conjunctive experimental and theoretical approaches provide a framework to explore the undetermined mechanism(s) regulating TRP channels that possess non-classical voltage-dependent properties.

  11. A novel mouse model of Warburg Micro syndrome reveals roles for RAB18 in eye development and organisation of the neuronal cytoskeleton

    Directory of Open Access Journals (Sweden)

    Sarah M. Carpanini

    2014-06-01

    Full Text Available Mutations in RAB18 have been shown to cause the heterogeneous autosomal recessive disorder Warburg Micro syndrome (WARBM. Individuals with WARBM present with a range of clinical symptoms, including ocular and neurological abnormalities. However, the underlying cellular and molecular pathogenesis of the disorder remains unclear, largely owing to the lack of any robust animal models that phenocopy both the ocular and neurological features of the disease. We report here the generation and characterisation of a novel Rab18-mutant mouse model of WARBM. Rab18-mutant mice are viable and fertile. They present with congenital nuclear cataracts and atonic pupils, recapitulating the characteristic ocular features that are associated with WARBM. Additionally, Rab18-mutant cells exhibit an increase in lipid droplet size following treatment with oleic acid. Lipid droplet abnormalities are a characteristic feature of cells taken from WARBM individuals, as well as cells taken from individuals with other neurodegenerative conditions. Neurological dysfunction is also apparent in Rab18-mutant mice, including progressive weakness of the hind limbs. We show that the neurological defects are, most likely, not caused by gross perturbations in synaptic vesicle recycling in the central or peripheral nervous system. Rather, loss of Rab18 is associated with widespread disruption of the neuronal cytoskeleton, including abnormal accumulations of neurofilament and microtubule proteins in synaptic terminals, and gross disorganisation of the cytoskeleton in peripheral nerves. Global proteomic profiling of peripheral nerves in Rab18-mutant mice reveals significant alterations in several core molecular pathways that regulate cytoskeletal dynamics in neurons. The apparent similarities between the WARBM phenotype and the phenotype that we describe here indicate that the Rab18-mutant mouse provides an important platform for investigation of the disease pathogenesis and therapeutic

  12. A novel mouse model of Warburg Micro syndrome reveals roles for RAB18 in eye development and organisation of the neuronal cytoskeleton.

    Science.gov (United States)

    Carpanini, Sarah M; McKie, Lisa; Thomson, Derek; Wright, Ann K; Gordon, Sarah L; Roche, Sarah L; Handley, Mark T; Morrison, Harris; Brownstein, David; Wishart, Thomas M; Cousin, Michael A; Gillingwater, Thomas H; Aligianis, Irene A; Jackson, Ian J

    2014-06-01

    Mutations in RAB18 have been shown to cause the heterogeneous autosomal recessive disorder Warburg Micro syndrome (WARBM). Individuals with WARBM present with a range of clinical symptoms, including ocular and neurological abnormalities. However, the underlying cellular and molecular pathogenesis of the disorder remains unclear, largely owing to the lack of any robust animal models that phenocopy both the ocular and neurological features of the disease. We report here the generation and characterisation of a novel Rab18-mutant mouse model of WARBM. Rab18-mutant mice are viable and fertile. They present with congenital nuclear cataracts and atonic pupils, recapitulating the characteristic ocular features that are associated with WARBM. Additionally, Rab18-mutant cells exhibit an increase in lipid droplet size following treatment with oleic acid. Lipid droplet abnormalities are a characteristic feature of cells taken from WARBM individuals, as well as cells taken from individuals with other neurodegenerative conditions. Neurological dysfunction is also apparent in Rab18-mutant mice, including progressive weakness of the hind limbs. We show that the neurological defects are, most likely, not caused by gross perturbations in synaptic vesicle recycling in the central or peripheral nervous system. Rather, loss of Rab18 is associated with widespread disruption of the neuronal cytoskeleton, including abnormal accumulations of neurofilament and microtubule proteins in synaptic terminals, and gross disorganisation of the cytoskeleton in peripheral nerves. Global proteomic profiling of peripheral nerves in Rab18-mutant mice reveals significant alterations in several core molecular pathways that regulate cytoskeletal dynamics in neurons. The apparent similarities between the WARBM phenotype and the phenotype that we describe here indicate that the Rab18-mutant mouse provides an important platform for investigation of the disease pathogenesis and therapeutic interventions.

