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Sample records for reveals cohesin functions

  1. Distinct Functions of Human Cohesin-SA1 and Cohesin-SA2 in Double-Strand Break Repair

    PubMed Central

    Kong, Xiangduo; Ball, Alexander R.; Pham, Hoang Xuan; Zeng, Weihua; Chen, Hsiao-Yuan; Schmiesing, John A.; Kim, Jong-Soo; Berns, Michael

    2014-01-01

    Cohesin is an essential multiprotein complex that mediates sister chromatid cohesion critical for proper segregation of chromosomes during cell division. Cohesin is also involved in DNA double-strand break (DSB) repair. In mammalian cells, cohesin is involved in both DSB repair and the damage checkpoint response, although the relationship between these two functions is unclear. Two cohesins differing by one subunit (SA1 or SA2) are present in somatic cells, but their functional specificities with regard to DNA repair remain enigmatic. We found that cohesin-SA2 is the main complex corecruited with the cohesin-loading factor NIPBL to DNA damage sites in an S/G2-phase-specific manner. Replacing the diverged C-terminal region of SA1 with the corresponding region of SA2 confers this activity on SA1. Depletion of SA2 but not SA1 decreased sister chromatid homologous recombination repair and affected repair pathway choice, indicating that DNA repair activity is specifically associated with cohesin recruited to damage sites. In contrast, both cohesin complexes function in the intra-S checkpoint, indicating that cell cycle-specific damage site accumulation is not a prerequisite for cohesin's intra-S checkpoint function. Our findings reveal the unique ways in which cohesin-SA1 and cohesin-SA2 participate in the DNA damage response, coordinately protecting genome integrity in human cells. PMID:24324008

  2. Combined Crystal Structure of a Type I Cohesin: MUTATION AND AFFINITY BINDING STUDIES REVEAL STRUCTURAL DETERMINANTS OF COHESIN-DOCKERIN SPECIFICITIES.

    PubMed

    Cameron, Kate; Weinstein, Jonathan Y; Zhivin, Olga; Bule, Pedro; Fleishman, Sarel J; Alves, Victor D; Gilbert, Harry J; Ferreira, Luís M A; Fontes, Carlos M G A; Bayer, Edward A; Najmudin, Shabir

    2015-06-26

    Cohesin-dockerin interactions orchestrate the assembly of one of nature's most elaborate multienzyme complexes, the cellulosome. Cellulosomes are produced exclusively by anaerobic microbes and mediate highly efficient hydrolysis of plant structural polysaccharides, such as cellulose and hemicellulose. In the canonical model of cellulosome assembly, type I dockerin modules of the enzymes bind to reiterated type I cohesin modules of a primary scaffoldin. Each type I dockerin contains two highly conserved cohesin-binding sites, which confer quaternary flexibility to the multienzyme complex. The scaffoldin also bears a type II dockerin that anchors the entire complex to the cell surface by binding type II cohesins of anchoring scaffoldins. In Bacteroides cellulosolvens, however, the organization of the cohesin-dockerin types is reversed, whereby type II cohesin-dockerin pairs integrate the enzymes into the primary scaffoldin, and type I modules mediate cellulosome attachment to an anchoring scaffoldin. Here, we report the crystal structure of a type I cohesin from B. cellulosolvens anchoring scaffoldin ScaB to 1.84-Å resolution. The structure resembles other type I cohesins, and the putative dockerin-binding site, centered at β-strands 3, 5, and 6, is likely to be conserved in other B. cellulosolvens type I cohesins. Combined computational modeling, mutagenesis, and affinity-based binding studies revealed similar hydrogen-bonding networks between putative Ser/Asp recognition residues in the dockerin at positions 11/12 and 45/46, suggesting that a dual-binding mode is not exclusive to the integration of enzymes into primary cellulosomes but can also characterize polycellulosome assembly and cell-surface attachment. This general approach may provide valuable structural information of the cohesin-dockerin interface, in lieu of a definitive crystal structure.

  3. Interallelic complementation provides functional evidence for cohesin-cohesin interactions on DNA.

    PubMed

    Eng, Thomas; Guacci, Vincent; Koshland, Douglas

    2015-11-15

    The cohesin complex (Mcd1p, Smc1p, Smc3p, and Scc3p) has multiple roles in chromosome architecture, such as promoting sister chromatid cohesion, chromosome condensation, DNA repair, and transcriptional regulation. The prevailing embrace model for sister chromatid cohesion posits that a single cohesin complex entraps both sister chromatids. We report interallelic complementation between pairs of nonfunctional mcd1 alleles (mcd1-1 and mcd1-Q266) or smc3 alleles (smc3-42 and smc3-K113R). Cells bearing individual mcd1 or smc3 mutant alleles are inviable and defective for both sister chromatid cohesion and condensation. However, cells coexpressing two defective mcd1 or two defective smc3 alleles are viable and have cohesion and condensation. Because cohesin contains only a single copy of Smc3p or Mcd1p, these examples of interallelic complementation must result from interplay or communication between the two defective cohesin complexes, each harboring one of the mutant allele products. Neither mcd1-1p nor smc3-42p is bound to chromosomes when expressed individually at its restrictive temperature. However, their chromosome binding is restored when they are coexpressed with their chromosome-bound interallelic complementing partner. Our results support a mechanism by which multiple cohesin complexes interact on DNA to mediate cohesion and condensation.

  4. The expanding universe of cohesin functions: a new genome stability caretaker involved in human disease and cancer.

    PubMed

    Mannini, Linda; Menga, Stefania; Musio, Antonio

    2010-06-01

    Cohesin is responsible for sister chromatid cohesion, ensuring the correct chromosome segregation. Beyond this role, cohesin and regulatory cohesin genes seem to play a role in preserving genome stability and gene transcription regulation. DNA damage is thought to be a major culprit for many human diseases, including cancer. Our present knowledge of the molecular basis underlying genome instability is extremely limited. Mutations in cohesin genes cause human diseases such as Cornelia de Lange syndrome and Roberts syndrome/SC phocomelia, and all the cell lines derived from affected patients show genome instability. Cohesin mutations have also been identified in colorectal cancer. Here, we will discuss the human disorders caused by alterations of cohesin function, with emphasis on the emerging role of cohesin as a genome stability caretaker.

  5. A Cohesin-Based Partitioning Mechanism Revealed upon Transcriptional Inactivation of Centromere

    PubMed Central

    Tsabar, Michael; Haase, Julian; Harrison, Benjamin; Snider, Chloe E.; Kaminsky, Lila; Hine, Rebecca M.; Haber, James E.; Bloom, Kerry

    2016-01-01

    Transcriptional inactivation of the budding yeast centromere has been a widely used tool in studies of chromosome segregation and aneuploidy. In haploid cells when an essential chromosome contains a single conditionally inactivated centromere (GAL-CEN), cell growth rate is slowed and segregation fidelity is reduced; but colony formation is nearly 100%. Pedigree analysis revealed that only 30% of the time both mother and daughter cell inherit the GAL-CEN chromosome. The reduced segregation capacity of the GAL-CEN chromosome is further compromised upon reduction of pericentric cohesin (mcm21∆), as reflected in a further diminishment of the Mif2 kinetochore protein at GAL-CEN. By redistributing cohesin from the nucleolus to the pericentromere (by deleting SIR2), there is increased presence of the kinetochore protein Mif2 at GAL-CEN and restoration of cell viability. These studies identify the ability of cohesin to promote chromosome segregation via kinetochore assembly, in a situation where the centromere has been severely compromised. PMID:27128635

  6. Structural basis of cohesin cleavage by separase

    PubMed Central

    Lin, Zhonghui; Luo, Xuelian; Yu, Hongtao

    2016-01-01

    Accurate chromosome segregation requires timely dissolution of chromosome cohesion after chromosomes are properly attached to the mitotic spindle. Separase is absolutely essential for cohesion dissolution in organisms from yeast to man1,2. It cleaves the kleisin subunit of cohesin and opens the cohesin ring to allow chromosome segregation. Cohesin cleavage is spatiotemporally controlled by separase-associated regulatory proteins, including the inhibitory chaperone securin3–6, and by phosphorylation of both the enzyme and substrates7–12. Dysregulation of this process causes chromosome missegregation and aneuploidy, contributing to cancer and birth defects. Despite its essential functions, atomic structures of separase have not been determined. Here, we report crystal structures of the separase protease domain from Chaetomium thermophilum, alone or covalently bound to unphosphorylated and phosphorylated inhibitory peptides derived from a cohesin cleavage site. These structures reveal how separase recognizes cohesin and how cohesin phosphorylation by polo-like kinase 1 (Plk1) enhances cleavage. Consistent with a previous cellular study13, mutating two securin residues in a conserved motif that partially matches the separase cleavage consensus converts securin from a separase inhibitor to a substrate. Our study establishes atomic mechanisms of substrate cleavage by separase and suggests competitive inhibition by securin. PMID:27027290

  7. Distinct Developmental Function of Two Caenorhabditis elegans Homologs of the Cohesin Subunit Scc1/Rad21V⃞

    PubMed Central

    Mito, Yoshiko; Sugimoto, Asako; Yamamoto, Masayuki

    2003-01-01

    Cohesin, which mediates sister chromatid cohesion, is composed of four subunits, named Scc1/Rad21, Scc3, Smc1, and Smc3 in yeast. Caenorhabditis elegans has a single homolog for each of Scc3, Smc1, and Smc3, but as many as four for Scc1/Rad21 (COH-1, SCC-1/COH-2, COH-3, and REC-8). Except for REC-8 required for meiosis, function of these C. elegans proteins remains largely unknown. Herein, we examined their possible involvement in mitosis and development. Embryos depleted of the homolog of either Scc3, or Smc1, or Smc3 by RNA interference revealed a defect in mitotic chromosome segregation but not in chromosome condensation and cytokinesis. Depletion of SCC-1/COH-2 caused similar phenotypes. SCC-1/COH-2 was present in cells destined to divide. It localized to chromosomes in a cell cycle-dependent manner. Worms depleted of COH-1 arrested at either the late embryonic or the larval stage, with no indication of mitotic dysfunction. COH-1 associated chromosomes throughout the cell cycle in all somatic cells undergoing late embryogenesis or larval development. Thus, SCC-1/COH-2 and the homologs of Scc3, Smc1, and Smc3 facilitate mitotic chromosome segregation during the development, presumably by forming a cohesin complex, whereas COH-1 seems to play a role important for development but unrelated to mitosis. PMID:12808038

  8. Chromosomes. A comprehensive Xist interactome reveals cohesin repulsion and an RNA-directed chromosome conformation.

    PubMed

    Minajigi, Anand; Froberg, John E; Wei, Chunyao; Sunwoo, Hongjae; Kesner, Barry; Colognori, David; Lessing, Derek; Payer, Bernhard; Boukhali, Myriam; Haas, Wilhelm; Lee, Jeannie T

    2015-07-17

    The inactive X chromosome (Xi) serves as a model to understand gene silencing on a global scale. Here, we perform "identification of direct RNA interacting proteins" (iDRiP) to isolate a comprehensive protein interactome for Xist, an RNA required for Xi silencing. We discover multiple classes of interactors-including cohesins, condensins, topoisomerases, RNA helicases, chromatin remodelers, and modifiers-that synergistically repress Xi transcription. Inhibiting two or three interactors destabilizes silencing. Although Xist attracts some interactors, it repels architectural factors. Xist evicts cohesins from the Xi and directs an Xi-specific chromosome conformation. Upon deleting Xist, the Xi acquires the cohesin-binding and chromosomal architecture of the active X. Our study unveils many layers of Xi repression and demonstrates a central role for RNA in the topological organization of mammalian chromosomes.

  9. A comprehensive Xist interactome reveals cohesin repulsion and an RNA-directed chromosome conformation

    PubMed Central

    Wei, Chunyao; Sunwoo, Hongjae; Kesner, Barry; Colognori, David; Lessing, Derek; Payer, Bernhard; Boukhali, Myriam; Haas, Wilhelm; Lee, Jeannie T.

    2016-01-01

    The inactive X chromosome (Xi) serves as a model to understand gene silencing on a global scale. Here, we perform “identification of direct RNA interacting proteins” (iDRiP) to isolate a comprehensive protein interactome for Xist, an RNA required for Xi silencing. We discover multiple classes of interactors, including cohesins, condensins, topoisomerases, RNA helicases, chromatin remodelers and modifiers, which synergistically repress Xi transcription. Inhibiting two or three interactors destabilizes silencing. While Xist attracts some interactors, it repels architectural factors. Xist evicts cohesins from the Xi and directs an Xi-specific chromosome conformation. Upon deleting Xist, the Xi acquires the cohesin-binding and chromosomal architecture of the active X. Our study unveils many layers of Xi repression and demonstrates a central role for RNA in the topological organization of mammalian chromosomes. PMID:26089354

  10. A Direct Role for Cohesin in Gene Regulation and Ecdysone Response in Drosophila Salivary Glands

    PubMed Central

    Pauli, Andrea; van Bemmel, Joke G.; Oliveira, Raquel A.; Itoh, Takehiko; Shirahige, Katsuhiko; van Steensel, Bas; Nasmyth, Kim

    2015-01-01

    Summary Background Developmental abnormalities observed in Cornelia de Lange syndrome have been genetically linked to mutations in the cohesin machinery. These and other recent experimental findings have led to the suggestion that cohesin, in addition to its canonical function of mediating sister chromatid cohesion, might also be involved in regulating gene expression. Results We report that cleavage of cohesin’s kleisin subunit in postmitotic Drosophila salivary glands induces major changes in the transcript levels of many genes. Kinetic analyses of changes in transcript levels upon cohesin cleavage reveal that a subset of genes responds to cohesin cleavage within a few hours. In addition, cohesin binds to most of these loci, suggesting that cohesin is directly regulating their expression. Among these genes are several that are regulated by the steroid hormone ecdysone. Cytological visualization of transcription at selected ecdysone-responsive genes reveals that puffing at Eip74EF ceases within an hour or two of cohesin cleavage, long before any decline in ecdysone receptor could be detected at this locus. Conclusion We conclude that cohesin regulates expression of a distinct set of genes, including those mediating the ecdysone response. PMID:20933422

  11. Structure of the cohesin loader Scc2

    PubMed Central

    Chao, William C. H.; Murayama, Yasuto; Muñoz, Sofía; Jones, Andrew W.; Wade, Benjamin O.; Purkiss, Andrew G.; Hu, Xiao-Wen; Borg, Aaron; Snijders, Ambrosius P.; Uhlmann, Frank; Singleton, Martin R.

    2017-01-01

    The functions of cohesin are central to genome integrity, chromosome organization and transcription regulation through its prevention of premature sister-chromatid separation and the formation of DNA loops. The loading of cohesin onto chromatin depends on the Scc2–Scc4 complex; however, little is known about how it stimulates the cohesion-loading activity. Here we determine the large ‘hook' structure of Scc2 responsible for catalysing cohesin loading. We identify key Scc2 surfaces that are crucial for cohesin loading in vivo. With the aid of previously determined structures and homology modelling, we derive a pseudo-atomic structure of the full-length Scc2–Scc4 complex. Finally, using recombinantly purified Scc2–Scc4 and cohesin, we performed crosslinking mass spectrometry and interaction assays that suggest Scc2–Scc4 uses its modular structure to make multiple contacts with cohesin. PMID:28059076

  12. Mechanism of Bacterial Cell-Surface Attachment Revealed by the Structure of Cellulosomal Type II Cohesin-dockerin Complex

    SciTech Connect

    Adams,J.; Pal, G.; Jia, Z.; Smith, S.

    2006-01-01

    Bacterial cell-surface attachment of macromolecular complexes maintains the microorganism in close proximity to extracellular substrates and allows for optimal uptake of hydrolytic byproducts. The cellulosome is a large multienzyme complex used by many anaerobic bacteria for the efficient degradation of plant cell-wall polysaccharides. The mechanism of cellulosome retention to the bacterial cell surface involves a calcium-mediated protein-protein interaction between the dockerin (Doc) module from the cellulosomal scaffold and a cohesin (Coh) module of cell-surface proteins located within the proteoglycan layer. Here, we report the structure of an ultra-high-affinity (K{sub a} = 1.44 x 10{sup 10} M{sup 1-}) complex between type II Doc, together with its neighboring X module from the cellulosome scaffold of Clostridium thermocellum, and a type II Coh module associated with the bacterial cell surface. Identification of X module-Doc and X module-Coh contacts reveal roles for the X module in Doc stability and enhanced Coh recognition. This extremely tight interaction involves one face of the Coh and both helices of the Doc and comprises significant hydrophobic character and a complementary extensive hydrogen-bond network. This structure represents a unique mechanism for cell-surface attachment in anaerobic bacteria and provides a rationale for discriminating between type I and type II Coh modules.

  13. Dose-dependent role of the cohesin complex in normal and malignant hematopoiesis

    PubMed Central

    Viny, Aaron D.; Ott, Christopher J.; Spitzer, Barbara; Rivas, Martin; Meydan, Cem; Papalexi, Efthymia; Yelin, Dana; Shank, Kaitlyn; Reyes, Jaime; Chiu, April; Romin, Yevgeniy; Boyko, Vitaly; Thota, Swapna; Maciejewski, Jaroslaw P.; Melnick, Ari

    2015-01-01

    Cohesin complex members have recently been identified as putative tumor suppressors in hematologic and epithelial malignancies. The cohesin complex guides chromosome segregation; however, cohesin mutant leukemias do not show genomic instability. We hypothesized that reduced cohesin function alters chromatin structure and disrupts cis-regulatory architecture of hematopoietic progenitors. We investigated the consequences of Smc3 deletion in normal and malignant hematopoiesis. Biallelic Smc3 loss induced bone marrow aplasia with premature sister chromatid separation and revealed an absolute requirement for cohesin in hematopoietic stem cell (HSC) function. In contrast, Smc3 haploinsufficiency increased self-renewal in vitro and in vivo, including competitive transplantation. Smc3 haploinsufficiency reduced coordinated transcriptional output, including reduced expression of transcription factors and other genes associated with lineage commitment. Smc3 haploinsufficiency cooperated with Flt3-ITD to induce acute leukemia in vivo, with potentiated Stat5 signaling and altered nucleolar topology. These data establish a dose dependency for cohesin in regulating chromatin structure and HSC function. PMID:26438361

  14. Cohesin embraces new phenotypes

    PubMed Central

    Krantz, Ian D

    2014-01-01

    A new study identifies homozygous missense mutations in SGOL1, which encodes a component of the cohesin complex, in a newly described disorder termed Chronic Atrial and Intestinal Dysrhythmia (CAID) syndrome. These findings implicate cohesin in the regulation of intrinsic cardiac and intestinal rhythm and further expand the growing group of disorders termed the cohesinopathies. PMID:25352100

  15. Cohesin Releases DNA through Asymmetric ATPase-Driven Ring Opening

    PubMed Central

    Elbatsh, Ahmed M.O.; Haarhuis, Judith H.I.; Petela, Naomi; Chapard, Christophe; Fish, Alexander; Celie, Patrick H.; Stadnik, Magda; Ristic, Dejan; Wyman, Claire; Medema, René H.; Nasmyth, Kim; Rowland, Benjamin D.

    2016-01-01

    Summary Cohesin stably holds together the sister chromatids from S phase until mitosis. To do so, cohesin must be protected against its cellular antagonist Wapl. Eco1 acetylates cohesin’s Smc3 subunit, which locks together the sister DNAs. We used yeast genetics to dissect how Wapl drives cohesin from chromatin and identified mutants of cohesin that are impaired in ATPase activity but remarkably confer robust cohesion that bypasses the need for the cohesin protectors Eco1 in yeast and Sororin in human cells. We uncover a functional asymmetry within the heart of cohesin’s highly conserved ABC-like ATPase machinery and find that both ATPase sites contribute to DNA loading, whereas DNA release is controlled specifically by one site. We propose that Smc3 acetylation locks cohesin rings around the sister chromatids by counteracting an activity associated with one of cohesin’s two ATPase sites. PMID:26895426

  16. Stage-Specific Binding Profiles of Cohesin in Resting and Activated B Lymphocytes Suggest a Role for Cohesin in Immunoglobulin Class Switching and Maturation

    PubMed Central

    Günal-Sadık, Gamze; Paszkowski-Rogacz, Maciej; Singaravelu, Kalaimathy; Beyer, Andreas; Buchholz, Frank; Jessberger, Rolf

    2014-01-01

    The immunoglobulin heavy chain locus (Igh) features higher-order chromosomal interactions to facilitate stage-specific assembly of the Ig molecule. Cohesin, a ring-like protein complex required for sister chromatid cohesion, shapes chromosome architecture and chromatin interactions important for transcriptional regulation and often acts together with CTCF. Cohesin is likely involved in B cell activation and Ig class switch recombination. Hence, binding profiles of cohesin in resting mature murine splenic B lymphocytes and at two stages after cell activation were elucidated by chromatin immunoprecipitation and deep sequencing. Comparative genomic analysis revealed cohesin extensively changes its binding to transcriptional control elements after 48 h of stimulation with LPS/IL-4. Cohesin was clearly underrepresented at switch regions regardless of their activation status, suggesting that switch regions need to be cohesin-poor. Specific binding changes of cohesin at B-cell specific gene loci Pax5 and Blimp-1 indicate new cohesin-dependent regulatory pathways. Together with conserved cohesin/CTCF sites at the Igh 3′RR, a prominent cohesin/CTCF binding site was revealed near the 3′ end of Cα where PolII localizes to 3′ enhancers. Our study shows that cohesin likely regulates B cell activation and maturation, including Ig class switching. PMID:25375358

  17. Of Rings and Rods: Regulating Cohesin Entrapment of DNA to Generate Intra- and Intermolecular Tethers

    PubMed Central

    2016-01-01

    The clinical relevance of cohesin in DNA repair, tumorigenesis, and severe birth defects continues to fuel efforts in understanding cohesin structure, regulation, and enzymology. Early models depicting huge cohesin rings that entrap two DNA segments within a single lumen are fading into obscurity based on contradictory findings, but elucidating cohesin structure amid a myriad of functions remains challenging. Due in large part to integrated uses of a wide range of methodologies, recent advances are beginning to cast light into the depths that previously cloaked cohesin structure. Additional efforts similarly provide new insights into cohesin enzymology: specifically, the discoveries of ATP-dependent transitions that promote cohesin binding and release from DNA. In combination, these efforts posit a new model that cohesin exists primarily as a relatively flattened structure that entraps only a single DNA molecule and that subsequent ATP hydrolysis, acetylation, and oligomeric assembly tether together individual DNA segments. PMID:27788133

  18. Cohesin in determining chromosome architecture

    SciTech Connect

    Haering, Christian H.; Jessberger, Rolf

    2012-07-15

    Cells use ring-like structured protein complexes for various tasks in DNA dynamics. The tripartite cohesin ring is particularly suited to determine chromosome architecture, for it is large and dynamic, may acquire different forms, and is involved in several distinct nuclear processes. This review focuses on cohesin's role in structuring chromosomes during mitotic and meiotic cell divisions and during interphase.

  19. Cohesin loss alters adult hematopoietic stem cell homeostasis, leading to myeloproliferative neoplasms

    PubMed Central

    Mullenders, Jasper; Aranda-Orgilles, Beatriz; Lhoumaud, Priscillia; Keller, Matthew; Pae, Juhee; Wang, Kun; Kayembe, Clarisse; Rocha, Pedro P.; Raviram, Ramya; Gong, Yixiao; Premsrirut, Prem K.; Tsirigos, Aristotelis; Bonneau, Richard; Skok, Jane A.; Cimmino, Luisa; Hoehn, Daniela

    2015-01-01

    The cohesin complex (consisting of Rad21, Smc1a, Smc3, and Stag2 proteins) is critically important for proper sister chromatid separation during mitosis. Mutations in the cohesin complex were recently identified in a variety of human malignancies including acute myeloid leukemia (AML). To address the potential tumor-suppressive function of cohesin in vivo, we generated a series of shRNA mouse models in which endogenous cohesin can be silenced inducibly. Notably, silencing of cohesin complex members did not have a deleterious effect on cell viability. Furthermore, knockdown of cohesin led to gain of replating capacity of mouse hematopoietic progenitor cells. However, cohesin silencing in vivo rapidly altered stem cells homeostasis and myelopoiesis. Likewise, we found widespread changes in chromatin accessibility and expression of genes involved in myelomonocytic maturation and differentiation. Finally, aged cohesin knockdown mice developed a clinical picture closely resembling myeloproliferative disorders/neoplasms (MPNs), including varying degrees of extramedullary hematopoiesis (myeloid metaplasia) and splenomegaly. Our results represent the first successful demonstration of a tumor suppressor function for the cohesin complex, while also confirming that cohesin mutations occur as an early event in leukemogenesis, facilitating the potential development of a myeloid malignancy. PMID:26438359

  20. Sister chromatid resolution: a cohesin releasing network and beyond.

    PubMed

    Shintomi, Keishi; Hirano, Tatsuya

    2010-10-01

    When chromosomes start to assemble in mitotic prophase, duplicated chromatids are not discernible within each chromosome. As condensation proceeds, they gradually show up, culminating in two rod-shaped structures apposed along their entire length within a metaphase chromosome. This process, known as sister chromatid resolution, is thought to be a prerequisite for rapid and synchronous separation of sister chromatids in anaphase. From a mechanistic point of view, the resolution process can be dissected into three distinct steps: (1) release of cohesin from chromosome arms; (2) formation of chromatid axes mediated by condensins; and (3) untanglement of inter-sister catenation catalyzed by topoisomerase II (topo II). In this review article, we summarize recent progress in our understanding the molecular mechanisms of sister chromatid resolution with a major focus on its first step, cohesin release. An emerging idea is that this seemingly simple step is regulated by an intricate network of positive and negative factors, including cohesin-binding proteins and mitotic kinases. Interestingly, some key factors responsible for cohesin release in early mitosis also play important roles in controlling cohesin functions during interphase. Finally, we discuss how the step of cohesin release might mechanistically be coordinated with the actions of condensins and topo II.

  1. Cohesins: chromatin architects in chromosome segregation, control of gene expression and much more.

    PubMed

    Barbero, José L

    2009-07-01

    Cells have evolved to develop molecules and control mechanisms that guarantee correct chromosome segregation and ensure the proper distribution of genetic material to daughter cells. In this sense, the establishment, maintenance, and removal of sister chromatid cohesion is one of the most fascinating and dangerous processes in the life of a cell because errors in the control of these processes frequently lead to cell death or aneuploidy. The main protagonist in this mechanism is a four-protein complex denominated the cohesin complex. In the last 10 years, we have improved our understanding of the key players in the regulation of sister chromatid cohesion during cell division in mitosis and meiosis. The last 2 years have seen an increase in evidence showing that cohesins have important functions in non-dividing cells, revealing new, unexplored roles for these proteins in the control of gene expression, development, and other essential cell functions in mammals.

  2. Specific Sites in the C Terminus of CTCF Interact with the SA2 Subunit of the Cohesin Complex and Are Required for Cohesin-Dependent Insulation Activity ▿

    PubMed Central

    Xiao, Tiaojiang; Wallace, Julie; Felsenfeld, Gary

    2011-01-01

    Recent studies have shown that the protein CTCF, which plays an important role in insulation and in large-scale organization of chromatin within the eukaryotic nucleus, depends for both activities on recruitment of the cohesin complex. We show here that the interaction of CTCF with the cohesin complex involves direct contacts between the cohesin subunit SA2 and specific regions of the C-terminal tail of CTCF. All other cohesin components are recruited through their interaction with SA2. Expression in vivo of CTCF mutants lacking the C-terminal domain, or with mutations at sites within it required for SA2 binding, disrupts the normal expression profile of the imprinted genes IGF2-H19 and also results in a loss of insulation activity. Taken together, our results demonstrate that specific sites on the C terminus of CTCF are essential for cohesin binding and insulator function. The only direct interaction between CTCF and cohesin involves contact with SA2, which is external to the cohesin ring. This suggests that in recruiting cohesin to CTCF, SA2 could bind first and the ring could assemble subsequently. PMID:21444719

  3. ASXL1 interacts with the cohesin complex to maintain chromatid separation and gene expression for normal hematopoiesis

    PubMed Central

    Li, Zhaomin; Zhang, Peng; Yan, Aimin; Guo, Zhengyu; Ban, Yuguang; Li, Jin; Chen, Shi; Yang, Hui; He, Yongzheng; Li, Jianping; Guo, Ying; Zhang, Wen; Hajiramezanali, Ehsan; An, Huangda; Fajardo, Darlene; Harbour, J. William; Ruan, Yijun; Nimer, Stephen D.; Yu, Peng; Chen, Xi; Xu, Mingjiang; Yang, Feng-Chun

    2017-01-01

    ASXL1 is frequently mutated in a spectrum of myeloid malignancies with poor prognosis. Loss of Asxl1 leads to myelodysplastic syndrome–like disease in mice; however, the underlying molecular mechanisms remain unclear. We report that ASXL1 interacts with the cohesin complex, which has been shown to guide sister chromatid segregation and regulate gene expression. Loss of Asxl1 impairs the cohesin function, as reflected by an impaired telophase chromatid disjunction in hematopoietic cells. Chromatin immunoprecipitation followed by DNA sequencing data revealed that ASXL1, RAD21, and SMC1A share 93% of genomic binding sites at promoter regions in Lin−cKit+ (LK) cells. We have shown that loss of Asxl1 reduces the genome binding of RAD21 and SMC1A and alters the expression of ASXL1/cohesin target genes in LK cells. Our study underscores the ASXL1-cohesin interaction as a novel means to maintain normal sister chromatid separation and regulate gene expression in hematopoietic cells. PMID:28116354

  4. DNA Entry into and Exit out of the Cohesin Ring by an Interlocking Gate Mechanism.

    PubMed

    Murayama, Yasuto; Uhlmann, Frank

    2015-12-17

    Structural maintenance of chromosome (SMC) complexes are proteinaceous rings that embrace DNA to enable vital chromosomal functions. The ring is formed by two SMC subunits, closed at a pair of ATPase heads, whose interaction is reinforced by a kleisin subunit. Using biochemical analysis of fission-yeast cohesin, we find that a similar series of events facilitates both topological entrapment and release of DNA. DNA-sensing lysines trigger ATP hydrolysis to open the SMC head interface, whereas the Wapl subunit disengages kleisin, but only after ATP rebinds. This suggests an interlocking gate mechanism for DNA transport both into and out of the cohesin ring. The entry direction is facilitated by a cohesin loader that appears to fold cohesin to expose the DNA sensor. Our results provide a model for dynamic DNA binding by all members of the SMC family and explain how lysine acetylation of cohesin establishes enduring sister chromatid cohesion.

  5. Genome-wide and parental allele-specific analysis of CTCF and cohesin DNA binding in mouse brain reveals a tissue-specific binding pattern and an association with imprinted differentially methylated regions.

    PubMed

    Prickett, Adam R; Barkas, Nikolaos; McCole, Ruth B; Hughes, Siobhan; Amante, Samuele M; Schulz, Reiner; Oakey, Rebecca J

    2013-10-01

    DNA binding factors are essential for regulating gene expression. CTCF and cohesin are DNA binding factors with central roles in chromatin organization and gene expression. We determined the sites of CTCF and cohesin binding to DNA in mouse brain, genome wide and in an allele-specific manner with high read-depth ChIP-seq. By comparing our results with existing data for mouse liver and embryonic stem (ES) cells, we investigated the tissue specificity of CTCF binding sites. ES cells have fewer unique CTCF binding sites occupied than liver and brain, consistent with a ground-state pattern of CTCF binding that is elaborated during differentiation. CTCF binding sites without the canonical consensus motif were highly tissue specific. In brain, a third of CTCF and cohesin binding sites coincide, consistent with the potential for many interactions between cohesin and CTCF but also many instances of independent action. In the context of genomic imprinting, CTCF and/or cohesin bind to a majority but not all differentially methylated regions, with preferential binding to the unmethylated parental allele. Whether the parental allele-specific methylation was established in the parental germlines or post-fertilization in the embryo is not a determinant in CTCF or cohesin binding. These findings link CTCF and cohesin with the control regions of a subset of imprinted genes, supporting the notion that imprinting control is mechanistically diverse.

  6. Divergent kleisin subunits of cohesin specify mechanisms to tether and release meiotic chromosomes.

    PubMed

    Severson, Aaron F; Meyer, Barbara J

    2014-08-29

    We show that multiple, functionally specialized cohesin complexes mediate the establishment and two-step release of sister chromatid cohesion that underlies the production of haploid gametes. In C. elegans, the kleisin subunits REC-8 and COH-3/4 differ between meiotic cohesins and endow them with distinctive properties that specify how cohesins load onto chromosomes and then trigger and release cohesion. Unlike REC-8 cohesin, COH-3/4 cohesin becomes cohesive through a replication-independent mechanism initiated by the DNA double-stranded breaks that induce crossover recombination. Thus, break-induced cohesion also tethers replicated meiotic chromosomes. Later, recombination stimulates separase-independent removal of REC-8 and COH-3/4 cohesins from reciprocal chromosomal territories flanking the crossover site. This region-specific removal likely underlies the two-step separation of homologs and sisters. Unexpectedly, COH-3/4 performs cohesion-independent functions in synaptonemal complex assembly. This new model for cohesin function diverges from that established in yeast but likely applies directly to plants and mammals, which utilize similar meiotic kleisins.

  7. Long-Range Chromosome Interactions Mediated by Cohesin Shape Circadian Gene Expression

    PubMed Central

    Xu, Yichi; Guo, Weimin; Li, Ping; Zhang, Yan; Zhao, Meng; Fan, Zenghua; Zhao, Zhihu; Yan, Jun

    2016-01-01

    Mammalian circadian rhythm is established by the negative feedback loops consisting of a set of clock genes, which lead to the circadian expression of thousands of downstream genes in vivo. As genome-wide transcription is organized under the high-order chromosome structure, it is largely uncharted how circadian gene expression is influenced by chromosome architecture. We focus on the function of chromatin structure proteins cohesin as well as CTCF (CCCTC-binding factor) in circadian rhythm. Using circular chromosome conformation capture sequencing, we systematically examined the interacting loci of a Bmal1-bound super-enhancer upstream of a clock gene Nr1d1 in mouse liver. These interactions are largely stable in the circadian cycle and cohesin binding sites are enriched in the interactome. Global analysis showed that cohesin-CTCF co-binding sites tend to insulate the phases of circadian oscillating genes while cohesin-non-CTCF sites are associated with high circadian rhythmicity of transcription. A model integrating the effects of cohesin and CTCF markedly improved the mechanistic understanding of circadian gene expression. Further experiments in cohesin knockout cells demonstrated that cohesin is required at least in part for driving the circadian gene expression by facilitating the enhancer-promoter looping. This study provided a novel insight into the relationship between circadian transcriptome and the high-order chromosome structure. PMID:27135601

  8. Rad61/Wpl1 (Wapl), a cohesin regulator, controls chromosome compaction during meiosis

    PubMed Central

    Challa, Kiran; Lee, Min-Su; Shinohara, Miki; Kim, Keun P.; Shinohara, Akira

    2016-01-01

    Meiosis-specific cohesin, required for the linking of the sister chromatids, plays a critical role in various chromosomal events during meiotic prophase I, such as chromosome morphogenesis and dynamics, as well as recombination. Rad61/Wpl1 (Wapl in other organisms) negatively regulates cohesin functions. In this study, we show that meiotic chromosome axes are shortened in the budding yeast rad61/wpl1 mutant, suggesting that Rad61/Wpl1 negatively regulates chromosome axis compaction. Rad61/Wpl1 is required for efficient resolution of telomere clustering during meiosis I, indicating a positive effect of Rad61/Wpl1 on the cohesin function required for telomere dynamics. Additionally, we demonstrate distinct activities of Rad61/Wpl1 during the meiotic recombination, including its effects on the efficient processing of intermediates. Thus, Rad61/Wpl1 both positively and negatively regulates various cohesin-mediated chromosomal processes during meiosis. PMID:26825462

  9. Atypical cohesin-dockerin complex responsible for cell surface attachment of cellulosomal components: binding fidelity, promiscuity, and structural buttresses.

    PubMed

    Salama-Alber, Orly; Jobby, Maroor K; Chitayat, Seth; Smith, Steven P; White, Bryan A; Shimon, Linda J W; Lamed, Raphael; Frolow, Felix; Bayer, Edward A

    2013-06-07

    The rumen bacterium Ruminococcus flavefaciens produces a highly organized multienzyme cellulosome complex that plays a key role in the degradation of plant cell wall polysaccharides, notably cellulose. The R. flavefaciens cellulosomal system is anchored to the bacterial cell wall through a relatively small ScaE scaffoldin subunit, which bears a single type IIIe cohesin responsible for the attachment of two major dockerin-containing scaffoldin proteins, ScaB and the cellulose-binding protein CttA. Although ScaB recruits the catalytic machinery onto the complex, CttA mediates attachment of the bacterial substrate via its two putative carbohydrate-binding modules. In an effort to understand the structural basis for assembly and cell surface attachment of the cellulosome in R. flavefaciens, we determined the crystal structure of the high affinity complex (Kd = 20.83 nM) between the cohesin module of ScaE (CohE) and its cognate X-dockerin (XDoc) modular dyad from CttA at 1.97-Å resolution. The structure reveals an atypical calcium-binding loop containing a 13-residue insert. The results further pinpoint two charged specificity-related residues on the surface of the cohesin module that are responsible for specific versus promiscuous cross-strain binding of the dockerin module. In addition, a combined functional role for the three enigmatic dockerin inserts was established whereby these extraneous segments serve as structural buttresses that reinforce the stalklike conformation of the X-module, thus segregating its tethered complement of cellulosomal components from the cell surface. The novel structure of the RfCohE-XDoc complex sheds light on divergent dockerin structure and function and provides insight into the specificity features of the type IIIe cohesin-dockerin interaction.

  10. Roberts syndrome: A deficit in acetylated cohesin leads to nucleolar dysfunction.

    PubMed

    Xu, Baoshan; Lu, Shuai; Gerton, Jennifer L

    2014-01-01

    All living organisms must go through cycles of replicating their genetic information and then dividing the copies between two new cells. This cyclical process, in cells from bacteria and human alike, requires a protein complex known as cohesin. Cohesin is a structural maintenance of chromosomes (SMC) complex. While bacteria have one form of this complex, yeast have several SMC complexes, and humans have at least a dozen cohesin complexes alone. Therefore the ancient structure and function of SMC complexes has been both conserved and specialized over the course of evolution. These complexes play roles in replication, repair, organization, and segregation of the genome. Mutations in the genes that encode cohesin and its regulatory factors are associated with developmental disorders such as Roberts syndrome, Cornelia de Lange syndrome, and cancer. In this review, we focus on how acetylation of cohesin contributes to its function. In Roberts syndrome, the lack of cohesin acetylation contributes to nucleolar defects and translational inhibition. An understanding of basic SMC complex function will be essential to unraveling the molecular etiology of human diseases associated with defective SMC function.

  11. Distinct Roles of Meiosis-Specific Cohesin Complexes in Mammalian Spermatogenesis

    PubMed Central

    Biswas, Uddipta; Hempel, Kai; Llano, Elena; Pendas, Alberto; Jessberger, Rolf

    2016-01-01

    Mammalian meiocytes feature four meiosis-specific cohesin proteins in addition to ubiquitous ones, but the roles of the individual cohesin complexes are incompletely understood. To decipher the functions of the two meiosis-specific kleisins, REC8 or RAD21L, together with the only meiosis-specific SMC protein SMC1β, we generated Smc1β-/-Rec8-/- and Smc1β-/-Rad21L-/- mouse mutants. Analysis of spermatocyte chromosomes revealed that besides SMC1β complexes, SMC1α/RAD21 and to a small extent SMC1α/REC8 contribute to chromosome axis length. Removal of SMC1β and RAD21L almost completely abolishes all chromosome axes. The sex chromosomes do not pair in single or double mutants, and autosomal synapsis is impaired in all mutants. Super resolution microscopy revealed synapsis-associated SYCP1 aberrantly deposited between sister chromatids and on single chromatids in Smc1β-/-Rad21L-/- cells. All mutants show telomere length reduction and structural disruptions, while wild-type telomeres feature a circular TRF2 structure reminiscent of t-loops. There is no loss of centromeric cohesion in both double mutants at leptonema/early zygonema, indicating that, at least in the mutant backgrounds, an SMC1α/RAD21 complex provides centromeric cohesion at this early stage. Thus, in early prophase I the most prominent roles of the meiosis-specific cohesins are in axis-related features such as axis length, synapsis and telomere integrity rather than centromeric cohesion. PMID:27792785

  12. The Drosophila Mi-2 Chromatin-Remodeling Factor Regulates Higher-Order Chromatin Structure and Cohesin Dynamics In Vivo

    PubMed Central

    Fasulo, Barbara; Deuring, Renate; Murawska, Magdalena; Gause, Maria; Dorighi, Kristel M.; Schaaf, Cheri A.; Dorsett, Dale; Brehm, Alexander; Tamkun, John W.