  13. H/D exchange mass spectrometry and statistical coupling analysis reveal a role for allostery in a ferredoxin-dependent bifurcating transhydrogenase catalytic cycle.

    Science.gov (United States)

    Berry, Luke; Poudel, Saroj; Tokmina-Lukaszewska, Monika; Colman, Daniel R; Nguyen, Diep M N; Schut, Gerrit J; Adams, Michael W W; Peters, John W; Boyd, Eric S; Bothner, Brian

    2018-01-01

    Recent investigations into ferredoxin-dependent transhydrogenases, a class of enzymes responsible for electron transport, have highlighted the biological importance of flavin-based electron bifurcation (FBEB). FBEB generates biomolecules with very low reduction potential by coupling the oxidation of an electron donor with intermediate potential to the reduction of high and low potential molecules. Bifurcating systems can generate biomolecules with very low reduction potentials, such as reduced ferredoxin (Fd), from species such as NADPH. Metabolic systems that use bifurcation are more efficient and confer a competitive advantage for the organisms that harbor them. Structural models are now available for two NADH-dependent ferredoxin-NADP + oxidoreductase (Nfn) complexes. These models, together with spectroscopic studies, have provided considerable insight into the catalytic process of FBEB. However, much about the mechanism and regulation of these multi-subunit proteins remains unclear. Using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and statistical coupling analysis (SCA), we identified specific pathways of communication within the model FBEB system, Nfn from Pyrococus furiosus, under conditions at each step of the catalytic cycle. HDX-MS revealed evidence for allosteric coupling across protein subunits upon nucleotide and ferredoxin binding. SCA uncovered a network of co-evolving residues that can provide connectivity across the complex. Together, the HDX-MS and SCA data show that protein allostery occurs across the ensemble of iron‑sulfur cofactors and ligand binding sites using specific pathways that connect domains allowing them to function as dynamically coordinated units. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Android Emotions Revealed

    DEFF Research Database (Denmark)

    Vlachos, Evgenios; Schärfe, Henrik

    2012-01-01

    This work presents a method for designing facial interfaces for sociable android robots with respect to the fundamental rules of human affect expression. Extending the work of Paul Ekman towards a robotic direction, we follow the judgment-based approach for evaluating facial expressions to test...... findings are based on the results derived from a number of judgments, and suggest that before programming the facial expressions of a Geminoid, the Original should pass through the proposed procedure. According to our recommendations, the facial expressions of an android should be tested by judges, even...... in which case an android robot like the Geminoid|DK –a duplicate of an Original person- reveals emotions convincingly; when following an empirical perspective, or when following a theoretical one. The methodology includes the processes of acquiring the empirical data, and gathering feedback on them. Our...

  15. Functional analysis of the Glucan Degradation Locus (GDL) in Caldicellulosiruptor bescii reveals essential roles of component glycoside hydrolases in plant biomass deconstruction.

    Science.gov (United States)

    Conway, Jonathan M; McKinley, Bennett S; Seals, Nathaniel L; Hernandez, Diana; Khatibi, Piyum A; Poudel, Suresh; Giannone, Richard J; Hettich, Robert L; Williams-Rhaesa, Amanda M; Lipscomb, Gina L; Adams, Michael W W; Kelly, Robert M

    2017-10-06

    The ability to hydrolyze microcrystalline cellulose is an uncommon feature in the microbial world, but one that can be exploited for conversion of lignocellulosic feedstocks into bio-based fuels and chemicals. Understanding the physiological and biochemical mechanisms by which microorganisms deconstruct cellulosic material is key to achieving this objective. The Glucan Degradation Locus (GDL) in the genomes of extremely thermophilic Caldicellulosiruptor species encodes polysaccharide lyases (PLs), unique cellulose binding proteins (tāpirins), and putative post-translational modifying enzymes, in addition to multi-domain, multi-functional glycoside hydrolases (GHs), thereby representing an alternative paradigm for plant biomass degradation, as compared to fungal or cellulosomal systems. To examine the individual and collective in vivo roles of the glycolytic enzymes, the six GHs in the GDL of Caldicellulosiruptor bescii were systematically deleted, and the extent to which the resulting mutant strains could solubilize microcrystalline cellulose (Avicel) and plant biomasses (switchgrass or poplar) was examined. Three of the GDL enzymes, Athe_1867 (CelA) (GH9-CBM3-CBM3-CBM3-GH48), Athe_1859 (GH5-CBM3-CBM3-GH44), and Athe_1857 (GH10-CBM3-CBM3-GH48), acted synergistically in vivo and accounted for 92% of naked microcellulose (Avicel) degradation. However, the relative importance of the GDL GHs varied for the plant biomass substrates tested. Furthermore, mixed cultures of mutant strains showed switchgrass solubilization depended on the secretome-bound enzymes collectively produced by the culture and not on the specific strain from which they came. These results demonstrate that certain GDL GHs are primarily responsible for the degradation of microcrystalline-containing substrates by C. bescii and provide new insights into the workings of a novel microbial mechanism for lignocellulose utilization. Importance The efficient and extensive degradation of complex