    2012-01-01

    dMi-2 is a highly conserved ATP-dependent chromatin-remodeling factor that regulates transcription and cell fates by altering the structure or positioning of nucleosomes. Here we report an unanticipated role for dMi-2 in the regulation of higher-order chromatin structure in Drosophila. Loss of dMi-2 function causes salivary gland polytene chromosomes to lose their characteristic banding pattern and appear more condensed than normal. Conversely, increased expression of dMi-2 triggers decondensation of polytene chromosomes accompanied by a significant increase in nuclear volume; this effect is relatively rapid and is dependent on the ATPase activity of dMi-2. Live analysis revealed that dMi-2 disrupts interactions between the aligned chromatids of salivary gland polytene chromosomes. dMi-2 and the cohesin complex are enriched at sites of active transcription; fluorescence-recovery after photobleaching (FRAP) assays showed that dMi-2 decreases stable association of cohesin with polytene chromosomes. These findings demonstrate that dMi-2 is an important regulator of both chromosome condensation and cohesin binding in interphase cells. PMID:22912596

  13. Altered cohesin gene dosage affects Mammalian meiotic chromosome structure and behavior.

    PubMed

    Murdoch, Brenda; Owen, Nichole; Stevense, Michelle; Smith, Helen; Nagaoka, So; Hassold, Terry; McKay, Michael; Xu, Huiling; Fu, Jun; Revenkova, Ekaterina; Jessberger, Rolf; Hunt, Patricia

    2013-01-01

    Based on studies in mice and humans, cohesin loss from chromosomes during the period of protracted meiotic arrest appears to play a major role in chromosome segregation errors during female meiosis. In mice, mutations in meiosis-specific cohesin genes cause meiotic disturbances and infertility. However, the more clinically relevant situation, heterozygosity for mutations in these genes, has not been evaluated. We report here evidence from the mouse that partial loss of gene function for either Smc1b or Rec8 causes perturbations in the formation of the synaptonemal complex (SC) and affects both synapsis and recombination between homologs during meiotic prophase. Importantly, these defects increase the frequency of chromosomally abnormal eggs in the adult female. These findings have important implications for humans: they suggest that women who carry mutations or variants that affect cohesin function have an elevated risk of aneuploid pregnancies and may even be at increased risk of transmitting structural chromosome abnormalities.

  14. The ATPases of cohesin interface with regulators to modulate cohesin-mediated DNA tethering

    PubMed Central

    Çamdere, Gamze; Guacci, Vincent; Stricklin, Jeremiah; Koshland, Douglas

    2015-01-01

    Cohesin tethers together regions of DNA, thereby mediating higher order chromatin organization that is critical for sister chromatid cohesion, DNA repair and transcriptional regulation. Cohesin contains a heterodimeric ATP-binding Cassette (ABC) ATPase comprised of Smc1 and Smc3 ATPase active sites. These ATPases are required for cohesin to bind DNA. Cohesin’s DNA binding activity is also promoted by the Eco1 acetyltransferase and inhibited by Wpl1. Recently we showed that after cohesin stably binds DNA, a second step is required for DNA tethering. This second step is also controlled by Eco1 acetylation. Here, we use genetic and biochemical analyses to show that this second DNA tethering step is regulated by cohesin ATPase. Furthermore, our results also suggest that Eco1 promotes cohesion by modulating the ATPase cycle of DNA-bound cohesin in a state that is permissive for DNA tethering and refractory to Wpl1 inhibition. DOI: http://dx.doi.org/10.7554/eLife.11315.001 PMID:26583750

  15. Cohesin regulates VSG monoallelic expression in trypanosomes.

    PubMed

    Landeira, David; Bart, Jean-Mathieu; Van Tyne, Daria; Navarro, Miguel

    2009-07-27

    Antigenic variation allows Trypanosoma brucei to evade the host immune response by switching the expression of 1 out of approximately 15 telomeric variant surface glycoprotein (VSG) expression sites (ESs). VSG ES transcription is mediated by RNA polymerase I in a discrete nuclear site named the ES body (ESB). However, nothing is known about how the monoallelic VSG ES transcriptional state is maintained over generations. In this study, we show that during S and G2 phases and early mitosis, the active VSG ES locus remains associated with the single ESB and exhibits a delay in the separation of sister chromatids relative to control loci. This delay is dependent on the cohesin complex, as partial knockdown of cohesin subunits resulted in premature separation of sister chromatids of the active VSG ES. Cohesin depletion also prompted transcriptional switching from the active to previously inactive VSG ESs. Thus, in addition to maintaining sister chromatid cohesion during mitosis, the cohesin complex plays an essential role in the correct epigenetic inheritance of the active transcriptional VSG ES state.

  16. FOXA and master transcription factors recruit Mediator and Cohesin to the core transcriptional regulatory circuitry of cancer cells

    NASA Astrophysics Data System (ADS)

    Fournier, Michèle; Bourriquen, Gaëlle; Lamaze, Fabien C.; Côté, Maxime C.; Fournier, Éric; Joly-Beauparlant, Charles; Caron, Vicky; Gobeil, Stéphane; Droit, Arnaud; Bilodeau, Steve

    2016-10-01

    Controlling the transcriptional program is essential to maintain the identity and the biological functions of a cell. The Mediator and Cohesin complexes have been established as central cofactors controlling the transcriptional program in normal cells. However, the distribution, recruitment and importance of these complexes in cancer cells have not been fully investigated. Here we show that FOXA and master transcription factors are part of the core transcriptional regulatory circuitry of cancer cells and are essential to recruit M ediator and Cohesin. Indeed, Mediator and Cohesin occupied the enhancer and promoter regions of actively transcribed genes and maintained the proliferation and colony forming potential. Through integration of publically available ChIP-Seq datasets, we predicted the core transcriptional regulatory circuitry of each cancer cell. Unexpectedly, for all cells investigated, the pioneer transcription factors FOXA1 and/or FOXA2 were identified in addition to cell-specific master transcription factors. Loss of both types of transcription factors phenocopied the loss of Mediator and Cohesin. Lastly, the master and pioneer transcription factors were essential to recruit Mediator and Cohesin to regulatory regions of actively transcribed genes. Our study proposes that maintenance of the cancer cell state is dependent on recruitment of Mediator and Cohesin through FOXA and master transcription factors.

  17. FOXA and master transcription factors recruit Mediator and Cohesin to the core transcriptional regulatory circuitry of cancer cells.

    PubMed

    Fournier, Michèle; Bourriquen, Gaëlle; Lamaze, Fabien C; Côté, Maxime C; Fournier, Éric; Joly-Beauparlant, Charles; Caron, Vicky; Gobeil, Stéphane; Droit, Arnaud; Bilodeau, Steve

    2016-10-14

    Controlling the transcriptional program is essential to maintain the identity and the biological functions of a cell. The Mediator and Cohesin complexes have been established as central cofactors controlling the transcriptional program in normal cells. However, the distribution, recruitment and importance of these complexes in cancer cells have not been fully investigated. Here we show that FOXA and master transcription factors are part of the core transcriptional regulatory circuitry of cancer cells and are essential to recruit M ediator and Cohesin. Indeed, Mediator and Cohesin occupied the enhancer and promoter regions of actively transcribed genes and maintained the proliferation and colony forming potential. Through integration of publically available ChIP-Seq datasets, we predicted the core transcriptional regulatory circuitry of each cancer cell. Unexpectedly, for all cells investigated, the pioneer transcription factors FOXA1 and/or FOXA2 were identified in addition to cell-specific master transcription factors. Loss of both types of transcription factors phenocopied the loss of Mediator and Cohesin. Lastly, the master and pioneer transcription factors were essential to recruit Mediator and Cohesin to regulatory regions of actively transcribed genes. Our study proposes that maintenance of the cancer cell state is dependent on recruitment of Mediator and Cohesin through FOXA and master transcription factors.

  18. FOXA and master transcription factors recruit Mediator and Cohesin to the core transcriptional regulatory circuitry of cancer cells

    PubMed Central

    Fournier, Michèle; Bourriquen, Gaëlle; Lamaze, Fabien C.; Côté, Maxime C.; Fournier, Éric; Joly-Beauparlant, Charles; Caron, Vicky; Gobeil, Stéphane; Droit, Arnaud; Bilodeau, Steve

    2016-01-01

    Controlling the transcriptional program is essential to maintain the identity and the biological functions of a cell. The Mediator and Cohesin complexes have been established as central cofactors controlling the transcriptional program in normal cells. However, the distribution, recruitment and importance of these complexes in cancer cells have not been fully investigated. Here we show that FOXA and master transcription factors are part of the core transcriptional regulatory circuitry of cancer cells and are essential to recruit M ediator and Cohesin. Indeed, Mediator and Cohesin occupied the enhancer and promoter regions of actively transcribed genes and maintained the proliferation and colony forming potential. Through integration of publically available ChIP-Seq datasets, we predicted the core transcriptional regulatory circuitry of each cancer cell. Unexpectedly, for all cells investigated, the pioneer transcription factors FOXA1 and/or FOXA2 were identified in addition to cell-specific master transcription factors. Loss of both types of transcription factors phenocopied the loss of Mediator and Cohesin. Lastly, the master and pioneer transcription factors were essential to recruit Mediator and Cohesin to regulatory regions of actively transcribed genes. Our study proposes that maintenance of the cancer cell state is dependent on recruitment of Mediator and Cohesin through FOXA and master transcription factors. PMID:27739523

  19. A Novel Acetivibrio cellulolyticus Anchoring Scaffoldin That Bears Divergent Cohesins

    PubMed Central

    Xu, Qi; Barak, Yoav; Kenig, Rina; Shoham, Yuval; Bayer, Edward A.; Lamed, Raphael

    2004-01-01

    Sequencing of a cellulosome-integrating gene cluster in Acetivibrio cellulolyticus was completed. The cluster contains four tandem scaffoldin genes (scaA, scaB, scaC, and scaD) bounded upstream and downstream, respectively, by a presumed cellobiose phosphorylase and a nucleotide methylase. The sequences and properties of scaA, scaB, and scaC were reported previously, and those of scaD are reported here. The scaD gene encodes an 852-residue polypeptide that includes a signal peptide, three cohesins, and a C-terminal S-layer homology (SLH) module. The calculated molecular weight of the mature ScaD is 88,960; a 67-residue linker segment separates cohesins 1 and 2, and two ∼30-residue linkers separate cohesin 2 from 3 and cohesin 3 from the SLH module. The presence of an SLH module in ScaD indicates its role as an anchoring protein. The first two ScaD cohesins can be classified as type II, similar to the four cohesins of ScaB. Surprisingly, the third ScaD cohesin belongs to the type I cohesins, like the seven ScaA cohesins. ScaD is the first scaffoldin to be described that contains divergent types of cohesins as integral parts of the polypeptide chain. The recognition properties among selected recombinant cohesins and dockerins from the different scaffoldins of the gene cluster were investigated by affinity blotting. The results indicated that the divergent types of ScaD cohesins also differ in their preference of dockerins. ScaD thus plays a dual role, both as a primary scaffoldin, capable of direct incorporation of a single dockerin-borne enzyme, and as a secondary scaffoldin that anchors the major primary scaffoldin, ScaA and its complement of enzymes to the cell surface. PMID:15317783

  20. A Scaffoldin of the Bacteroides cellulosolvens Cellulosome That Contains 11 Type II Cohesins

    PubMed Central

    Ding, Shi-You; Bayer, Edward A.; Steiner, David; Shoham, Yuval; Lamed, Raphael

    2000-01-01

    A cellulosomal scaffoldin gene, termed cipBc, was identified and sequenced from the mesophilic cellulolytic anaerobe Bacteroides cellulosolvens. The gene encodes a 2,292-residue polypeptide (excluding the signal sequence) with a calculated molecular weight of 242,437. CipBc contains an N-terminal signal peptide, 11 type II cohesin domains, an internal family III cellulose-binding domain (CBD), and a C-terminal dockerin domain. Its CBD belongs to family IIIb, like that of CipV from Acetivibrio cellulolyticus but unlike the family IIIa CBDs of other clostridial scaffoldins. In contrast to all other scaffoldins thus far described, CipBc lacks a hydrophilic domain or domain X of unknown function. The singularity of CipBc, however, lies in its numerous type II cohesin domains, all of which are very similar in sequence. One of the latter cohesin domains was expressed, and the expressed protein interacted selectively with cellulosomal enzymes, one of which was identified as a family 48 glycosyl hydrolase on the basis of partial sequence alignment. By definition, the dockerins, carried by the cellulosomal enzymes of this species, would be considered to be type II. This is the first example of authentic type II cohesins that are confirmed components of a cellulosomal scaffoldin subunit rather than a cell surface anchoring component. The results attest to the emerging diversity of cellulosomes and their component sequences in nature. PMID:10940036

  1. Mutant Cohesin in Premature Ovarian Failure

    PubMed Central

    Overbeek, Paul A.; Barbero, Jose Luis; Oka, Kazuhiro; Harrison, Wilbur; Vaiman, Daniel; Ben-Neriah, Ziva; García-Tuñón, Ignacio; Fellous, Marc; Pendás, Alberto M.; Veitia, Reiner A.; Vilain, Eric

    2014-01-01

    Summary Premature ovarian failure is a major cause of female infertility. The genetic causes of this disorder remain unknown in most patients. Using whole-exome sequence analysis of a large consanguineous family with inherited premature ovarian failure, we identified a homozygous 1-bp deletion inducing a frameshift mutation in STAG3 on chromosome 7. STAG3 encodes a meiosis-specific subunit of the cohesin ring, which ensures correct sister chromatid cohesion. Female mice devoid of Stag3 are sterile, and their fetal oocytes are arrested at early prophase I, leading to oocyte depletion at 1 week of age. PMID:24597867

  2. HDAC8 Inhibition Blocks SMC3 Deacetylation and Delays Cell Cycle Progression without Affecting Cohesin-dependent Transcription in MCF7 Cancer Cells.

    PubMed

    Dasgupta, Tanushree; Antony, Jisha; Braithwaite, Antony W; Horsfield, Julia A

    2016-06-10

    Cohesin, a multi-subunit protein complex involved in chromosome organization, is frequently mutated or aberrantly expressed in cancer. Multiple functions of cohesin, including cell division and gene expression, highlight its potential as a novel therapeutic target. The SMC3 subunit of cohesin is acetylated (ac) during S phase to establish cohesion between replicated chromosomes. Following anaphase, ac-SMC3 is deacetylated by HDAC8. Reversal of SMC3 acetylation is imperative for recycling cohesin so that it can be reloaded in interphase for both non-mitotic and mitotic functions. We blocked deacetylation of ac-SMC3 using an HDAC8-specific inhibitor PCI-34051 in MCF7 breast cancer cells, and examined the effects on transcription of cohesin-dependent genes that respond to estrogen. HDAC8 inhibition led to accumulation of ac-SMC3 as expected, but surprisingly, had no influence on the transcription of estrogen-responsive genes that are altered by siRNA targeting of RAD21 or SMC3. Knockdown of RAD21 altered estrogen receptor α (ER) recruitment at SOX4 and IL20, and affected transcription of these genes, while HDAC8 inhibition did not. Rather, inhibition of HDAC8 delayed cell cycle progression, suppressed proliferation and induced apoptosis in a concentration-dependent manner. We conclude that HDAC8 inhibition does not change the estrogen-specific transcriptional role of cohesin in MCF7 cells, but instead, compromises cell cycle progression and cell survival. Our results argue that candidate inhibitors of cohesin function may differ in their effects depending on the cellular genotype and should be thoroughly tested for predicted effects on cohesin's mechanistic roles.

  3. Lampbrush chromosomes enable study of cohesin dynamics.

    PubMed

    Austin, Christopher; Novikova, Natalya; Guacci, Vincent; Bellini, Michel

    2009-01-01

    The lampbrush chromosomes present in the nuclei of amphibian oocytes offer unique biological approaches for study of the mechanisms that regulate chromatin structure with high spatial resolution. We discuss fundamental aspects of the remarkable organization and plasticity exhibited by lampbrush chromosomes. We then utilize lampbrush chromosomes to characterize the chromosomal distribution and dynamics of cohesin, the four-protein complex (RAD21/MCD1/SCC1, SMC1, SMC3, SCC3/SA2) responsible for sister chromatid cohesion. We find that endogenous SMC3 and newly expressed hRAD21 co-localize on chromosomal axes, sites where sister chromatids are tightly paired. We present evidence suggesting that hRAD21 recruitment to lampbrush chromosomes is modulated by chromosomal SMC1 and SMC3. Notably, using a technique for de novo chromosome assembly, we demonstrate that both SMC3 and hRAD21 are recruited to single, unreplicated lampbrush chromatids. Finally, we used our novel method of analyzing the oocyte nucleus under oil combined with fluorescence recovery after photobleaching, to provide direct evidence that cohesin is highly dynamic at discrete, condensed chromosomal regions. Collectively, these data demonstrate that lampbrush chromosomes provide a unique and powerful tool for combining biochemical and cytological analyses for dissection of complex chromosomal processes.

  4. CTCF and CohesinSA-1 Mark Active Promoters and Boundaries of Repressive Chromatin Domains in Primary Human Erythroid Cells

    PubMed Central

    Steiner, Laurie A.; Schulz, Vincent; Makismova, Yelena; Lezon-Geyda, Kimberly; Gallagher, Patrick G.

    2016-01-01

    Background CTCF and cohesinSA-1 are regulatory proteins involved in a number of critical cellular processes including transcription, maintenance of chromatin domain architecture, and insulator function. To assess changes in the CTCF and cohesinSA-1 interactomes during erythropoiesis, chromatin immunoprecipitation coupled with high throughput sequencing and mRNA transcriptome analyses via RNA-seq were performed in primary human hematopoietic stem and progenitor cells (HSPC) and primary human erythroid cells from single donors. Results Sites of CTCF and cohesinSA-1 co-occupancy were enriched in gene promoters in HSPC and erythroid cells compared to single CTCF or cohesin sites. Cell type-specific CTCF sites in erythroid cells were linked to highly expressed genes, with the opposite pattern observed in HSPCs. Chromatin domains were identified by ChIP-seq with antibodies against trimethylated lysine 27 histone H3, a modification associated with repressive chromatin. Repressive chromatin domains increased in both number and size during hematopoiesis, with many more repressive domains in erythroid cells than HSPCs. CTCF and cohesinSA-1 marked the boundaries of these repressive chromatin domains in a cell-type specific manner. Conclusion These genome wide data, changes in sites of protein occupancy, chromatin architecture, and related gene expression, support the hypothesis that CTCF and cohesinSA-1 have multiple roles in the regulation of gene expression during erythropoiesis including transcriptional regulation at gene promoters and maintenance of chromatin architecture. These data from primary human erythroid cells provide a resource for studies of normal and perturbed erythropoiesis. PMID:27219007

  5. Cohesin organizes chromatin loops at DNA replication factories

    PubMed Central

    Guillou, Emmanuelle; Ibarra, Arkaitz; Coulon, Vincent; Casado-Vela, Juan; Rico, Daniel; Casal, Ignacio; Schwob, Etienne; Losada, Ana; Méndez, Juan

    2010-01-01

    Genomic DNA is packed in chromatin fibers organized in higher-order structures within the interphase nucleus. One level of organization involves the formation of chromatin loops that may provide a favorable environment to processes such as DNA replication, transcription, and repair. However, little is known about the mechanistic basis of this structuration. Here we demonstrate that cohesin participates in the spatial organization of DNA replication factories in human cells. Cohesin is enriched at replication origins and interacts with prereplication complex proteins. Down-regulation of cohesin slows down S-phase progression by limiting the number of active origins and increasing the length of chromatin loops that correspond with replicon units. These results give a new dimension to the role of cohesin in the architectural organization of interphase chromatin, by showing its participation in DNA replication. PMID:21159821

  6. Cohesin controls planar cell polarity by regulating the level of the seven-pass transmembrane cadherin Flamingo.

    PubMed

    Mouri, Kousuke; Horiuchi, Shin-ya; Uemura, Tadashi

    2012-06-01

    Planar cell polarity (PCP) refers to the coordination of global organ axes and individual cell polarity in vertebrate and invertebrate epithelia. Mechanisms of PCP have been best studied in the Drosophila wing, in which each epidermal cell produces a single wing hair at the distal cell edge, and this spatial specification is mediated by redistribution of the core group proteins, including the seven-pass transmembrane cadherin Flamingo/Starry night (Fmi/Stan), to selective plasma membrane domains. Through genetic screening, we found that a mutation of the SMC3 gene caused dramatic misspecification of wing hair positions. SMC3 protein is one subunit of the cohesin complex, which regulates sister chromatid cohesion and also plays a role in transcriptional control of gene expression. In the SMC3 mutant cells, Fmi appeared to be upregulated by a posttranscriptional mechanism(s), and this elevation of Fmi was at least one cause of the PCP defect. In addition to the PCP phenotype, the loss of the cohesin function affected wing morphogenesis at multiple levels: one malformation was loss of the wing margin, and this was most likely a result of downregulation of the homeodomain protein Cut. At the cellular level, apical cell size and hexagonal packing were affected in the mutant wing. Dysfunction of cohesin in humans results in Cornelia de Lange syndrome (CdLS), which is characterized by various developmental abnormalities and mental retardation. Our analysis of cohesin in epithelia may provide new insight into cellular and molecular mechanisms of CdLS.

  7. Polo kinase Cdc5 associates with centromeres to facilitate the removal of centromeric cohesin during mitosis

    PubMed Central

    Mishra, Prashant K.; Ciftci-Yilmaz, Sultan; Reynolds, David; Au, Wei-Chun; Boeckmann, Lars; Dittman, Lauren E.; Jowhar, Ziad; Pachpor, Tejaswini; Yeh, Elaine; Baker, Richard E.; Hoyt, M. Andrew; D’Amours, Damien; Bloom, Kerry; Basrai, Munira A.

    2016-01-01

    Sister chromatid cohesion is essential for tension-sensing mechanisms that monitor bipolar attachment of replicated chromatids in metaphase. Cohesion is mediated by the association of cohesins along the length of sister chromatid arms. In contrast, centromeric cohesin generates intrastrand cohesion and sister centromeres, while highly cohesin enriched, are separated by >800 nm at metaphase in yeast. Removal of cohesin is necessary for sister chromatid separation during anaphase, and this is regulated by evolutionarily conserved polo-like kinase (Cdc5 in yeast, Plk1 in humans). Here we address how high levels of cohesins at centromeric chromatin are removed. Cdc5 associates with centromeric chromatin and cohesin-associated regions. Maximum enrichment of Cdc5 in centromeric chromatin occurs during the metaphase-to-anaphase transition and coincides with the removal of chromosome-associated cohesin. Cdc5 interacts with cohesin in vivo, and cohesin is required for association of Cdc5 at centromeric chromatin. Cohesin removal from centromeric chromatin requires Cdc5 but removal at distal chromosomal arm sites does not. Our results define a novel role for Cdc5 in regulating removal of centromeric cohesins and faithful chromosome segregation. PMID:27226485

  8. ChromoShake: a chromosome dynamics simulator reveals that chromatin loops stiffen centromeric chromatin

    PubMed Central

    Lawrimore, Josh; Aicher, Joseph K.; Hahn, Patrick; Fulp, Alyona; Kompa, Ben; Vicci, Leandra; Falvo, Michael; Taylor, Russell M.; Bloom, Kerry

    2016-01-01

    ChromoShake is a three-dimensional simulator designed to find the thermodynamically favored states for given chromosome geometries. The simulator has been applied to a geometric model based on experimentally determined positions and fluctuations of DNA and the distribution of cohesin and condensin in the budding yeast centromere. Simulations of chromatin in differing initial configurations reveal novel principles for understanding the structure and function of a eukaryotic centromere. The entropic position of DNA loops mirrors their experimental position, consistent with their radial displacement from the spindle axis. The barrel-like distribution of cohesin complexes surrounding the central spindle in metaphase is a consequence of the size of the DNA loops within the pericentromere to which cohesin is bound. Linkage between DNA loops of different centromeres is requisite to recapitulate experimentally determined correlations in DNA motion. The consequences of radial loops and cohesin and condensin binding are to stiffen the DNA along the spindle axis, imparting an active function to the centromere in mitosis. PMID:26538024

  9. Chromosome Cohesion Established by Rec8-Cohesin in Fetal Oocytes Is Maintained without Detectable Turnover in Oocytes Arrested for Months in Mice.

    PubMed

    Burkhardt, Sabrina; Borsos, Máté; Szydlowska, Anna; Godwin, Jonathan; Williams, Suzannah A; Cohen, Paula E; Hirota, Takayuki; Saitou, Mitinori; Tachibana-Konwalski, Kikuë

    2016-03-07

    Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation in mitosis and meiosis [1]. Rec8-containing cohesin, bound to Smc3/Smc1α or Smc3/Smc1β, maintains bivalent cohesion in mammalian meiosis [2-6]. In females, meiotic DNA replication and recombination occur in fetal oocytes. After birth, oocytes arrest at the prolonged dictyate stage until recruited to grow into mature oocytes that divide at ovulation. How cohesion is maintained in arrested oocytes remains a pivotal question relevant to maternal age-related aneuploidy. Hypothetically, cohesin turnover regenerates cohesion in oocytes. Evidence for post-replicative cohesion establishment mechanism exists, in yeast and invertebrates [7, 8]. In mouse fetal oocytes, cohesin loading factor Nipbl/Scc2 localizes to chromosome axes during recombination [9, 10]. Alternatively, cohesion is maintained without turnover. Consistent with this, cohesion maintenance does not require Smc1β transcription, but unlike Rec8, Smc1β is not required for establishing bivalent cohesion [11, 12]. Rec8 maintains cohesion without turnover during weeks of oocyte growth [3]. Whether the same applies to months or decades of arrest is unknown. Here, we test whether Rec8 activated in arrested mouse oocytes builds cohesion revealed by TEV cleavage and live-cell imaging. Rec8 establishes cohesion when activated during DNA replication in fetal oocytes using tamoxifen-inducible Cre. In contrast, no new cohesion is detected when Rec8 is activated in arrested oocytes by tamoxifen despite cohesin synthesis. We conclude that cohesion established in fetal oocytes is maintained for months without detectable turnover in dictyate-arrested oocytes. This implies that women's fertility depends on the longevity of cohesin proteins that established cohesion in utero.

  10. Proteomics Analysis with a Nano Random Forest Approach Reveals Novel Functional Interactions Regulated by SMC Complexes on Mitotic Chromosomes*

    PubMed Central

    Ohta, Shinya; Montaño-Gutierrez, Luis F.; de Lima Alves, Flavia; Ogawa, Hiromi; Toramoto, Iyo; Sato, Nobuko; Morrison, Ciaran G.; Takeda, Shunichi; Hudson, Damien F.; Earnshaw, William C.

    2016-01-01

    Packaging of DNA into condensed chromosomes during mitosis is essential for the faithful segregation of the genome into daughter nuclei. Although the structure and composition of mitotic chromosomes have been studied for over 30 years, these aspects are yet to be fully elucidated. Here, we used stable isotope labeling with amino acids in cell culture to compare the proteomes of mitotic chromosomes isolated from cell lines harboring conditional knockouts of members of the condensin (SMC2, CAP-H, CAP-D3), cohesin (Scc1/Rad21), and SMC5/6 (SMC5) complexes. Our analysis revealed that these complexes associate with chromosomes independently of each other, with the SMC5/6 complex showing no significant dependence on any other chromosomal proteins during mitosis. To identify subtle relationships between chromosomal proteins, we employed a nano Random Forest (nanoRF) approach to detect protein complexes and the relationships between them. Our nanoRF results suggested that as few as 113 of 5058 detected chromosomal proteins are functionally linked to chromosome structure and segregation. Furthermore, nanoRF data revealed 23 proteins that were not previously suspected to have functional interactions with complexes playing important roles in mitosis. Subsequent small-interfering-RNA-based validation and localization tracking by green fluorescent protein-tagging highlighted novel candidates that might play significant roles in mitotic progression. PMID:27231315

  11. Open chromatin reveals the functional maize genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Every cellular process mediated through nuclear DNA must contend with chromatin. As results from ENCODE show, open chromatin assays can efficiently integrate across diverse regulatory elements, revealing functional non-coding genome. In this study, we use a MNase hypersensitivity assay to discover o...

  12. RSC facilitates Rad59-dependent homologous recombination between sister chromatids by promoting cohesin loading at DNA double-strand breaks.

    PubMed

    Oum, Ji-Hyun; Seong, Changhyun; Kwon, Youngho; Ji, Jae-Hoon; Sid, Amy; Ramakrishnan, Sreejith; Ira, Grzegorz; Malkova, Anna; Sung, Patrick; Lee, Sang Eun; Shim, Eun Yong

    2011-10-01

    Homologous recombination repairs DNA double-strand breaks by searching for, invading, and copying information from a homologous template, typically the homologous chromosome or sister chromatid. Tight wrapping of DNA around histone octamers, however, impedes access of repair proteins to DNA damage. To facilitate DNA repair, modifications of histones and energy-dependent remodeling of chromatin are required, but the precise mechanisms by which chromatin modification and remodeling enzymes contribute to homologous DNA repair are unknown. Here we have systematically assessed the role of budding yeast RSC (remodel structure of chromatin), an abundant, ATP-dependent chromatin-remodeling complex, in the cellular response to spontaneous and induced DNA damage. RSC physically interacts with the recombination protein Rad59 and functions in homologous recombination. Multiple recombination assays revealed that RSC is uniquely required for recombination between sister chromatids by virtue of its ability to recruit cohesin at DNA breaks and thereby promoting sister chromatid cohesion. This study provides molecular insights into how chromatin remodeling contributes to DNA repair and maintenance of chromatin fidelity in the face of DNA damage.

  13. Checks and balances between cohesin and polycomb in gene silencing and transcription.

    PubMed

    Dorsett, Dale; Kassis, Judith A

    2014-06-02

    The cohesin protein complex was discovered for its roles in sister chromatid cohesion and segregation, and the Polycomb group (PcG) proteins for their roles in epigenetic gene silencing during development. Cohesin also controls gene transcription via multiple mechanisms. Genetic and molecular evidence from Drosophila argue that cohesin and the PRC1 PcG complex interact to control transcription of many active genes that are critical for development, and that via these interactions cohesin also controls the availability of PRC1 for gene silencing.

  14. Open chromatin reveals the functional maize genome

    PubMed Central

    Rodgers-Melnick, Eli; Vera, Daniel L.; Bass, Hank W.

    2016-01-01

    Cellular processes mediated through nuclear DNA must contend with chromatin. Chromatin structural assays can efficiently integrate information across diverse regulatory elements, revealing the functional noncoding genome. In this study, we use a differential nuclease sensitivity assay based on micrococcal nuclease (MNase) digestion to discover open chromatin regions in the maize genome. We find that maize MNase-hypersensitive (MNase HS) regions localize around active genes and within recombination hotspots, focusing biased gene conversion at their flanks. Although MNase HS regions map to less than 1% of the genome, they consistently explain a remarkably large amount (∼40%) of heritable phenotypic variance in diverse complex traits. MNase HS regions are therefore on par with coding sequences as annotations that demarcate the functional parts of the maize genome. These results imply that less than 3% of the maize genome (coding and MNase HS regions) may give rise to the overwhelming majority of phenotypic variation, greatly narrowing the scope of the functional genome. PMID:27185945

  15. Mutations in Cohesin Complex Members SMC3 and SMC1A Cause a Mild Variant of Cornelia de Lange Syndrome with Predominant Mental Retardation

    PubMed Central

    Deardorff, Matthew A.; Kaur, Maninder; Yaeger, Dinah; Rampuria, Abhinav; Korolev, Sergey; Pie, Juan; Gil-Rodríguez, Concepcion; Arnedo, María; Loeys, Bart; Kline, Antonie D.; Wilson, Meredith; Lillquist, Kaj; Siu, Victoria; Ramos, Feliciano J.; Musio, Antonio; Jackson, Laird S.; Dorsett, Dale; Krantz, Ian D.

    2007-01-01

    Mutations in the cohesin regulators NIPBL and ESCO2 are causative of the Cornelia de Lange syndrome (CdLS) and Roberts or SC phocomelia syndrome, respectively. Recently, mutations in the cohesin complex structural component SMC1A have been identified in two probands with features of CdLS. Here, we report the identification of a mutation in the gene encoding the complementary subunit of the cohesin heterodimer, SMC3, and 14 additional SMC1A mutations. All mutations are predicted to retain an open reading frame, and no truncating mutations were identified. Structural analysis of the mutant SMC3 and SMC1A proteins indicate that all are likely to produce functional cohesin complexes, but we posit that they may alter their chromosome binding dynamics. Our data indicate that SMC3 and SMC1A mutations (1) contribute to ∼5% of cases of CdLS, (2) result in a consistently mild phenotype with absence of major structural anomalies typically associated with CdLS, and (3) in some instances, result in a phenotype that approaches that of apparently nonsyndromic mental retardation. PMID:17273969

  16. The roles of cohesins in mitosis, meiosis, and human health and disease.

    PubMed

    Brooker, Amanda S; Berkowitz, Karen M

    2014-01-01

    Mitosis and meiosis are essential processes that occur during development. Throughout these processes, cohesion is required to keep the sister chromatids together until their separation at anaphase. Cohesion is created by multiprotein subunit complexes called cohesins. Although the subunits differ slightly in mitosis and meiosis, the canonical cohesin complex is composed of four subunits that are quite diverse. The cohesin complexes are also important for DNA repair, gene expression, development, and genome integrity. Here we provide an overview of the roles of cohesins during these different events as well as their roles in human health and disease, including the cohesinopathies. Although the exact roles and mechanisms of these proteins are still being elucidated, this review serves as a guide for the current knowledge of cohesins.

  17. Structural Basis of Eco1-Mediated Cohesin Acetylation

    PubMed Central

    Chao, William C. H.; Wade, Benjamin O.; Bouchoux, Céline; Jones, Andrew W.; Purkiss, Andrew G.; Federico, Stefania; O’Reilly, Nicola; Snijders, Ambrosius P.; Uhlmann, Frank; Singleton, Martin R.

    2017-01-01

    Sister-chromatid cohesion is established by Eco1-mediated acetylation on two conserved tandem lysines in the cohesin Smc3 subunit. However, the molecular basis of Eco1 substrate recognition and acetylation in cohesion is not fully understood. Here, we discover and rationalize the substrate specificity of Eco1 using mass spectrometry coupled with in-vitro acetylation assays and crystallography. Our structures of the X. laevis Eco2 (xEco2) bound to its primary and secondary Smc3 substrates demonstrate the plasticity of the substrate-binding site, which confers substrate specificity by concerted conformational changes of the central β hairpin and the C-terminal extension. PMID:28290497

  18. Topology and structure of an engineered human cohesin complex bound to Pds5B

    PubMed Central

    Hons, Michael T.; Huis in ‘t Veld, Pim J.; Kaesler, Jan; Rombaut, Pascaline; Schleiffer, Alexander; Herzog, Franz; Stark, Holger; Peters, Jan-Michael

    2016-01-01

    The cohesin subunits Smc1, Smc3 and Scc1 form large tripartite rings which mediate sister chromatid cohesion and chromatin structure. These are thought to entrap DNA with the help of the associated proteins SA1/2 and Pds5A/B. Structural information is available for parts of cohesin, but analyses of entire cohesin complexes are limited by their flexibility. Here we generated a more rigid ‘bonsai' cohesin by truncating the coiled coils of Smc1 and Smc3 and used single-particle electron microscopy, chemical crosslinking-mass spectrometry and in silico modelling to generate three-dimensional models of cohesin bound to Pds5B. The HEAT-repeat protein Pds5B forms a curved structure around the nucleotide-binding domains of Smc1 and Smc3 and bridges the Smc3-Scc1 and SA1-Scc1 interfaces. These results indicate that Pds5B forms an integral part of the cohesin ring by contacting all other cohesin subunits, a property that may reflect the complex role of Pds5 proteins in controlling cohesin–DNA interactions. PMID:27549742

  19. The cohesin complex prevents the end-joining of distant DNA double-strand ends in S phase: Consequences on genome stability maintenance.

    PubMed

    Gelot, Camille; Guirouilh-Barbat, Josée; Lopez, Bernard S

    2016-07-03

    DNA double-strand break (DSB) repair is essential for genome stability maintenance, but the joining of distant DNA double strand ends (DSEs) inevitably leads to genome rearrangements. Therefore, DSB repair should be tightly controlled to secure genome stability while allowing genetic variability. Tethering of the proximal ends of a 2-ended DSB limits their mobility, protecting thus against their joining with a distant DSE. However, replication stress generates DSBs with only one DSE, on which tethering is impossible. Consistently, we demonstrated that the joining of 2 DSBs only 3.2 kb apart is repressed in the S, but not the G1, phase, revealing an additional mechanism limiting DNA ends mobility in S phase. The cohesin complex, by maintaining the 2 sister chromatids linked, limits DSEs mobility and thus represses the joining of distant DSEs, while allowing that of adjacent DSEs. At the genome scale, the cohesin complex protects against deletions, inversions, translocations and chromosome fusion.

  20. A cohesin-based structural platform supporting homologous chromosome pairing in meiosis.

    PubMed

    Ding, Da-Qiao; Haraguchi, Tokuko; Hiraoka, Yasushi

    2016-08-01

    The pairing and recombination of homologous chromosomes during the meiotic prophase is necessary for the accurate segregation of chromosomes in meiosis. However, the mechanism by which homologous chromosomes achieve this pairing has remained an open question. Meiotic cohesins have been shown to affect chromatin compaction; however, the impact of meiotic cohesins on homologous pairing and the fine structures of cohesion-based chromatin remain to be determined. A recent report using live-cell imaging and super-resolution microscopy demonstrated that the lack of meiotic cohesins alters the chromosome axis structures and impairs the pairing of homologous chromosomes. These results suggest that meiotic cohesin-based chromosome axis structures are crucial for the pairing of homologous chromosomes.

  1. Recruitment of the cohesin loading factor NIPBL to DNA double-strand breaks depends on MDC1, RNF168 and HP1{gamma} in human cells

    SciTech Connect

    Oka, Yasuyoshi; Suzuki, Keiji; Yamauchi, Motohiro; Mitsutake, Norisato; Yamashita, Shunichi

    2011-08-12

    Highlights: {yields} NIPBL is recruited to DSBs. {yields} Localization of NIPBL to DSBs is regulated by MDC1 and RNF168. {yields} HP1{gamma} is required for NIPBL accumulation at DSBs. -- Abstract: The cohesin loading factor NIPBL is required for cohesin to associate with chromosomes and plays a role in DNA double-strand break (DSB) repair. Although the NIPBL homolog Scc2 is recruited to an enzymatically generated DSB and promotes cohesin-dependent DSB repair in yeast, the mechanism of the recruitment remains poorly understood. Here we show that the human NIPBL is recruited to the sites of DNA damage generated by micro-irradiation as well as to the sites of DSBs induced by homing endonuclease, I-PpoI. The recruitment of NIPBL was impaired by RNAi-mediated knockdown of MDC1 or RNF168, both of which also accumulate at DSBs. We also show that the recruitment of NIPBL to the sites of DNA damage is mediated by its C-terminal region containing HEAT repeats and Heterochromatin protein 1 (HP1) interacting motif. Furthermore, NIPBL accumulation at damaged sites was also compromised by HP1{gamma} depletion. Taken together, our study reveals that human NIPBL is a novel protein recruited to DSB sites, and the recruitment is controlled by MDC1, RNF168 and HP1{gamma}.