  16. The potential role of microbiota for controlling the spread of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE in neonatal population [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Thibaud Delerue

    2017-07-01

    Full Text Available The spread of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE in the hospital and also the community is worrisome. Neonates particularly are exposed to the risk of ESBL-PE acquisition and, owing to the immaturity of their immune system, to a higher secondary risk of ESBL-PE-related infection. Reducing the risk of acquisition in the hospital is usually based on a bundle of measures, including screening policies at admission, improving hand hygiene compliance, and decreasing antibiotic consumption. However, recent scientific data suggest new prevention opportunities based on microbiota modifications.

  17. Genome-Wide Identification, Characterization, and Expression Profiling of Glutathione S-Transferase (GST) Family in Pumpkin Reveals Likely Role in Cold-Stress Tolerance

    Science.gov (United States)

    Abdul Kayum, Md.; Nath, Ujjal Kumar; Park, Jong-In; Choi, Eung Kyoo; Song, Jae-Young; Kim, Hoy-Taek; Nou, Ill-Sup

    2018-01-01

    Plant growth and development can be adversely affected by cold stress, limiting productivity. The glutathione S-transferase (GST) family comprises important detoxifying enzymes, which play major roles in biotic and abiotic stress responses by reducing the oxidative damage caused by reactive oxygen species. Pumpkins (Cucurbita maxima) are widely grown, economically important, and nutritious; however, their yield can be severely affected by cold stress. The identification of putative candidate genes responsible for cold-stress tolerance, including the GST family genes, is therefore vital. For the first time, we identified 32 C. maxima GST (CmaGST) genes using a combination of bioinformatics approaches and characterized them by expression profiling. These CmaGST genes represent seven of the 14 known classes of plant GSTs, with 18 CmaGSTs categorized into the tau class. The CmaGSTs were distributed across 13 of pumpkin’s 20 chromosomes, with the highest numbers found on chromosomes 4 and 6. The large number of CmaGST genes resulted from gene duplication; 11 and 5 pairs of CmaGST genes were segmental- and tandem-duplicated, respectively. In addition, all CmaGST genes showed organ-specific expression. The expression of the putative GST genes in pumpkin was examined under cold stress in two lines with contrasting cold tolerance: cold-tolerant CP-1 (C. maxima) and cold-susceptible EP-1 (Cucurbita moschata). Seven genes (CmaGSTU3, CmaGSTU7, CmaGSTU8, CmaGSTU9, CmaGSTU11, CmaGSTU12, and CmaGSTU14) were highly expressed in the cold-tolerant line and are putative candidates for use in breeding cold-tolerant crop varieties. These results increase our understanding of the cold-stress-related functions of the GST family, as well as potentially enhancing pumpkin breeding programs. PMID:29439434

  18. A buoyancy-based screen of Drosophila larvae for fat-storage mutants reveals a role for Sir2 in coupling fat storage to nutrient availability.

    Directory of Open Access Journals (Sweden)