  2. Structure of cohesin subcomplex pinpoints direct shugoshin–Wapl antagonism in centromeric cohesion

    PubMed Central

    Hara, Kodai; Zheng, Ge; Qu, Qianhui; Liu, Hong; Ouyang, Zhuqing; Chen, Zhe; Tomchick, Diana R.; Yu, Hongtao

    2014-01-01

    Orderly termination of sister-chromatid cohesion during mitosis is critical for accurate chromosome segregation. During prophase, mitotic kinases phosphorylate cohesin and its protector sororin, triggering Wapl-dependent cohesin release from chromosome arms. The shugoshin (Sgo1)–PP2A complex protects centromeric cohesin until its cleavage by separase at anaphase onset. Here, we report the crystal structure of a human cohesin subcomplex comprising SA2 and Scc1. Multiple HEAT repeats of SA2 form a dragon-shaped structure. Scc1 makes extensive contacts with SA2, with one binding hotspot. Sgo1 and Wapl compete for binding to a conserved site on SA2–Scc1. Mutations of SA2 residues at this site that disrupt Wapl binding bypass Sgo1 requirement in cohesion protection. Thus, besides recruiting PP2A to dephosphorylate cohesin and sororin, Sgo1 physically shields cohesin from Wapl. This unexpected, direct antagonism between Sgo1 and Wapl augments centromeric cohesion protection. PMID:25173175

  3. Identification and Characterization of Sa/Scc3p Subunits in the Xenopus and Human Cohesin Complexes

    PubMed Central

    Losada, Ana; Yokochi, Tomoki; Kobayashi, Ryuji; Hirano, Tatsuya

    2000-01-01

    A multisubunit protein complex, termed cohesin, plays an essential role in sister chromatid cohesion in yeast and in Xenopus laevis cell-free extracts. We report here that two distinct cohesin complexes exist in Xenopus egg extracts. A 14S complex (x-cohesinSA1) contains XSMC1, XSMC3, XRAD21, and a newly identified subunit, XSA1. In a second 12.5S complex (x-cohesinSA2), XSMC1, XSMC3, and XRAD21 associate with a different subunit, XSA2. Both XSA1 and XSA2 belong to the SA family of mammalian proteins and exhibit similarity to Scc3p, a recently identified component of yeast cohesin. In Xenopus egg extracts, x-cohesinSA1 is predominant, whereas x-cohesinSA2 constitutes only a very minor population. Human cells have a similar pair of cohesin complexes, but the SA2-type is the dominant form in somatic tissue culture cells. Immunolocalization experiments suggest that chromatin association of cohesinSA1 and cohesinSA2 may be differentially regulated. Dissociation of x-cohesinSA1 from chromatin correlates with phosphorylation of XSA1 in the cell-free extracts. Purified cdc2-cyclin B can phosphorylate XSA1 in vitro and reduce the ability of x-cohesinSA1 to bind to DNA or chromatin. These results shed light on the mechanism by which sister chromatid cohesion is partially dissolved in early mitosis, far before the onset of anaphase, in vertebrate cells. PMID:10931856

  4. Meiotic cohesin subunits RAD21L and REC8 are positioned at distinct regions between lateral elements and transverse filaments in the synaptonemal complex of mouse spermatocytes

    PubMed Central

    RONG, Mei; MATSUDA, Atsushi; HIRAOKA, Yasushi; LEE, Jibak

    2016-01-01

    Cohesins containing a meiosis-specific α-kleisin subunit, RAD21L or REC8, play roles in diverse aspects of meiotic chromosome dynamics including formation of axial elements (AEs), assembly of the synaptonemal complex (SC), recombination of homologous chromosomes (homologs), and cohesion of sister chromatids. However, the exact functions of individual α-kleisins remain to be elucidated. Here, we examined the localization of RAD21L and REC8 within the SC by super-resolution microscopy, 3D-SIM. We found that both RAD21L and REC8 were localized at the connection sites between lateral elements (LEs) and transverse filaments (TFs) of pachynema with RAD21L locating interior to REC8 sites. RAD21L and REC8 were not symmetrical in terms of synaptic homologs, suggesting that the arrangement of different cohesins is not strictly fixed along all chromosome axes. Intriguingly, some RAD21L signals, but not REC8 signals, were observed between unsynapsed regions of AEs of zygonema as if they formed a bridge between homologs. Furthermore, the signals of recombination intermediates overlapped with those of RAD21L to a greater degree than with those of REC8. These results highlight the different properties of two meiotic α-kleisins, and strongly support the previous proposition that RAD21L is an atypical cohesin that establishes the association between homologs rather than sister chromatids. PMID:27665783

  5. Revealing neuronal function through microelectrode array recordings

    PubMed Central

    Obien, Marie Engelene J.; Deligkaris, Kosmas; Bullmann, Torsten; Bakkum, Douglas J.; Frey, Urs

    2015-01-01

    Microelectrode arrays and microprobes have been widely utilized to measure neuronal activity, both in vitro and in vivo. The key advantage is the capability to record and stimulate neurons at multiple sites simultaneously. However, unlike the single-cell or single-channel resolution of intracellular recording, microelectrodes detect signals from all possible sources around every sensor. Here, we review the current understanding of microelectrode signals and the techniques for analyzing them. We introduce the ongoing advancements in microelectrode technology, with focus on achieving higher resolution and quality of recordings by means of monolithic integration with on-chip circuitry. We show how recent advanced microelectrode array measurement methods facilitate the understanding of single neurons as well as network function. PMID:25610364

  6. Revealing remodeler function: Varied and unique

    NASA Astrophysics Data System (ADS)

    Eastlund, Allen

    Chromatin remodelers perform a necessary and required function for the successful expression of our genetic code. By modifying, shifting, or ejecting nucleosomes from the chromatin structure they allow access to the underlying DNA to the rest of the cell's machinery. This research has focused on two major remodeler motors from major families of chromatin remodelers: the trimeric motor domain of RSC and the motor domain of the ISWI family, ISWI. Using primarily stopped-flow spectrofluorometry, I have categorized the time-dependent motions of these motor domains along their preferred substrate, double-stranded DNA. Combined with collected ATP utilization data, I present the subsequent analysis and associated conclusions that stem from the underlying assumptions and models. Interestingly, there is little in common between the investigated proteins aside from their favored medium. While RSC exhibits modest translocation characteristics and highly effective motion with the ability for large molecular forces, ISWI is not only structurally different but highly inefficient in its motion leading to difficulties in determining its specific translocation mechanics. While chromatin remodeling is a ubiquitous facet of eukaryotic life, there remains much to be understood about their general mechanisms.

  7. SMC1B is present in mammalian somatic cells and interacts with mitotic cohesin proteins

    PubMed Central

    Mannini, Linda; Cucco, Francesco; Quarantotti, Valentina; Amato, Clelia; Tinti, Mara; Tana, Luigi; Frattini, Annalisa; Delia, Domenico; Krantz, Ian D.; Jessberger, Rolf; Musio, Antonio

    2015-01-01

    Cohesin is an evolutionarily conserved protein complex that plays a role in many biological processes: it ensures faithful chromosome segregation, regulates gene expression and preserves genome stability. In mammalian cells, the mitotic cohesin complex consists of two structural maintenance of chromosome proteins, SMC1A and SMC3, the kleisin protein RAD21 and a fourth subunit either STAG1 or STAG2. Meiotic paralogs in mammals were reported for SMC1A, RAD21 and STAG1/STAG2 and are called SMC1B, REC8 and STAG3 respectively. It is believed that SMC1B is only a meiotic-specific cohesin member, required for sister chromatid pairing and for preventing telomere shortening. Here we show that SMC1B is also expressed in somatic mammalian cells and is a member of a mitotic cohesin complex. In addition, SMC1B safeguards genome stability following irradiation whereas its ablation has no effect on chromosome segregation. Finally, unexpectedly SMC1B depletion impairs gene transcription, particularly at genes mapping to clusters such as HOX and PCDHB. Genome-wide analyses show that cluster genes changing in expression are enriched for cohesin-SMC1B binding. PMID:26673124

  8. Single versus dual-binding conformations in cellulosomal cohesin-dockerin complexes.

    PubMed

    Nash, Michael A; Smith, Steven P; Fontes, Carlos Mga; Bayer, Edward A

    2016-10-01

    Cohesins and dockerins are complementary interacting protein modules that form stable and highly specific receptor-ligand complexes. They play a crucial role in the assembly of cellulose-degrading multi-enzyme complexes called cellulosomes and have potential applicability in several technology areas, including biomass conversion processes. Here, we describe several exceptional properties of cohesin-dockerin complexes, including their tenacious biochemical affinity, remarkably high mechanostability and a dual-binding mode of recognition that is contrary to the conventional lock-and-key model of receptor-ligand interactions. We focus on structural aspects of the dual mode of cohesin-dockerin binding, highlighting recent single-molecule analysis techniques for its explicit characterization.

  9. Role of Securin, Separase and Cohesins in female meiosis and polar body formation in Drosophila.

    PubMed

    Guo, Zhihao; Batiha, Osamah; Bourouh, Mohammed; Fifield, Eric; Swan, Andrew

    2016-02-01

    Chromosome segregation in meiosis is controlled by a conserved pathway that culminates in Separase-mediated cleavage of the α-kleisin Rec8, leading to dissolution of cohesin rings. Drosophila has no gene encoding Rec8, and the absence of a known Separase target raises the question of whether Separase and its regulator Securin (Pim in Drosophila) are important in Drosophila meiosis. Here, we investigate the role of Securin, Separase and the cohesin complex in female meiosis using fluorescence in situ hybridization against centromeric and arm-specific sequences to monitor cohesion. We show that Securin destruction and Separase activity are required for timely release of arm cohesion in anaphase I and centromere-proximal cohesion in anaphase II. They are also required for release of arm cohesion on polar body chromosomes. Cohesion on polar body chromosomes depends on the cohesin components SMC3 and the mitotic α-kleisin Rad21 (also called Vtd in Drosophila). We provide cytological evidence that SMC3 is required for arm cohesion in female meiosis, whereas Rad21, in agreement with recent findings, is not. We conclude that in Drosophila meiosis, cohesion is regulated by a conserved Securin-Separase pathway that targets a diverged Separase target, possibly within the cohesin complex.

  10. Key herbivores reveal limited functional redundancy on inshore coral reefs

    NASA Astrophysics Data System (ADS)

    Johansson, C. L.; van de Leemput, I. A.; Depczynski, M.; Hoey, A. S.; Bellwood, D. R.

    2013-12-01

    Marine ecosystems are facing increasing exposure to a range of stressors and declines in critical ecological functions. The likelihood of further loss of functions and resilience is dependent, in part, on the extent of functional redundancy (i.e. the capacity of one species to functionally compensate for the loss of another species) within critical functional groups. We used multiple metrics; species richness, generic richness, abundance and reserve capacity (i.e. the relative number of individuals available to fulfil the function if the numerically dominant species is lost), as indicators to assess the potential functional redundancy of four functional groups of herbivorous fishes (browsers, excavators, grazers and scrapers) in two of the worlds' most intact coral reef ecosystems: the Great Barrier Reef (GBR) and Ningaloo Reef in Western Australia. We found marked variations in potential redundancy among habitats within each reef system and functional groups. Despite negligible fishing of herbivorous fishes, coastal habitats in both reef systems had lower functional redundancy compared to offshore locations for all herbivorous fishes collectively and the four functional groups independently. This pattern was consistent in all four indicators of redundancy. The potential vulnerability of these coastal habitats is highlighted by recent shifts from coral to macroalgal dominance on several coastal reefs of the GBR. Our approach provides a simple yet revealing evaluation of potential functional redundancy. Moreover, it highlights the spatial variation in potential vulnerability and resilience of reef systems.

  11. Revealing quantum correlation by negativity of the Wigner function

    NASA Astrophysics Data System (ADS)

    Taghiabadi, Razieh; Akhtarshenas, Seyed Javad; Sarbishaei, Mohsen

    2016-05-01

    We analyze two two-mode continuous variable separable states with the same marginal states. We adopt the definition of classicality in the form of well-defined positive Wigner function describing the state and find that although the states possess positive local Wigner functions, they exhibit negative Wigner functions for the global states. Using the negativity of Wigner function as an indicator of nonclassicality, we show that despite these states possess different negativities of the Wigner function, they do not reveal this difference as phase space nonclassicalities such as negativity of the Mandel Q parameter or quadrature squeezing. We then concentrate on quantum correlation of these states and show that quantum discord and local quantum uncertainty, as two well-defined measures of quantum correlation, manifest the difference between negativity of the Wigner functions. The non-Gaussianity of these states is also examined and show that the difference in behavior of their non-Gaussianity is the same as the difference between negativity of their Wigner functions. We also investigate the influence of correlation rank criterion and find that when the states can be produced locally from classical states, the Wigner functions cannot reveal their quantum correlations.

  12. Sequence Analysis of Scaffolding Protein CipC and ORFXp, a New Cohesin-Containing Protein in Clostridium cellulolyticum: Comparison of Various Cohesin Domains and Subcellular Localization of ORFXp

    PubMed Central

    Pagès, Sandrine; Bélaïch, Anne; Fierobe, Henri-Pierre; Tardif, Chantal; Gaudin, Christian; Bélaïch, Jean-Pierre

    1999-01-01

    The gene encoding the scaffolding protein of the cellulosome from Clostridium cellulolyticum, whose partial sequence was published earlier (S. Pagès, A. Bélaïch, C. Tardif, C. Reverbel-Leroy, C. Gaudin, and J.-P. Bélaïch, J. Bacteriol. 178:2279–2286, 1996; C. Reverbel-Leroy, A. Bélaïch, A. Bernadac, C. Gaudin, J. P. Bélaïch, and C. Tardif, Microbiology 142:1013–1023, 1996), was completely sequenced. The corresponding protein, CipC, is composed of a cellulose binding domain at the N terminus followed by one hydrophilic domain (HD1), seven highly homologous cohesin domains (cohesin domains 1 to 7), a second hydrophilic domain, and a final cohesin domain (cohesin domain 8) which is only 57 to 60% identical to the seven other cohesin domains. In addition, a second gene located 8.89 kb downstream of cipC was found to encode a three-domain protein, called ORFXp, which includes a cohesin domain. By using antiserum raised against the latter, it was observed that ORFXp is associated with the membrane of C. cellulolyticum and is not detected in the cellulosome fraction. Western blot and BIAcore experiments indicate that cohesin domains 1 and 8 from CipC recognize the same dockerins and have similar affinity for CelA (Ka = 4.8 × 109 M−1) whereas the cohesin from ORFXp, although it is also able to bind all cellulosome components containing a dockerin, has a 19-fold lower Ka for CelA (2.6 × 108 M−1). Taken together, these data suggest that ORFXp may play a role in cellulosome assembly. PMID:10074072

  13. Genetic Interactions Between the Meiosis-Specific Cohesin Components, STAG3, REC8, and RAD21L

    PubMed Central

    Ward, Ayobami; Hopkins, Jessica; Mckay, Matthew; Murray, Steve; Jordan, Philip W.

    2016-01-01

    Cohesin is an essential structural component of chromosomes that ensures accurate chromosome segregation during mitosis and meiosis. Previous studies have shown that there are cohesin complexes specific to meiosis, required to mediate homologous chromosome pairing, synapsis, recombination, and segregation. Meiosis-specific cohesin complexes consist of two structural maintenance of chromosomes proteins (SMC1α/SMC1β and SMC3), an α-kleisin protein (RAD21, RAD21L, or REC8), and a stromal antigen protein (STAG1, 2, or 3). STAG3 is exclusively expressed during meiosis, and is the predominant STAG protein component of cohesin complexes in primary spermatocytes from mouse, interacting directly with each α-kleisin subunit. REC8 and RAD21L are also meiosis-specific cohesin components. Stag3 mutant spermatocytes arrest in early prophase (“zygotene-like” stage), displaying failed homolog synapsis and persistent DNA damage, as a result of unstable loading of cohesin onto the chromosome axes. Interestingly, Rec8, Rad21L double mutants resulted in an earlier “leptotene-like” arrest, accompanied by complete absence of STAG3 loading. To assess genetic interactions between STAG3 and α-kleisin subunits RAD21L and REC8, our lab generated Stag3, Rad21L, and Stag3, Rec8 double knockout mice, and compared them to the Rec8, Rad21L double mutant. These double mutants are phenotypically distinct from one another, and more severe than each single knockout mutant with regards to chromosome axis formation, cohesin loading, and sister chromatid cohesion. The Stag3, Rad21L, and Stag3, Rec8 double mutants both progress further into prophase I than the Rec8, Rad21L double mutant. Our genetic analysis demonstrates that cohesins containing STAG3 and REC8 are the main complex required for centromeric cohesion, and RAD21L cohesins are required for normal clustering of pericentromeric heterochromatin. Furthermore, the STAG3/REC8 and STAG3/RAD21L cohesins are the primary cohesins required

  14. Loss of cohesin complex components STAG2 or STAG3 confers resistance to BRAF inhibition in melanoma

    PubMed Central

    Shen, Che-Hung; Kim, Sun Hye; Trousil, Sebastian; Frederick, Dennie T.; Piris, Adriano; Yuan, Ping; Cai, Li; Gu, Lei; Li, Man; Lee, Jung Hyun; Mitra, Devarati; Fisher, David E.; Sullivan, Ryan J.; Flaherty, Keith T.; Zheng, Bin

    2016-01-01

    The protein kinase V-Raf murine sarcoma viral oncogene homolog B (BRAF) is an oncogenic driver and therapeutic target in melanoma. Inhibitors of BRAF (BRAFi) have shown high response rates and extended survival in melanoma patients bearing tumors that express BRAF Val600 mutations, but a vast majority of these patients develop drug resistance. Here we show that loss of Stromal antigen 2 or 3 (STAG2 or STAG3), which encode subunits of the cohesin complex, in melanoma cells results in resistance to BRAFi. We identified loss-of-function mutations in STAG2 as well as decreased expression of STAG2 or STAG3 proteins in several tumor samples from patients with acquired resistance to BRAFi and in BRAFi-resistant melanoma cell lines. Knockdown of STAG2 or STAG3 decreased sensitivity of Val600Glu BRAF-mutant melanoma cells and xenograft tumors to BRAFi. Loss of STAG2 inhibited CCCTC-binding factor (CTCF)-mediated expression of dual specificity phosphatase 6 (DUSP6), leading to reactivation of ERK signaling. Our studies unveil a previously unknown genetic mechanism of BRAFi resistance and provide new insights into the tumor suppressor function of STAG2 and STAG3. PMID:27500726

  15. Phosphorylation of cohesin Rec11/SA3 by casein kinase 1 promotes homologous recombination by assembling the meiotic chromosome axis.

    PubMed

    Sakuno, Takeshi; Watanabe, Yoshinori

    2015-01-26

    In meiosis, cohesin is required for sister chromatid cohesion, as well as meiotic chromosome axis assembly and recombination. However, mechanisms underlying the multifunctional nature of cohesin remain elusive. Here, we show that fission yeast casein kinase 1 (CK1) plays a crucial role in assembling the meiotic chromosome axis (so-called linear element: LinE) and promoting recombination. An in vitro phosphorylation screening assay identified meiotic cohesin subunit Rec11/SA3 as an excellent substrate of CK1. The phosphorylation of Rec11 by CK1 mediates the interaction with the Rec10/Red1/SCP2 axis component, a key step in meiotic chromosome axis assembly, and is dispensable for sister chromatid cohesion. Crucially, the expression of Rec11-Rec10 fusion protein nearly completely bypasses the requirement for CK1 or cohesin phosphorylation for LinE assembly and recombination. This study uncovers a central mechanism of the cohesin-dependent assembly of the meiotic chromosome axis and recombination apparatus that acts independently of sister chromatid cohesion.

  16. The cohesin subunit Rad21 is required for synaptonemal complex maintenance, but not sister chromatid cohesion, during Drosophila female meiosis.

    PubMed

    Urban, Evelin; Nagarkar-Jaiswal, Sonal; Lehner, Christian F; Heidmann, Stefan K

    2014-08-01

    Replicated sister chromatids are held in close association from the time of their synthesis until their separation during the next mitosis. This association is mediated by the ring-shaped cohesin complex that appears to embrace the sister chromatids. Upon proteolytic cleavage of the α-kleisin cohesin subunit at the metaphase-to-anaphase transition by separase, sister chromatids are separated and segregated onto the daughter nuclei. The more complex segregation of chromosomes during meiosis is thought to depend on the replacement of the mitotic α-kleisin cohesin subunit Rad21/Scc1/Mcd1 by the meiotic paralog Rec8. In Drosophila, however, no clear Rec8 homolog has been identified so far. Therefore, we have analyzed the role of the mitotic Drosophila α-kleisin Rad21 during female meiosis. Inactivation of an engineered Rad21 variant by premature, ectopic cleavage during oogenesis results not only in loss of cohesin from meiotic chromatin, but also in precocious disassembly of the synaptonemal complex (SC). We demonstrate that the lateral SC component C(2)M can interact directly with Rad21, potentially explaining why Rad21 is required for SC maintenance. Intriguingly, the experimentally induced premature Rad21 elimination, as well as the expression of a Rad21 variant with destroyed separase consensus cleavage sites, do not interfere with chromosome segregation during meiosis, while successful mitotic divisions are completely prevented. Thus, chromatid cohesion during female meiosis does not depend on Rad21-containing cohesin.

  17. The Cohesin Subunit Rad21 Is Required for Synaptonemal Complex Maintenance, but Not Sister Chromatid Cohesion, during Drosophila Female Meiosis

    PubMed Central

    Lehner, Christian F.; Heidmann, Stefan K.