    Tânia Reis

    2010-11-01

    Full Text Available Obesity has a strong genetic component, but few of the genes that predispose to obesity are known. Genetic screens in invertebrates have the potential to identify genes and pathways that regulate the levels of stored fat, many of which are likely to be conserved in humans. To facilitate such screens, we have developed a simple buoyancy-based screening method for identifying mutant Drosophila larvae with increased levels of stored fat. Using this approach, we have identified 66 genes that when mutated increase organismal fat levels. Among these was a sirtuin family member, Sir2. Sirtuins regulate the storage and metabolism of carbohydrates and lipids by deacetylating key regulatory proteins. However, since mammalian sirtuins function in many tissues in different ways, it has been difficult to define their role in energy homeostasis accurately under normal feeding conditions. We show that knockdown of Sir2 in the larval fat body results in increased fat levels. Moreover, using genetic mosaics, we demonstrate that Sir2 restricts fat accumulation in individual cells of the fat body in a cell-autonomous manner. Consistent with this function, changes in the expression of metabolic enzymes in Sir2 mutants point to a shift away from catabolism. Surprisingly, although Sir2 is typically upregulated under conditions of starvation, Sir2 mutant larvae survive better than wild type under conditions of amino-acid starvation as long as sugars are provided. Our findings point to a Sir2-mediated pathway that activates a catabolic response to amino-acid starvation irrespective of the sugar content of the diet.

  19. Functional genomics reveals an essential and specific role for Stat1 in protection of the central nervous system following herpes simplex virus corneal infection.

    Science.gov (United States)

    Pasieka, Tracy Jo; Cilloniz, Cristian; Carter, Victoria S; Rosato, Pamela; Katze, Michael G; Leib, David A

    2011-12-01

    indicated an essential role for normal Stat1-dependent signaling in mediating a nonpathological immune response to viral CNS infection.

  20. Proteomic investigation of Vibrio alginolyticus challenged Caenorhabditis elegans revealed regulation of cellular homeostasis proteins and their role in supporting innate immune system.

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    Durai, Sellegounder; Singh, Nirpendra; Kundu, Suman; Balamurugan, Krishnaswamy

    2014-08-01

    Caenorhabditis elegans has been the preferred model system for many investigators to study pathogenesis. In the present investigation, regulation of C. elegans proteome was explored against V. alginolyticus infection using quantitative proteomics approach. Proteins were separated using 2D-DIGE and the differentially regulated proteins were identified using PMF and MALDI TOF/TOF analysis. The results thus obtained were validated using Western blotting for candidate proteins. The corresponding transcriptional regulation was quantified subsequently using real-time PCR. Interaction network for candidate proteins was predicted using search tool for the retrieval of interacting genes/proteins (STRING) and functional validation was performed using respective mutant strains. Out of the 25 proteins identified, 21 proteins appeared to be upregulated while four were downregulated. Upregulated proteins included those involved in stress-response (PDI-2, HSP-6), immune-response (protein kinase -18, GST-8) and energy-production (ATP-2) while proteins involved in structural maintenance (IFB-2) and lipid metabolism (SODH-1) were downregulated. The roles of these players in the host system during Vibrio infection was analyzed in vivo using wild type and mutant C. elegans. Survival assays using mutants lacking pdi-2, ire-1, and xbp-1 displayed enhanced susceptibility to V. alginolyticus. Cellular stress generated by V. alginolyticus was determined using ROS assay. This is the first report of proteome changes in C. elegans against V. alginolyticus challenge and highlights the significance of unfolded protein response (UPR) pathway during bacterial infection. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Transcriptomic analysis reveals the roles of gibberellin-regulated genes and transcription factors in regulating bolting in lettuce (Lactuca sativa L.).

    Science.gov (United States)

    Liu, Xueying; Lv, Shanshan; Liu, Ran; Fan, Shuangxi; Liu, Chaojie; Liu, Renyi; Han, Yingyan

    2018-01-01

    A cool temperature is preferred for lettuce cultivation, as high temperatures cause premature bolting. Accordingly, exploring the mechanism of bolting and preventing premature bolting is important for agriculture. To explore this relationship in depth, morphological, physiological, and transcriptomic analyses of the bolting-sensitive line S39 at the five-leaf stage grown at 37°C were performed in the present study. Based on paraffin section results, we observed that S39 began bolting on the seventh day at 37°C. During bolting in the heat-treated plants, GA3 and GA4 levels in leaves and the indoleacetic acid (IAA) level in the stem reached a maximum on the sixth day, and these high contents were maintained. Additionally, bolting begins in the fifth day after GA3 treatment in S39 plants, GA3 and GA4 increased and then decreased, reaching a maximum on the fourth day in leaves. Similarly, IAA contents reached a maximum in the stem on the fifth day. No bolting was observed in the control group grown at 25°C, and significant changes were not observed in GA3 and GA4 levels in the controls during the observation period. RNA-sequencing data implicated transcription factors (TFs) in regulating bolting in lettuce, suggesting that the high GA contents in the leaves and IAA in the stem promote bolting. TFs possibly modulate the expression of related genes, such as those encoding hormones, potentially regulating bolting in lettuce. Compared to the control group, 258 TFs were identified in the stem of the treatment group, among which 98 and 156 were differentially up- and down-regulated, respectively; in leaves, 202 and 115 TFs were differentially up- and down-regulated, respectively. Significant changes in the treated group were observed for C2H2 zinc finger, AP2-EREBP, and WRKY families, indicating that these TFs may play important roles in regulating bolting.