    2014-01-01

    Replicated sister chromatids are held in close association from the time of their synthesis until their separation during the next mitosis. This association is mediated by the ring-shaped cohesin complex that appears to embrace the sister chromatids. Upon proteolytic cleavage of the α-kleisin cohesin subunit at the metaphase-to-anaphase transition by separase, sister chromatids are separated and segregated onto the daughter nuclei. The more complex segregation of chromosomes during meiosis is thought to depend on the replacement of the mitotic α-kleisin cohesin subunit Rad21/Scc1/Mcd1 by the meiotic paralog Rec8. In Drosophila, however, no clear Rec8 homolog has been identified so far. Therefore, we have analyzed the role of the mitotic Drosophila α-kleisin Rad21 during female meiosis. Inactivation of an engineered Rad21 variant by premature, ectopic cleavage during oogenesis results not only in loss of cohesin from meiotic chromatin, but also in precocious disassembly of the synaptonemal complex (SC). We demonstrate that the lateral SC component C(2)M can interact directly with Rad21, potentially explaining why Rad21 is required for SC maintenance. Intriguingly, the experimentally induced premature Rad21 elimination, as well as the expression of a Rad21 variant with destroyed separase consensus cleavage sites, do not interfere with chromosome segregation during meiosis, while successful mitotic divisions are completely prevented. Thus, chromatid cohesion during female meiosis does not depend on Rad21-containing cohesin. PMID:25101996

  18. Temporal changes in milk proteomes reveal developing milk functions.

    PubMed

    Gao, Xinliu; McMahon, Robert J; Woo, Jessica G; Davidson, Barbara S; Morrow, Ardythe L; Zhang, Qiang

    2012-07-06

    Human milk proteins provide essential nutrition for growth and development, and support a number of vital developmental processes in the neonate. A complete understanding of the possible functions of human milk proteins has been limited by incomplete knowledge of the human milk proteome. In this report, we have analyzed the proteomes of whey from human transitional and mature milk using ion-exchange and SDS-PAGE based protein fractionation methods. With a larger-than-normal sample loading approach, we are able to largely extend human milk proteome to 976 proteins. Among them, 152 proteins are found to render significant regulatory changes between transitional milk and mature milk. We further found that immunoglobulins sIgA and IgM are more abundant in transitional milk, whereas IgG is more abundant in mature milk, suggesting a transformation in defense mechanism from newborns to young infants. Additionally, we report a more comprehensive view of a complement system and associated regulatory apparatus in human milk, demonstrating the presence and function of a system similar to that found in the circulation but prevailed by alternative pathway in complement activation. Proteins involved in various aspects of carbohydrate metabolism are also described, revealing either a transition in milk functionality to accommodate carbohydrate-rich secretions as lactation progresses, or a potentially novel way of looking at the metabolic state of the mammary tissue. Lately, a number of extracellular matrix (ECM) proteins are found to be in higher abundance in transitional milk and may be relevant to the development of infants' gastrointestinal tract in early life. In contrast, the ECM protein fibronectin and several of the actin cytoskeleton proteins that it regulates are more abundant in mature milk, which may indicate the important functional role for milk in regulating reactive oxygen species.

  19. The cohesin subunit Rad21 is a negative regulator of hematopoietic self-renewal through epigenetic repression of HoxA7 and HoxA9

    PubMed Central

    Fisher, Joseph B.; Peterson, Jonathan; Reimer, Michael; Stelloh, Cary; Pulakanti, Kirthi; Gerbec, Zachary J.; Abel, Alex M.; Strouse, Jennifer Miksanek; Strouse, Christopher; McNulty, Maureen; Malarkannan, Subramaniam; Crispino, John D.; Milanovich, Samuel; Rao, Sridhar

    2016-01-01

    Acute myelogenous leukemia (AML) is a high-risk hematopoietic malignancy caused by a variety of mutations, including genes encoding the cohesin complex. Recent studies have demonstrated that reduction in cohesin complex levels leads to enhanced self-renewal in hematopoietic stem and progenitors (HSPCs). We sought to delineate the molecular mechanisms by which cohesin mutations promote enhanced HSPC self-renewal since this represents a critical initial step during leukemic transformation. We verified that RNAi against the cohesin subunit Rad21 causes enhanced self-renewal of HSPCs in vitro through derepression of Polycomb Repressive Complex 2 (PRC2) target genes, including Hoxa7 and Hoxa9. Importantly, knockdown of either Hoxa7 or Hoxa9 suppressed self-renewal, implying both are critical downstream effectors of reduced cohesin levels. We further demonstrate that the cohesin and PRC2 complexes interact and are bound in close proximity to Hoxa7 and Hoxa9. Rad21 depletion resulted in decreased levels of H3K27me3 at the Hoxa7 and Hoxa9 promoters, consistent with Rad21 being critical to proper gene silencing by recruiting the PRC2 complex. Our data demonstrates that the cohesin complex regulates PRC2 targeting to silence Hoxa7 and Hoxa9 and negatively regulate self-renewal. Our studies identify a novel epigenetic mechanism underlying leukemogenesis in AML patients with cohesin mutations. PMID:27554164

  20. Knock-Out Models Reveal New Aquaporin Functions

    PubMed Central

    Verkman, Alan S.

    2013-01-01

    Knockout mice have been informative in the discovery of unexpected biological functions of aquaporins. Knockout mice have confirmed the predicted roles of aquaporins in transepithelial fluid transport, as in the urinary concentrating mechanism and glandular fluid secretion. A less obvious, though predictable role of aquaporins is in tissue swelling under stress, as in the brain in stroke, tumor and infection. Phenotype analysis of aquaporin knockout mice has revealed several unexpected cellular roles of aquaporins whose mechanisms are being elucidated. Aquaporins facilitate cell migration, as seen in aquaporin-dependent tumor angiogenesis and tumor metastasis, by a mechanism that may involve facilitated water transport in lamellipodia of migrating cells. The ‘aquaglyceroporins’, aquaporins that transport both glycerol and water, regulate glycerol content in epidermis, fat and other tissues, and lead to a multiplicity of interesting consequences of gene disruption including dry skin, resistance to skin carcinogenesis, impaired cell proliferation and altered fat metabolism. An even more surprising role of a mammalian aquaporin is in neural signal transduction in the central nervous system. The many roles of aquaporins might be exploited for clinical benefit by modulation of aquaporin expression/function – as diuretics, and in the treatment of brain swelling, glaucoma, epilepsy, obesity and cancer. PMID:19096787

  1. Cohesin Is limiting for the suppression of DNA damage-induced recombination between homologous chromosomes.

    PubMed

    Covo, Shay; Westmoreland, James W; Gordenin, Dmitry A; Resnick, Michael A

    2010-07-01

    Double-strand break (DSB) repair through homologous recombination (HR) is an evolutionarily conserved process that is generally error-free. The risk to genome stability posed by nonallelic recombination or loss-of-heterozygosity could be reduced by confining HR to sister chromatids, thereby preventing recombination between homologous chromosomes. Here we show that the sister chromatid cohesion complex (cohesin) is a limiting factor in the control of DSB repair and genome stability and that it suppresses DNA damage-induced interactions between homologues. We developed a gene dosage system in tetraploid yeast to address limitations on various essential components in DSB repair and HR. Unlike RAD50 and RAD51, which play a direct role in HR, a 4-fold reduction in the number of essential MCD1 sister chromatid cohesion subunit genes affected survival of gamma-irradiated G(2)/M cells. The decreased survival reflected a reduction in DSB repair. Importantly, HR between homologous chromosomes was strongly increased by ionizing radiation in G(2)/M cells with a single copy of MCD1 or SMC3 even at radiation doses where survival was high and DSB repair was efficient. The increased recombination also extended to nonlethal doses of UV, which did not induce DSBs. The DNA damage-induced recombinants in G(2)/M cells included crossovers. Thus, the cohesin complex has a dual role in protecting chromosome integrity: it promotes DSB repair and recombination between sister chromatids, and it suppresses damage-induced recombination between homologues. The effects of limited amounts of Mcd1and Smc3 indicate that small changes in cohesin levels may increase the risk of genome instability, which may lead to genetic diseases and cancer.

  2. Independent mechanisms recruit the cohesin loader protein NIPBL to sites of DNA damage

    PubMed Central

    Bot, Christopher; Pfeiffer, Annika; Giordano, Fosco; Dantuma, Nico P.; Ström, Lena

    2017-01-01

    ABSTRACT NIPBL is required to load the cohesin complex on to DNA. While the canonical role of cohesin is to couple replicated sister chromatids together until the onset of mitosis, it also promotes tolerance to DNA damage. Here, we show that NIPBL is recruited to DNA damage throughout the cell cycle via independent mechanisms, influenced by type of damage. First, the heterochromatin protein HP1γ (also known as CBX3) recruits NIPBL to DNA double-strand breaks (DSBs) through the corresponding HP1-binding motif within the N-terminus. By contrast, the C-terminal HEAT repeat domain is unable to recruit NIPBL to DSBs but independently targets NIPBL to laser microirradiation-induced DNA damage. Each mechanism is dependent on the RNF8 and RNF168 ubiquitylation pathway, while the recruitment of the HEAT repeat domain requires further ATM or ATR activity. Thus, NIPBL has evolved a sophisticated response to damaged DNA that is influenced by the form of damage, suggesting a highly dynamic role for NIPBL in maintaining genomic stability. PMID:28167679

  3. Independent mechanisms recruit the cohesin loader protein NIPBL to sites of DNA damage.

    PubMed

    Bot, Christopher; Pfeiffer, Annika; Giordano, Fosco; Manjeera, Dharani E; Dantuma, Nico P; Ström, Lena

    2017-03-15

    NIPBL is required to load the cohesin complex on to DNA. While the canonical role of cohesin is to couple replicated sister chromatids together until the onset of mitosis, it also promotes tolerance to DNA damage. Here, we show that NIPBL is recruited to DNA damage throughout the cell cycle via independent mechanisms, influenced by type of damage. First, the heterochromatin protein HP1γ (also known as CBX3) recruits NIPBL to DNA double-strand breaks (DSBs) through the corresponding HP1-binding motif within the N-terminus. By contrast, the C-terminal HEAT repeat domain is unable to recruit NIPBL to DSBs but independently targets NIPBL to laser microirradiation-induced DNA damage. Each mechanism is dependent on the RNF8 and RNF168 ubiquitylation pathway, while the recruitment of the HEAT repeat domain requires further ATM or ATR activity. Thus, NIPBL has evolved a sophisticated response to damaged DNA that is influenced by the form of damage, suggesting a highly dynamic role for NIPBL in maintaining genomic stability.

  4. Cohesin phosphorylation and mobility of SMC1 at ionizing radiation-induced DNA double-strand breaks in human cells

    SciTech Connect

    Bauerschmidt, Christina; Helleday, Thomas

    2011-02-01

    Cohesin, a hetero-tetrameric complex of SMC1, SMC3, Rad21 and Scc3, associates with chromatin after mitosis and holds sister chromatids together following DNA replication. Following DNA damage, cohesin accumulates at and promotes the repair of DNA double-strand breaks. In addition, phosphorylation of the SMC1/3 subunits contributes to DNA damage-induced cell cycle checkpoint regulation. The aim of this study was to determine the regulation and consequences of SMC1/3 phosphorylation as part of the cohesin complex. We show here that the ATM-dependent phosphorylation of SMC1 and SMC3 is mediated by H2AX, 53BP1 and MDC1. Depletion of RAD21 abolishes these phosphorylations, indicating that only the fully assembled complex is phosphorylated. Comparison of wild type SMC1 and SMC1S966A in fluorescence recovery after photo-bleaching experiments shows that phosphorylation of SMC1 is required for an increased mobility after DNA damage in G2-phase cells, suggesting that ATM-dependent phosphorylation facilitates mobilization of the cohesin complex after DNA damage.

  5. Roles of cohesin and condensin in chromosome dynamics during mammalian meiosis.

    PubMed

    Lee, Jibak

    2013-10-01

    Meiosis is a key step for sexual reproduction in which chromosome number is halved by two successive meiotic divisions after a single round of DNA replication. In the first meiotic division (meiosis I), homologous chromosomes pair, synapse, and recombine with their partners in prophase I. As a result, homologous chromosomes are physically connected until metaphase I and then segregated from each other at the onset of anaphase I. In the subsequent second meiotic division (meiosis II), sister chromatids are segregated. Chromosomal abnormality arising during meiosis is one of the major causes of birth defects and congenital disorders in mammals including human and domestic animals. Hence understanding of the mechanism underlying these unique chromosome behavior in meiosis is of great importance. This review focuses on the roles of cohesin and condensin, and their regulation in chromosome dynamics during mammalian meiosis.

  6. Unsuspected functional disparity in Devonian fishes revealed by tooth morphometrics?

    NASA Astrophysics Data System (ADS)

    Gauchey, Samuel; Girard, Catherine; Adnet, Sylvain; Renaud, Sabrina

    2014-09-01

    The shape of features involved in key biological functions, such as teeth in nutrition, can provide insights into ecological processes even in ancient time, by linking the occupation of the morphological space (disparity) to the occupation of the ecological space. Investigating disparity in radiating groups may provide insights into the ecological diversification underlying evolution of morphological diversity. Actinopterygian fishes initiated their radiation in the Devonian, a period characterized by the diversification of marine ecosystem. Although a former morpho-functional analysis of jaw shape concluded to conservative and poorly diversified morphologies in this early part of their history, fish tooth disparity evidenced here an unsuspected diversity of possible functional significance in the pivotal period of the Late Devonian (Famennian). All teeth being caniniforms, some were stocky and robust, in agreement with expectations for active generalist predators. More surprisingly, elongated teeth also occurred at the beginning of Famennian. Their needle-like shape challenges morpho-functional interpretations by making them fragile in response to bending or torsion. The occurrence of both types of fish teeth during the beginning of the Famennian points to a discrete but real increase in disparity, thus testifying a first burst of feeding specialization despite overall conservative jaw morphology. The disappearance of these needle-like teeth in the Late Famennian might have been related to a relay in dental diversity with abundant co-occurring groups, namely conodonts and chondrichthyans (sharks).

  7. A selective screen reveals discrete functional domains in Drosophila Nanos.

    PubMed Central

    Arrizabalaga, G; Lehmann, R

    1999-01-01

    The Drosophila protein Nanos encodes an evolutionarily conserved protein with two zinc finger motifs. In the embryo, Nanos protein function is required for establishment of the anterior-posterior body pattern and for the migration of primordial germ cells. During oogenesis, Nanos protein is involved in the establishment and maintenance of germ-line stem cells and the differentiation of oocyte precursor cells. To establish proper embryonic patterning, Nanos acts as a translational regulator of hunchback RNA. Nanos' targets for germ cell migration and development are not known. Here, we describe a selective genetic screen aimed at isolating new nanos alleles. The molecular and genetic analysis of 68 new alleles has allowed us to identify amino acids critical for nanos function. This analysis shows that the CCHC motifs, which coordinate two metal ions, are essential for all known functions of Nanos protein. Furthermore, a region C-terminal to the zinc fingers seems to constitute a novel functional domain within the Nanos protein. This "tail region" of Nanos is required for abdomen formation and germ cell migration, but not for oogenesis. PMID:10581288

  8. Advances in cell surface glycoengineering reveal biological function.

    PubMed

    Nischan, Nicole; Kohler, Jennifer J

    2016-08-01

    Cell surface glycans are critical mediators of cell-cell, cell-ligand, and cell-pathogen interactions. By controlling the set of glycans displayed on the surface of a cell, it is possible to gain insight into the biological functions of glycans. Moreover, control of glycan expression can be used to direct cellular behavior. While genetic approaches to manipulate glycosyltransferase gene expression are available, their utility in glycan engineering has limitations due to the combinatorial nature of glycan biosynthesis and the functional redundancy of glycosyltransferase genes. Biochemical and chemical strategies offer valuable complements to these genetic approaches, notably by enabling introduction of unnatural functionalities, such as fluorophores, into cell surface glycans. Here, we describe some of the most recent developments in glycoengineering of cell surfaces, with an emphasis on strategies that employ novel chemical reagents. We highlight key examples of how these advances in cell surface glycan engineering enable study of cell surface glycans and their function. Exciting new technologies include synthetic lipid-glycans, new chemical reporters for metabolic oligosaccharide engineering to allow tandem and in vivo labeling of glycans, improved chemical and enzymatic methods for glycoproteomics, and metabolic glycosyltransferase inhibitors. Many chemical and biochemical reagents for glycan engineering are commercially available, facilitating their adoption by the biological community.

  9. Genome-Wide Association and Functional Follow-Up Reveals New Loci for Kidney Function

    PubMed Central

    Fuchsberger, Christian; Olden, Matthias; Chen, Ming-Huei; Tin, Adrienne; Taliun, Daniel; Li, Man; Gao, Xiaoyi; Gorski, Mathias; Yang, Qiong; Hundertmark, Claudia; Foster, Meredith C.; O'Seaghdha, Conall M.; Glazer, Nicole; Isaacs, Aaron; Liu, Ching-Ti; Smith, Albert V.; O'Connell, Jeffrey R.; Struchalin, Maksim; Tanaka, Toshiko; Li, Guo; Johnson, Andrew D.; Gierman, Hinco J.; Feitosa, Mary; Hwang, Shih-Jen; Atkinson, Elizabeth J.; Lohman, Kurt; Cornelis, Marilyn C.; Johansson, Åsa; Tönjes, Anke; Dehghan, Abbas; Chouraki, Vincent; Holliday, Elizabeth G.; Sorice, Rossella; Kutalik, Zoltan; Lehtimäki, Terho; Esko, Tõnu; Deshmukh, Harshal; Ulivi, Sheila; Chu, Audrey Y.; Murgia, Federico; Trompet, Stella; Imboden, Medea; Kollerits, Barbara; Pistis, Giorgio; Harris, Tamara B.; Launer, Lenore J.; Aspelund, Thor; Eiriksdottir, Gudny; Mitchell, Braxton D.; Boerwinkle, Eric; Schmidt, Helena; Cavalieri, Margherita; Rao, Madhumathi; Hu, Frank B.; Demirkan, Ayse; Oostra, Ben A.; de Andrade, Mariza; Turner, Stephen T.; Ding, Jingzhong; Andrews, Jeanette S.; Freedman, Barry I.; Koenig, Wolfgang; Illig, Thomas; Döring, Angela; Wichmann, H.-Erich; Kolcic, Ivana; Zemunik, Tatijana; Boban, Mladen; Minelli, Cosetta; Wheeler, Heather E.; Igl, Wilmar; Zaboli, Ghazal; Wild, Sarah H.; Wright, Alan F.; Campbell, Harry; Ellinghaus, David; Nöthlings, Ute; Jacobs, Gunnar; Biffar, Reiner; Endlich, Karlhans; Ernst, Florian; Homuth, Georg; Kroemer, Heyo K.; Nauck, Matthias; Stracke, Sylvia; Völker, Uwe; Völzke, Henry; Kovacs, Peter; Stumvoll, Michael; Mägi, Reedik; Hofman, Albert; Uitterlinden, Andre G.; Rivadeneira, Fernando; Aulchenko, Yurii S.; Polasek, Ozren; Hastie, Nick; Vitart, Veronique; Helmer, Catherine; Wang, Jie Jin; Ruggiero, Daniela; Bergmann, Sven; Kähönen, Mika; Viikari, Jorma; Nikopensius, Tiit; Province, Michael; Ketkar, Shamika; Colhoun, Helen; Doney, Alex; Robino, Antonietta; Giulianini, Franco; Krämer, Bernhard K.; Portas, Laura; Ford, Ian; Buckley, Brendan M.; Adam, Martin; Thun, Gian-Andri; Paulweber, Bernhard; Haun, Margot; Sala, Cinzia; Metzger, Marie; Mitchell, Paul; Ciullo, Marina; Kim, Stuart K.; Vollenweider, Peter; Raitakari, Olli; Metspalu, Andres; Palmer, Colin; Gasparini, Paolo; Pirastu, Mario; Jukema, J. Wouter; Probst-Hensch, Nicole M.; Kronenberg, Florian; Toniolo, Daniela; Gudnason, Vilmundur; Shuldiner, Alan R.; Coresh, Josef; Schmidt, Reinhold; Ferrucci, Luigi; Siscovick, David S.; van Duijn, Cornelia M.; Borecki, Ingrid; Kardia, Sharon L. R.; Liu, Yongmei; Curhan, Gary C.; Rudan, Igor; Gyllensten, Ulf; Wilson, James F.; Franke, Andre; Pramstaller, Peter P.; Rettig, Rainer; Prokopenko, Inga; Witteman, Jacqueline C. M.; Hayward, Caroline; Ridker, Paul; Parsa, Afshin; Bochud, Murielle; Heid, Iris M.; Goessling, Wolfram; Chasman, Daniel I.; Kao, W. H. Linda; Fox, Caroline S.