  2. Differential microRNA Analysis of Glandular Trichomes and Young Leaves in Xanthium strumarium L. Reveals Their Putative Roles in Regulating Terpenoid Biosynthesis.

    Science.gov (United States)

    Fan, Rongyan; Li, Yuanjun; Li, Changfu; Zhang, Yansheng

    2015-01-01

    The medicinal plant Xanthium strumarium L. (X. strumarium) is covered with glandular trichomes, which are the sites for synthesizing pharmacologically active terpenoids such as xanthatin. MicroRNAs (miRNAs) are a class of 21-24 nucleotide (nt) non-coding RNAs, most of which are identified as regulators of plant growth development. Identification of miRNAs involved in the biosynthesis of plant secondary metabolites remains limited. In this study, high-throughput Illumina sequencing, combined with target gene prediction, was performed to discover novel and conserved miRNAs with potential roles in regulating terpenoid biosynthesis in X. strumarium glandular trichomes. Two small RNA libraries from leaves and glandular trichomes of X. strumarium were established. In total, 1,185 conserved miRNAs and 37 novel miRNAs were identified, with 494 conserved miRNAs and 18 novel miRNAs being differentially expressed between the two tissue sources. Based on the X. strumarium transcriptome data that we recently constructed, 3,307 annotated mRNA transcripts were identified as putative targets of the differentially expressed miRNAs. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis suggested that some of the differentially expressed miRNAs, including miR6435, miR5021 and miR1134, might be involved in terpenoid biosynthesis in the X. strumarium glandular trichomes. This study provides the first comprehensive analysis of miRNAs in X. strumarium, which forms the basis for further understanding of miRNA-based regulation on terpenoid biosynthesis.

  3. Differential microRNA Analysis of Glandular Trichomes and Young Leaves in Xanthium strumarium L. Reveals Their Putative Roles in Regulating Terpenoid Biosynthesis.

    Directory of Open Access Journals (Sweden)

    Rongyan Fan

    Full Text Available The medicinal plant Xanthium strumarium L. (X. strumarium is covered with glandular trichomes, which are the sites for synthesizing pharmacologically active terpenoids such as xanthatin. MicroRNAs (miRNAs are a class of 21-24 nucleotide (nt non-coding RNAs, most of which are identified as regulators of plant growth development. Identification of miRNAs involved in the biosynthesis of plant secondary metabolites remains limited. In this study, high-throughput Illumina sequencing, combined with target gene prediction, was performed to discover novel and conserved miRNAs with potential roles in regulating terpenoid biosynthesis in X. strumarium glandular trichomes. Two small RNA libraries from leaves and glandular trichomes of X. strumarium were established. In total, 1,185 conserved miRNAs and 37 novel miRNAs were identified, with 494 conserved miRNAs and 18 novel miRNAs being differentially expressed between the two tissue sources. Based on the X. strumarium transcriptome data that we recently constructed, 3,307 annotated mRNA transcripts were identified as putative targets of the differentially expressed miRNAs. KEGG (Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested that some of the differentially expressed miRNAs, including miR6435, miR5021 and miR1134, might be involved in terpenoid biosynthesis in the X. strumarium glandular trichomes. This study provides the first comprehensive analysis of miRNAs in X. strumarium, which forms the basis for further understanding of miRNA-based regulation on terpenoid biosynthesis.