    2012-01-01

    Chronic kidney disease (CKD) is an important public health problem with a genetic component. We performed genome-wide association studies in up to 130,600 European ancestry participants overall, and stratified for key CKD risk factors. We uncovered 6 new loci in association with estimated glomerular filtration rate (eGFR), the primary clinical measure of CKD, in or near MPPED2, DDX1, SLC47A1, CDK12, CASP9, and INO80. Morpholino knockdown of mpped2 and casp9 in zebrafish embryos revealed podocyte and tubular abnormalities with altered dextran clearance, suggesting a role for these genes in renal function. By providing new insights into genes that regulate renal function, these results could further our understanding of the pathogenesis of CKD. PMID:22479191

  10. Conformational diversity analysis reveals three functional mechanisms in proteins

    PubMed Central

    Fornasari, María Silvina

    2017-01-01

    Protein motions are a key feature to understand biological function. Recently, a large-scale analysis of protein conformational diversity showed a positively skewed distribution with a peak at 0.5 Å C-alpha root-mean-square-deviation (RMSD). To understand this distribution in terms of structure-function relationships, we studied a well curated and large dataset of ~5,000 proteins with experimentally determined conformational diversity. We searched for global behaviour patterns studying how structure-based features change among the available conformer population for each protein. This procedure allowed us to describe the RMSD distribution in terms of three main protein classes sharing given properties. The largest of these protein subsets (~60%), which we call “rigid” (average RMSD = 0.83 Å), has no disordered regions, shows low conformational diversity, the largest tunnels and smaller and buried cavities. The two additional subsets contain disordered regions, but with differential sequence composition and behaviour. Partially disordered proteins have on average 67% of their conformers with disordered regions, average RMSD = 1.1 Å, the highest number of hinges and the longest disordered regions. In contrast, malleable proteins have on average only 25% of disordered conformers and average RMSD = 1.3 Å, flexible cavities affected in size by the presence of disordered regions and show the highest diversity of cognate ligands. Proteins in each set are mostly non-homologous to each other, share no given fold class, nor functional similarity but do share features derived from their conformer population. These shared features could represent conformational mechanisms related with biological functions. PMID:28192432

  11. Functional asymmetries revealed in visually guided saccades: an FMRI study.

    PubMed

    Petit, Laurent; Zago, Laure; Vigneau, Mathieu; Andersson, Frédéric; Crivello, Fabrice; Mazoyer, Bernard; Mellet, Emmanuel; Tzourio-Mazoyer, Nathalie

    2009-11-01

    Because eye movements are a fundamental tool for spatial exploration, we hypothesized that the neural bases of these movements in humans should be under right cerebral dominance, as already described for spatial attention. We used functional magnetic resonance imaging in 27 right-handed participants who alternated central fixation with either large or small visually guided saccades (VGS), equally performed in both directions. Hemispheric functional asymmetry was analyzed to identify whether brain regions showing VGS activation elicited hemispheric asymmetries. Hemispheric anatomical asymmetry was also estimated to assess its influence on the VGS functional lateralization. Right asymmetrical activations of a saccadic/attentional system were observed in the lateral frontal eye fields (FEF), the anterior part of the intraparietal sulcus (aIPS), the posterior third of the superior temporal sulcus (STS), the occipitotemporal junction (MT/V5 area), the middle occipital gyrus, and medially along the calcarine fissure (V1). The present rightward functional asymmetries were not related to differences in gray matter (GM) density/sulci positions between right and left hemispheres in the precentral, intraparietal, superior temporal, and extrastriate regions. Only V1 asymmetries were explained for almost 20% of the variance by a difference in the position of the right and left calcarine fissures. Left asymmetrical activations of a saccadic motor system were observed in the medial FEF and in the motor strip eye field along the Rolando sulcus. They were not explained by GM asymmetries. We suggest that the leftward saccadic motor asymmetry is part of a general dominance of the left motor cortex in right-handers, which must include an effect of sighting dominance. Our results demonstrate that, although bilateral by nature, the brain network involved in the execution of VGSs, irrespective of their direction, presented specific right and left asymmetries that were not related to

  12. Yeast studies reveal moonlighting functions of the ancient actin cytoskeleton

    PubMed Central

    Sattlegger, Evelyn; Chernova, Tatiana A.; Gogoi, Neeku M.; Pillai, Indu V.; Chernoff, Yury O.; Munn, Alan L.

    2014-01-01

    Classic functions of the actin cytoskeleton include control of cell size and shape and the internal organisation of cells. These functions are manifest in cellular processes of fundamental importance throughout biology such as the generation of cell polarity, cell migration, cell adhesion and cell division. However, studies in the unicellular model eukaryote Saccharomyces cerevisiae (Baker's yeast) are giving insights into other functions in which the actin cytoskeleton plays a critical role. These include endocytosis, control of protein translation and determination of protein 3-dimensional shape (especially conversion of normal cellular proteins into prions). Here we present a concise overview of these new "moonlighting" roles for the actin cytoskeleton and how some of these roles might lie at the heart of important molecular switches. This is an exciting time for researchers interested in the actin cytoskeleton. We show here how studies of actin are leading us into many new and exciting realms at the interface of genetics, biochemistry and cell biology. While many of the pioneering studies have been conducted using yeast, the conservation of the actin cytoskeleton and its component proteins throughout eukaryotes suggests that these new roles for the actin cytoskeleton may not be restricted to yeast cells but rather may reflect new roles for the actin cytoskeleton of all eukaryotes. PMID:25138357

  13. Primer Sets Developed for Functional Genes Reveal Shifts in Functionality of Fungal Community in Soils

    PubMed Central

    Hannula, S. Emilia; van Veen, Johannes A.

    2016-01-01

    Phylogenetic diversity of soil microbes is a hot topic at the moment. However, the molecular tools for the assessment of functional diversity in the fungal community are less developed than tools based on genes encoding the ribosomal operon. Here 20 sets of primers targeting genes involved mainly in carbon cycling were designed and/or validated and the functioning of soil fungal communities along a chronosequence of land abandonment from agriculture was evaluated using them. We hypothesized that changes in fungal community structure during secondary succession would lead to difference in the types of genes present in soils and that these changes would be directional. We expected an increase in genes involved in degradation of recalcitrant organic matter in time since agriculture. Out of the investigated genes, the richness of the genes related to carbon cycling was significantly higher in fields abandoned for longer time. The composition of six of the genes analyzed revealed significant differences between fields abandoned for shorter and longer time. However, all genes revealed significant variance over the fields studied, and this could be related to other parameters than the time since agriculture such as pH, organic matter, and the amount of available nitrogen. Contrary to our initial hypothesis, the genes significantly different between fields were not related to the decomposition of more recalcitrant matter but rather involved in degradation of cellulose and hemicellulose. PMID:27965632

  14. Primer Sets Developed for Functional Genes Reveal Shifts in Functionality of Fungal Community in Soils.

    PubMed

    Hannula, S Emilia; van Veen, Johannes A

    2016-01-01

    Phylogenetic diversity of soil microbes is a hot topic at the moment. However, the molecular tools for the assessment of functional diversity in the fungal community are less developed than tools based on genes encoding the ribosomal operon. Here 20 sets of primers targeting genes involved mainly in carbon cycling were designed and/or validated and the functioning of soil fungal communities along a chronosequence of land abandonment from agriculture was evaluated using them. We hypothesized that changes in fungal community structure during secondary succession would lead to difference in the types of genes present in soils and that these changes would be directional. We expected an increase in genes involved in degradation of recalcitrant organic matter in time since agriculture. Out of the investigated genes, the richness of the genes related to carbon cycling was significantly higher in fields abandoned for longer time. The composition of six of the genes analyzed revealed significant differences between fields abandoned for shorter and longer time. However, all genes revealed significant variance over the fields studied, and this could be related to other parameters than the time since agriculture such as pH, organic matter, and the amount of available nitrogen. Contrary to our initial hypothesis, the genes significantly different between fields were not related to the decomposition of more recalcitrant matter but rather involved in degradation of cellulose and hemicellulose.

  15. Remote Synchronization Reveals Network Symmetries and Functional Modules

    NASA Astrophysics Data System (ADS)

    Nicosia, Vincenzo; Valencia, Miguel; Chavez, Mario; Díaz-Guilera, Albert; Latora, Vito

    2013-04-01

    We study a Kuramoto model in which the oscillators are associated with the nodes of a complex network and the interactions include a phase frustration, thus preventing full synchronization. The system organizes into a regime of remote synchronization where pairs of nodes with the same network symmetry are fully synchronized, despite their distance on the graph. We provide analytical arguments to explain this result, and we show how the frustration parameter affects the distribution of phases. An application to brain networks suggests that anatomical symmetry plays a role in neural synchronization by determining correlated functional modules across distant locations.

  16. Nucleome Analysis Reveals Structure-function Relationships for Colon Cancer.

    PubMed

    Seaman, Laura; Chen, Haiming; Brown, Markus; Wangsa, Darawalee; Patterson, Geoff; Camps, Jordi; Omenn, Gilbert S; Ried, Thomas; Rajapakse, Indika

    2017-03-03

    Chromosomal translocations and aneuploidy are hallmarks of cancer genomes; however, the impact of these aberrations on the nucleome (i.e., nuclear structure and gene expression) are not yet understood. Here, the nucleome of the colorectal cancer cell line HT-29 was analyzed using chromosome conformation capture (Hi-C) to study genome structure, complemented by RNA sequencing (RNA-seq) to determine consequent changes in genome function. Importantly, translocations and copy number changes were identified at high resolution from Hi-C data and the structure-function relationships present in normal cells were maintained in cancer. In addition, a new copy number-based normalization method for Hi-C data was developed to analyze the effect of chromosomal aberrations on local chromatin structure. The data demonstrate that at the site of translocations the correlation between chromatin organization and gene expression increases; thus, chromatin accessibility more directly reflects transcription. Additionally, the homogeneously staining region of chromosome band 8q24 of HT-29, which includes the MYC oncogene, interacts with various loci throughout the genome and is composed of open chromatin. The methods, described herein, can be applied to the assessment of the nucleome in other cell types with chromosomal aberrations.

  17. Statistical universals reveal the structures and functions of human music.

    PubMed

    Savage, Patrick E; Brown, Steven; Sakai, Emi; Currie, Thomas E

    2015-07-21

    Music has been called "the universal language of mankind." Although contemporary theories of music evolution often invoke various musical universals, the existence of such universals has been disputed for decades and has never been empirically demonstrated. Here we combine a music-classification scheme with statistical analyses, including phylogenetic comparative methods, to examine a well-sampled global set of 304 music recordings. Our analyses reveal no absolute universals but strong support for many statistical universals that are consistent across all nine geographic regions sampled. These universals include 18 musical features that are common individually as well as a network of 10 features that are commonly associated with one another. They span not only features related to pitch and rhythm that are often cited as putative universals but also rarely cited domains including performance style and social context. These cross-cultural structural regularities of human music may relate to roles in facilitating group coordination and cohesion, as exemplified by the universal tendency to sing, play percussion instruments, and dance to simple, repetitive music in groups. Our findings highlight the need for scientists studying music evolution to expand the range of musical cultures and musical features under consideration. The statistical universals we identified represent important candidates for future investigation.

  18. Widespread distribution of encapsulin nanocompartments reveals functional diversity.

    PubMed

    Giessen, Tobias W; Silver, Pamela A

    2017-03-06

    Cells organize and regulate their metabolism via membrane- or protein-bound organelles. In this way, incompatible processes can be spatially separated and controlled. In prokaryotes, protein-based compartments are used to sequester harmful reactions and store useful compounds. These protein compartments play key roles in various metabolic and ecological processes, ranging from iron homeostasis to carbon fixation. One of the newest types of protein organelle are encapsulin nanocompartments. They are able to encapsulate specific protein cargo and are proposed to be involved in redox-related processes. We identified more than 900 putative encapsulin systems in bacterial and archaeal genomes. Encapsulins can be found in fifteen bacterial and two archaeal phyla. Our analysis reveals one new capsid type and nine previously unknown cargo proteins targeted to the interior of encapsulins. We experimentally characterize three newly identified encapsulin systems and illustrate their probable involvement in iron mineralization, oxidative and nitrosative stress resistance and anaerobic ammonium oxidation, a process responsible for 30% of the nitrogen lost from the oceans.

  19. Statistical universals reveal the structures and functions of human music

    PubMed Central

    Savage, Patrick E.; Brown, Steven; Sakai, Emi; Currie, Thomas E.

    2015-01-01

    Music has been called “the universal language of mankind.” Although contemporary theories of music evolution often invoke various musical universals, the existence of such universals has been disputed for decades and has never been empirically demonstrated. Here we combine a music-classification scheme with statistical analyses, including phylogenetic comparative methods, to examine a well-sampled global set of 304 music recordings. Our analyses reveal no absolute universals but strong support for many statistical universals that are consistent across all nine geographic regions sampled. These universals include 18 musical features that are common individually as well as a network of 10 features that are commonly associated with one another. They span not only features related to pitch and rhythm that are often cited as putative universals but also rarely cited domains including performance style and social context. These cross-cultural structural regularities of human music may relate to roles in facilitating group coordination and cohesion, as exemplified by the universal tendency to sing, play percussion instruments, and dance to simple, repetitive music in groups. Our findings highlight the need for scientists studying music evolution to expand the range of musical cultures and musical features under consideration. The statistical universals we identified represent important candidates for future investigation. PMID:26124105

  20. Targeted deletion of Wwox reveals a tumor suppressor function.

    PubMed

    Aqeilan, Rami I; Trapasso, Francesco; Hussain, Sadiq; Costinean, Stefan; Marshall, Dean; Pekarsky, Yuri; Hagan, John P; Zanesi, Nicola; Kaou, Mohamed; Stein, Gary S; Lian, Jane B; Croce, Carlo M

    2007-03-06

    The WW domain-containing oxidoreductase (WWOX) spans the second most common fragile site of the human genome, FRA16D, located at 16q23, and its expression is altered in several types of human cancer. We have previously shown that restoration of WWOX expression in cancer cells suppresses tumorigenicity. To investigate WWOX tumor suppressor function in vivo, we generated mice carrying a targeted deletion of the Wwox gene and monitored incidence of tumor formation. Osteosarcomas in juvenile Wwox(-/-) and lung papillary carcinoma in adult Wwox(+/-) mice occurred spontaneously. In addition, Wwox(+/-) mice develop significantly more ethyl nitrosourea-induced lung tumors and lymphomas in comparison to wild-type littermate mice. Intriguingly, these tumors still express Wwox protein, suggesting haploinsuffiency of WWOX itself is cancer predisposing. These results indicate that WWOX is a bona fide tumor suppressor.

  1. Revealing the structural and functional diversity of plant cell walls.

    PubMed

    Knox, J Paul

    2008-06-01

    The extensive knowledge of the chemistry of isolated cell wall polymers, and that relating to the identification and partial annotation of gene families involved in their synthesis and modification, is not yet matched by a sophisticated understanding of the occurrence of the polymers within cell walls of the diverse cell types within a growing organ. Currently, the main sets of tools that are used to determine cell-type-specific configurations of cell wall polymers and aspects of cell wall microstructures are antibodies, carbohydrate-binding modules (CBMs) and microspectroscopies. As these tools are applied we see that cell wall polymers are extensively developmentally regulated and that there is a range of structurally distinct primary and secondary cell walls within organs and across species. The challenge now is to document cell wall structures in relation to diverse cell biological events and to integrate this knowledge with the emerging understanding of polymer functions.

  2. Global Analysis of ATM Polymorphism Reveals Significant Functional Constraint

    PubMed Central

    Thorstenson, Yvonne R.; Shen, Peidong; Tusher, Virginia G.; Wayne, Tierney L.; Davis, Ronald W.; Chu, Gilbert; Oefner, Peter J.

    2001-01-01

    ATM, the gene that is mutated in ataxia-telangiectasia, is associated with cerebellar degeneration, abnormal proliferation of small blood vessels, and cancer. These clinically important manifestations have stimulated interest in defining the sequence variation in the ATM gene. Therefore, we undertook a comprehensive survey of sequence variation in ATM in diverse human populations. The protein-encoding exons of the gene (9,168 bp) and the adjacent intron and untranslated sequences (14,661 bp) were analyzed in 93 individuals from seven major human populations. In addition, the coding sequence was analyzed in one chimpanzee, one gorilla, one orangutan, and one Old World monkey. In human ATM, 88 variant sites were discovered by denaturing high-performance liquid chromatography, which is 96%–100% sensitive for detection of DNA sequence variation. ATM was compared to 14 other autosomal genes for nucleotide diversity. The noncoding regions of ATM had diversity values comparable to other genes, but the coding regions had very low diversity, especially in the last 29% of the protein sequence. A test of the neutral evolution hypothesis, through use of the Hudson/Kreitman/Aguadé statistic, revealed that this region of the human ATM gene was significantly constrained relative to that of the orangutan, the Old World monkey, and the mouse, but not relative to that of the chimpanzee or the gorilla. ATM displayed extensive linkage disequilibrium, consistent with suppression of meiotic recombination at this locus. Seven haplotypes were defined. Two haplotypes accounted for 82% of all chromosomes analyzed in all major populations; two others carrying the same D126E missense polymorphism accounted for 33% of chromosomes in Africa but were never observed outside of Africa. The high frequency of this polymorphism may be due either to a population expansion within Africa or to selective pressure. PMID:11443540

  3. P-wave Receiver Functions reveal the Bohemian Massif crust

    NASA Astrophysics Data System (ADS)

    Kampfova Exnerova, Hana; Plomerova, Jaroslava; Vecsey, Ludek

    2015-04-01

    In this study we present initial results of P-wave Receiver Functions (RF) calculated from broad-band waveforms of teleseismic events recorded by temporary and permanent stations in the Bohemian Massif (BM, Central Europe). Temporary arrays BOHEMA I (2001-2003), BOHEMA II (2004-2005) and BOHEMA III (2005-2006) operated during passive seismic experiments oriented towards studying velocity structure of the lithosphere and the upper mantle. Receiver Functions show relative response of the Earth structure under a seismic station and nowadays represent frequently-used method to retrieve structure of the crust, whose knowledge is needed in various studies of the upper mantle. The recorded waveforms are composites of direct P and P-to-S converted waves that reverberate in the structure beneath the receiver (Ammon, 1997). The RFs are sensitive to seismic velocity contrast and are thus suited to identifying velocity discontinuities in the crust, including the Mohorovičić discontinuity (Moho). Relative travel-time delays of the converted phases detected in the RFs are transformed into estimates of discontinuity depths assuming external information on the vp/vs and P velocity. To evaluate RFs we use the Multiple-taper spectral correlation (MTC) method (Park and Levin, 2000) and process signals from teleseismic events at epicentral distances of 30 - 100° with magnitude Mw > 5.5. Recordings are filtered with Butterworth band-pass filter of 2 - 8 s. To select automatically signals which are strong enough, we calculate signal-to-noise ratios (SNR) in two steps. In the first step we calculate SNR for signals from intervals (-1s, 3s)/(-10s, -2s), where P-arrival time represent time zero. In the second step we broaden the intervals and calculate SNR for (-1s, 9s)/(-60s, -2s). We also employ forward modelling of the RFs using Interactive Receiver Functions Forward Modeller (IRFFM) (Tkalčić et al., 2010) to produce, in the first step, one-dimensional velocity models under

  4. Proteomic profiling of high risk medulloblastoma reveals functional biology.

    PubMed

    Staal, Jerome A; Lau, Ling San; Zhang, Huizhen; Ingram, Wendy J; Hallahan, Andrew R; Northcott, Paul A; Pfister, Stefan M; Wechsler-Reya, Robert J; Rusert, Jessica M; Taylor, Michael D; Cho, Yoon-Jae; Packer, Roger J; Brown, Kristy J; Rood, Brian R

    2015-06-10

    Genomic characterization of medulloblastoma has improved molecular risk classification but struggles to define functional biological processes, particularly for the most aggressive subgroups. We present here a novel proteomic approach to this problem using a reference library of stable isotope labeled medulloblastoma-specific proteins as a spike-in standard for accurate quantification of the tumor proteome. Utilizing high-resolution mass spectrometry, we quantified the tumor proteome of group 3 medulloblastoma cells and demonstrate that high-risk MYC amplified tumors can be segregated based on protein expression patterns. We cross-validated the differentially expressed protein candidates using an independent transcriptomic data set and further confirmed them in a separate cohort of medulloblastoma tissue samples to identify the most robust proteogenomic differences. Interestingly, highly expressed proteins associated with MYC-amplified tumors were significantly related to glycolytic metabolic pathways via alternative splicing of pyruvate kinase (PKM) by heterogeneous ribonucleoproteins (HNRNPs). Furthermore, when maintained under hypoxic conditions, these MYC-amplified tumors demonstrated increased viability compared to non-amplified tumors within the same subgroup. Taken together, these findings highlight the power of proteomics as an integrative platform to help prioritize genetic and molecular drivers of cancer biology and behavior.