  4. Sequence analysis of two alleles reveals that intra-and intergenic recombination played a role in the evolution of the radish fertility restorer (Rfo

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    Budar Françoise

    2010-02-01

    Full Text Available Abstract Background Land plant genomes contain multiple members of a eukaryote-specific gene family encoding proteins with pentatricopeptide repeat (PPR motifs. Some PPR proteins were shown to participate in post-transcriptional events involved in organellar gene expression, and this type of function is now thought to be their main biological role. Among PPR genes, restorers of fertility (Rf of cytoplasmic male sterility systems constitute a peculiar subgroup that is thought to evolve in response to the presence of mitochondrial sterility-inducing genes. Rf genes encoding PPR proteins are associated with very close relatives on complex loci. Results We sequenced a non-restoring allele (L7rfo of the Rfo radish locus whose restoring allele (D81Rfo was previously described, and compared the two alleles and their PPR genes. We identified a ca 13 kb long fragment, likely originating from another part of the radish genome, inserted into the L7rfo sequence. The L7rfo allele carries two genes (PPR-1 and PPR-2 closely related to the three previously described PPR genes of the restorer D81Rfo allele (PPR-A, PPR-B, and PPR-C. Our results indicate that alleles of the Rfo locus have experienced complex evolutionary events, including recombination and insertion of extra-locus sequences, since they diverged. Our analyses strongly suggest that present coding sequences of Rfo PPR genes result from intragenic recombination. We found that the 10 C-terminal PPR repeats in Rfo PPR gene encoded proteins result from the tandem duplication of a 5 PPR repeat block. Conclusions The Rfo locus appears to experience more complex evolution than its flanking sequences. The Rfo locus and PPR genes therein are likely to evolve as a result of intergenic and intragenic recombination. It is therefore not possible to determine which genes on the two alleles are direct orthologs. Our observations recall some previously reported data on pathogen resistance complex loci.

  5. Roles of NlAKTIP in the Growth and Eclosion of the Rice Brown Planthopper, Nilaparvata lugens Stål, as Revealed by RNA Interference

    Science.gov (United States)

    Hao, Peiying; Lu, Chaofeng; Ma, Yan; Xu, Lingbo; Zhu, Jiajun; Yu, Xiaoping

    2015-01-01

    AKT-interacting protein (AKTIP) interacts with serine/threonine protein kinase B (PKB)/AKT. AKTIP modulates AKT’s activity by enhancing the phosphorylation of the regulatory site and plays a crucial role in multiple biological processes. In this study, the full length cDNA of NlAKTIP, a novel AKTIP gene in the brown planthopper (BPH) Nilaparvata lugens, was cloned. The reverse transcription quantitive PCR (RT-qPCR) results showed that the NlAKTIP gene was strongly expressed in gravid female adults, but was relatively weakly expressed in nymphs and male adult BPH. In female BPH, treatment with dsAKTIP resulted in the efficient silencing of NlAKTIP, leading to a significant reduction of mRNA levels, about 50% of those of the untreated control group at day 7 of the study. BPH fed with dsAKTIP had reduced growth with lower body weights and smaller sizes, and the body weight of BPH treated with dsAKTIP at day 7 decreased to about 30% of that of the untreated control. Treatment of dsAKTIP significantly delayed the eclosion for over 7 days relative to the control group and restricted ovarian development to Grade I (transparent stage), whereas the controls developed to Grade IV (matured stage). These results indicated that NlAKTIP is crucial to the growth and development of female BPH. This study provided a valuable clue of a potential target NlAKTIP for inhibiting the BPH, and also provided a new point of view on the interaction between BPH and resistant rice. PMID:26402675

  6. Reconstitution of proapoptotic BAK function in liposomes reveals a dual role for mitochondrial lipids in the BAK-driven membrane permeabilization process.

    Science.gov (United States)

    Landeta, Olatz; Landajuela, Ane; Gil, David; Taneva, Stefka; Di Primo, Carmelo; Sot, Begoña; Valle, Mikel; Frolov, Vadim A; Basañez, Gorka

    2011-03-11

    BAK is a key effector of mitochondrial outer membrane permeabilization (MOMP) whose molecular mechanism of action remains to be fully dissected in intact cells, mainly due to the inherent complexity of the intracellular apoptotic machinery. Here we show that the core features of the BAK-driven MOMP pathway can be reproduced in a highly simplified in vitro system consisting of recombinant human BAK lacking the carboxyl-terminal 21 residues (BAKΔC) and tBID in combination with liposomes bearing an appropriate lipid environment. Using this minimalist reconstituted system we established that tBID suffices to trigger BAKΔC membrane insertion, oligomerization, and pore formation. Furthermore, we demonstrate that tBID-activated BAKΔC permeabilizes the membrane by forming structurally dynamic p