  5. Revealing humans’ sensorimotor functions with electrical cortical stimulation

    PubMed Central

    Desmurget, Michel; Sirigu, Angela

    2015-01-01

    Direct electrical stimulation (DES) of the human brain has been used by neurosurgeons for almost a century. Although this procedure serves only clinical purposes, it generates data that have a great scientific interest. Had DES not been employed, our comprehension of the organization of the sensorimotor systems involved in movement execution, language production, the emergence of action intentionality or the subjective feeling of movement awareness would have been greatly undermined. This does not mean, of course, that DES is a gold standard devoid of limitations and that other approaches are not of primary importance, including electrophysiology, modelling, neuroimaging or psychophysics in patients and healthy subjects. Rather, this indicates that the contribution of DES cannot be restricted, in humans, to the ubiquitous concepts of homunculus and somatotopy. DES is a fundamental tool in our attempt to understand the human brain because it represents a unique method for mapping sensorimotor pathways and interfering with the functioning of localized neural populations during the performance of well-defined behavioural tasks. PMID:26240422

  6. Epistatic relationships reveal the functional organization of yeast transcription factors.

    PubMed

    Zheng, Jiashun; Benschop, Joris J; Shales, Michael; Kemmeren, Patrick; Greenblatt, Jack; Cagney, Gerard; Holstege, Frank; Li, Hao; Krogan, Nevan J

    2010-10-05

    The regulation of gene expression is, in large part, mediated by interplay between the general transcription factors (GTFs) that function to bring about the expression of many genes and site-specific DNA-binding transcription factors (STFs). Here, quantitative genetic profiling using the epistatic miniarray profile (E-MAP) approach allowed us to measure 48 391 pairwise genetic interactions, both negative (aggravating) and positive (alleviating), between and among genes encoding STFs and GTFs in Saccharomyces cerevisiae. This allowed us to both reconstruct regulatory models for specific subsets of transcription factors and identify global epistatic patterns. Overall, there was a much stronger preference for negative relative to positive genetic interactions among STFs than there was among GTFs. Negative genetic interactions, which often identify factors working in non-essential, redundant pathways, were also enriched for pairs of STFs that co-regulate similar sets of genes. Microarray analysis demonstrated that pairs of STFs that display negative genetic interactions regulate gene expression in an independent rather than coordinated manner. Collectively, these data suggest that parallel/compensating relationships between regulators, rather than linear pathways, often characterize transcriptional circuits.

  7. Synthetic actin-binding domains reveal compositional constraints for function.

    PubMed

    Lorenzi, Maria; Gimona, Mario

    2008-01-01

    The actin-binding domains of many proteins consist of a canonical type 1/type 2 arrangement of the structurally conserved calponin homology domain. Using the actin-binding domain of alpha-actinin-1 as a scaffold we have generated synthetic actin-binding domains by altering position and composition of the calponin homology domains. We show that the presence of two calponin homology domains alone and in the context of an actin-binding domain is not sufficient for actin-binding, and that both single and homotypic type 2 calponin homology domain tandems fail to bind to actin in vitro and in transfected cells. In contrast, single and tandem type 1 calponin homology domain arrays bind actin directly but result in defective turnover rates on actin filaments, and in aberrant actin bundling when introduced into the full-length alpha-actinin molecule. An actin-binding domain harboring the calponin homology domains in an inverted position, however, functions both in isolation and in the context of the dimeric alpha-actinin molecule. Our data demonstrate that the dynamics and specificity of actin-binding via actin-binding domains requires both the filament binding properties of the type 1, and regulation by type 2 calponin homology domains, and appear independent of their position.

  8. An acetyltransferase-independent function of Eso1 regulates centromere cohesion

    PubMed Central

    Lin, Su-Jiun; Tapia-Alveal, Claudia; Jabado, Omar J.; Germain, Doris; O’Connell, Matthew J.

    2016-01-01

    Eukaryotes contain three essential Structural Maintenance of Chromosomes (SMC) complexes: cohesin, condensin, and Smc5/6. Cohesin forms a ring-shaped structure that embraces sister chromatids to promote their cohesion. The cohesiveness of cohesin is promoted by acetylation of N-terminal lysines of the Smc3 subunit by the acetyltransferases Eco1 in Saccharomyces cerevisiae and the homologue, Eso1, in Schizosaccharomyces pombe. In both yeasts, these acetyltransferases are essential for cell viability. However, whereas nonacetylatable Smc3 mutants are lethal in S. cerevisiae, they are not in S. pombe. We show that the lethality of a temperature-sensitive allele of eso1 (eso1-H17) is due to activation of the spindle assembly checkpoint (SAC) and is associated with premature centromere separation. The lack of cohesion at the centromeres does not correlate with Psm3 acetylation or cohesin levels at the centromeres, but is associated ith significantly reduced recruitment of the cohesin regulator Pds5. The SAC activation in this context is dependent on Smc5/6 function, which is required to remove cohesin from chromosome arms but not centromeres. The mitotic defects caused by Smc5/6 and Eso1 dysfunction are cosuppressed in double mutants. This identifies a novel function (or functions) for Eso1 and Smc5/6 at centromeres and extends the functional relationships between these SMC complexes. PMID:27798241

  9. Functional Tissue Analysis Reveals Successful Cryopreservation of Human Osteoarthritic Synovium

    PubMed Central

    de Vries, Marieke; Bennink, Miranda B.; van Lent, Peter L. E. M.; van der Kraan, Peter M.; Koenders, Marije I.; Thurlings, Rogier M.; van de Loo, Fons A. J.

    2016-01-01

    Osteoarthritis (OA) is a degenerative joint disease affecting cartilage and is the most common form of arthritis worldwide. One third of OA patients have severe synovitis and less than 10% have no evidence of synovitis. Moreover, synovitis is predictive for more severe disease progression. This offers a target for therapy but more research on the pathophysiological processes in the synovial tissue of these patients is needed. Functional studies performed with synovial tissue will be more approachable when this material, that becomes available by joint replacement surgery, can be stored for later use. We set out to determine the consequences of slow-freezing of human OA synovial tissue. Therefore, we validated a method that can be applied in every routine laboratory and performed a comparative study of five cryoprotective agent (CPA) solutions. To determine possible deleterious cryopreservation-thaw effects on viability, the synovial tissue architecture, metabolic activity, RNA quality, expression of cryopreservation associated stress genes, and expression of OA characteristic disease genes was studied. Furthermore, the biological activity of the cryopreserved tissue was determined by measuring cytokine secretion induced by the TLR ligands lipopolysaccharides and Pam3Cys. Compared to non frozen synovium, no difference in cell and tissue morphology could be identified in the conditions using the CS10, standard and CryoSFM CPA solution for cryopreservation. However, we observed significantly lower preservation of tissue morphology with the Biofreeze and CS2 media. The other viability assays showed trends in the same direction but were not sensitive enough to detect significant differences between conditions. In all assays tested a clearly lower viability was detected in the condition in which synovium was frozen without CPA solution. This detailed analysis showed that OA synovial tissue explants can be cryopreserved while maintaining the morphology, viability and

  10. Physical Association of Saccharomyces cerevisiae Polo-like Kinase Cdc5 with Chromosomal Cohesin Facilitates DNA Damage Response.

    PubMed

    Pakchuen, Sujiraporn; Ishibashi, Mai; Takakusagi, Emi; Shirahige, Katsuhiko; Sutani, Takashi

    2016-08-12

    At the onset of anaphase, a protease called separase breaks the link between sister chromatids by cleaving the cohesin subunit Scc1. This irreversible step in the cell cycle is promoted by degradation of the separase inhibitor, securin, and polo-like kinase (Plk) 1-dependent phosphorylation of the Scc1 subunit. Plk could recognize substrates through interaction between its phosphopeptide interaction domain, the polo-box domain, and a phosphorylated priming site in the substrate, which has been generated by a priming kinase beforehand. However, the physiological relevance of this targeting mechanism remains to be addressed for many of the Plk1 substrates. Here, we show that budding yeast Plk1, Cdc5, is pre-deposited onto cohesin engaged in cohesion on chromosome arms in G2/M phase cells. The Cdc5-cohesin association is mediated by direct interaction between the polo-box domain of Cdc5 and Scc1 phosphorylated at multiple sites in its middle region. Alanine substitutions of the possible priming phosphorylation sites (scc1-15A) impair Cdc5 association with chromosomal cohesin, but they make only a moderate impact on mitotic cell growth even in securin-deleted cells (pds1Δ), where Scc1 phosphorylation by Cdc5 is indispensable. The same scc1-15A pds1Δ double mutant, however, exhibits marked sensitivity to the DNA-damaging agent phleomycin, suggesting that the priming phosphorylation of Scc1 poses an additional layer of regulation that enables yeast cells to adapt to genotoxic environments.

  11. Aurora-A mediated histone H3 phosphorylation of threonine 118 controls condensin I and cohesin occupancy in mitosis

    PubMed Central

    Wike, Candice L; Graves, Hillary K; Hawkins, Reva; Gibson, Matthew D; Ferdinand, Michelle B; Zhang, Tao; Chen, Zhihong; Hudson, Damien F; Ottesen, Jennifer J; Poirier, Michael G; Schumacher, Jill; Tyler, Jessica K

    2016-01-01

    Phosphorylation of histone H3 threonine 118 (H3 T118ph) weakens histone DNA-contacts, disrupting the nucleosome structure. We show that Aurora-A mediated H3 T118ph occurs at pericentromeres and chromosome arms during prophase and is lost upon chromosome alignment. Expression of H3 T118E or H3 T118I (a SIN mutation that bypasses the need for the ATP-dependent nucleosome remodeler SWI/SNF) leads to mitotic problems including defects in spindle attachment, delayed cytokinesis, reduced chromatin packaging, cohesion loss, cohesin and condensin I loss in human cells. In agreement, overexpression of Aurora-A leads to increased H3 T118ph levels, causing cohesion loss, and reduced levels of cohesin and condensin I on chromatin. Normal levels of H3 T118ph are important because it is required for development in fruit flies. We propose that H3 T118ph alters the chromatin structure during specific phases of mitosis to promote timely condensin I and cohesin disassociation, which is essential for effective chromosome segregation. DOI: http://dx.doi.org/10.7554/eLife.11402.001 PMID:26878753

  12. Dynamic functional connectivity revealed by resting-state functional near-infrared spectroscopy

    PubMed Central

    Li, Zhen; Liu, Hanli; Liao, Xuhong; Xu, Jingping; Liu, Wenli; Tian, Fenghua; He, Yong; Niu, Haijing

    2015-01-01

    The brain is a complex network with time-varying functional connectivity (FC) and network organization. However, it remains largely unknown whether resting-state fNIRS measurements can be used to characterize dynamic characteristics of intrinsic brain organization. In this study, for the first time, we used the whole-cortical fNIRS time series and a sliding-window correlation approach to demonstrate that fNIRS measurement can be ultimately used to quantify the dynamic characteristics of resting-state brain connectivity. Our results reveal that the fNIRS-derived FC is time-varying, and the variability strength (Q) is correlated negatively with the time-averaged, static FC. Furthermore, the Q values also show significant differences in connectivity between different spatial locations (e.g., intrahemispheric and homotopic connections). The findings are reproducible across both sliding-window lengths and different brain scanning sessions, suggesting that the dynamic characteristics in fNIRS-derived cerebral functional correlation results from true cerebral fluctuation. PMID:26203365

  13. The protease activity of yeast separase (esp1) is required for anaphase spindle elongation independently of its role in cleavage of cohesin.

    PubMed

    Baskerville, Chris; Segal, Marisa; Reed, Steven I

    2008-04-01

    Separase is a caspase-family protease required for the metaphase-anaphase transition in eukaryotes. In budding yeast, the separase ortholog, Esp1, has been shown to cleave a subunit of cohesin, Mcd1 (Scc1), thereby releasing sister chromatids from cohesion and allowing anaphase. However, whether Esp1 has other substrates required for anaphase has been controversial. Whereas it has been reported that cleavage of Mcd1 is sufficient to trigger anaphase in the absence of Esp1 activation, another study using a temperature-sensitive esp1 mutant concluded that depletion of Mcd1 was not sufficient for anaphase in the absence of Esp1 function. Here we revisit the issue and demonstrate that neither depletion of Mcd1 nor ectopic cleavage of Mcd1 by Tev1 protease is sufficient to support anaphase in an esp1 temperature-sensitive mutant. Furthermore, we demonstrate that the catalytic activity of the Esp1 protease is required for this Mcd1-independent anaphase function. These data suggest that another protein, possibly a spindle-associated protein, is cleaved by Esp1 to allow anaphase. Such a function is consistent with the previous observation that Esp1 localizes to the mitotic spindle during anaphase.

  14. Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy.

    PubMed

    Jobst, Markus A; Milles, Lukas F; Schoeler, Constantin; Ott, Wolfgang; Fried, Daniel B; Bayer, Edward A; Gaub, Hermann E; Nash, Michael A

    2015-10-31

    Receptor-ligand pairs are ordinarily thought to interact through a lock and key mechanism, where a unique molecular conformation is formed upon binding. Contrary to this paradigm, cellulosomal cohesin-dockerin (Coh-Doc) pairs are believed to interact through redundant dual binding modes consisting of two distinct conformations. Here, we combined site-directed mutagenesis and single-molecule force spectroscopy (SMFS) to study the unbinding of Coh:Doc complexes under force. We designed Doc mutations to knock out each binding mode, and compared their single-molecule unfolding patterns as they were dissociated from Coh using an atomic force microscope (AFM) cantilever. Although average bulk measurements were unable to resolve the differences in Doc binding modes due to the similarity of the interactions, with a single-molecule method we were able to discriminate the two modes based on distinct differences in their mechanical properties. We conclude that under native conditions wild-type Doc from Clostridium thermocellum exocellulase Cel48S populates both binding modes with similar probabilities. Given the vast number of Doc domains with predicted dual binding modes across multiple bacterial species, our approach opens up new possibilities for understanding assembly and catalytic properties of a broad range of multi-enzyme complexes.

  15. Imprinting at the PLAGL1 domain is contained within a 70-kb CTCF/cohesin-mediated non-allelic chromatin loop

    PubMed Central

    Iglesias-Platas, Isabel; Court, Franck; Camprubi, Cristina; Sparago, Angela; Guillaumet-Adkins, Amy; Martin-Trujillo, Alex; Riccio, Andrea; Moore, Gudrun E.; Monk, David

    2013-01-01

    Paternal duplications of chromosome 6q24, a region that contains the imprinted PLAGL1 and HYMAI transcripts, are associated with transient neonatal diabetes mellitus. A common feature of imprinted genes is that they tend to cluster together, presumably as a result of sharing common cis-acting regulatory elements. To determine the extent of this imprinted cluster in human and mouse, we have undertaken a systematic analysis of allelic expression and DNA methylation of the genes mapping within an ∼1.4-Mb region flanking PLAGL1/Plagl1. We confirm that all nine neighbouring genes are biallelically expressed in both species. In human we identify two novel paternally expressed PLAGL1 coding transcripts that originate from unique promoter regions. Chromatin immunoprecipitation for CTCF and the cohesin subunits RAD21 and SMC3 reveals evolutionarily conserved binding sites within unmethylated regions ∼5 kb downstream of the PLAGL1 differentially methylated region and within the PLAGL1 3′ untranslated region (UTR). Higher-order chromatin looping occurs between these regions in both expressing and non-expressing tissues, forming a non-allelic chromatin loop around the PLAGL1/Plagl1 gene. In placenta and brain tissues, we identify an additional interaction between the PLAGL1 P3/P4 promoters and the unmethylated element downstream of the PLAGL1 differentially methylated region that we propose facilitates imprinted expression of these alternative isoforms. PMID:23295672

  16. Meiosis-Specific Cohesin Component, Stag3 Is Essential for Maintaining Centromere Chromatid Cohesion, and Required for DNA Repair and Synapsis between Homologous Chromosomes

    PubMed Central

    Hopkins, Jessica; Bedigian, Rick; Oka, Kazuhiro; Overbeek, Paul; Murray, Steve; Jordan, Philip W.

    2014-01-01

    Cohesins are important for chromosome structure and chromosome segregation during mitosis and meiosis. Cohesins are composed of two structural maintenance of chromosomes (SMC1-SMC3) proteins that form a V-shaped heterodimer structure, which is bridged by a α-kleisin protein and a stromal antigen (STAG) protein. Previous studies in mouse have shown that there is one SMC1 protein (SMC1β), two α-kleisins (RAD21L and REC8) and one STAG protein (STAG3) that are meiosis-specific. During meiosis, homologous chromosomes must recombine with one another in the context of a tripartite structure known as the synaptonemal complex (SC). From interaction studies, it has been shown that there are at least four meiosis-specific forms of cohesin, which together with the mitotic cohesin complex, are lateral components of the SC. STAG3 is the only meiosis-specific subunit that is represented within all four meiosis-specific cohesin complexes. In Stag3 mutant germ cells, the protein level of other meiosis-specific cohesin subunits (SMC1β, RAD21L and REC8) is reduced, and their localization to chromosome axes is disrupted. In contrast, the mitotic cohesin complex remains intact and localizes robustly to the meiotic chromosome axes. The instability of meiosis-specific cohesins observed in Stag3 mutants results in aberrant DNA repair processes, and disruption of synapsis between homologous chromosomes. Furthermore, mutation of Stag3 results in perturbation of pericentromeric heterochromatin clustering, and disruption of centromere cohesion between sister chromatids during meiotic prophase. These defects result in early prophase I arrest and apoptosis in both male and female germ cells. The meiotic defects observed in Stag3 mutants are more severe when compared to single mutants for Smc1β, Rec8 and Rad21l, however they are not as severe as the Rec8, Rad21l double mutants. Taken together, our study demonstrates that STAG3 is required for the stability of all meiosis-specific cohesin

  17. Meiosis-specific cohesin component, Stag3 is essential for maintaining centromere chromatid cohesion, and required for DNA repair and synapsis between homologous chromosomes.

    PubMed

    Hopkins, Jessica; Hwang, Grace; Jacob, Justin; Sapp, Nicklas; Bedigian, Rick; Oka, Kazuhiro; Overbeek, Paul; Murray, Steve; Jordan, Philip W

    2014-07-01

    Cohesins are important for chromosome structure and chromosome segregation during mitosis and meiosis. Cohesins are composed of two structural maintenance of chromosomes (SMC1-SMC3) proteins that form a V-shaped heterodimer structure, which is bridged by a α-kleisin protein and a stromal antigen (STAG) protein. Previous studies in mouse have shown that there is one SMC1 protein (SMC1β), two α-kleisins (RAD21L and REC8) and one STAG protein (STAG3) that are meiosis-specific. During meiosis, homologous chromosomes must recombine with one another in the context of a tripartite structure known as the synaptonemal complex (SC). From interaction studies, it has been shown that there are at least four meiosis-specific forms of cohesin, which together with the mitotic cohesin complex, are lateral components of the SC. STAG3 is the only meiosis-specific subunit that is represented within all four meiosis-specific cohesin complexes. In Stag3 mutant germ cells, the protein level of other meiosis-specific cohesin subunits (SMC1β, RAD21L and REC8) is reduced, and their localization to chromosome axes is disrupted. In contrast, the mitotic cohesin complex remains intact and localizes robustly to the meiotic chromosome axes. The instability of meiosis-specific cohesins observed in Stag3 mutants results in aberrant DNA repair processes, and disruption of synapsis between homologous chromosomes. Furthermore, mutation of Stag3 results in perturbation of pericentromeric heterochromatin clustering, and disruption of centromere cohesion between sister chromatids during meiotic prophase. These defects result in early prophase I arrest and apoptosis in both male and female germ cells. The meiotic defects observed in Stag3 mutants are more severe when compared to single mutants for Smc1β, Rec8 and Rad21l, however they are not as severe as the Rec8, Rad21l double mutants. Taken together, our study demonstrates that STAG3 is required for the stability of all meiosis-specific cohesin

  18. Interspecies activity correlations reveal functional correspondence between monkey and human brain areas.

    PubMed

    Mantini, Dante; Hasson, Uri; Betti, Viviana; Perrucci, Mauro G; Romani, Gian Luca; Corbetta, Maurizio; Orban, Guy A; Vanduffel, Wim

    2012-02-05

    Evolution-driven functional changes in the primate brain are typically assessed by aligning monkey and human activation maps using cortical surface expansion models. These models use putative homologous areas as registration landmarks, assuming they are functionally correspondent. For cases in which functional changes have occurred in an area, this assumption prohibits to reveal whether other areas may have assumed lost functions. Here we describe a method to examine functional correspondences across species. Without making spatial assumptions, we assessed similarities in sensory-driven functional magnetic resonance imaging responses between monkey (Macaca mulatta) and human brain areas by temporal correlation. Using natural vision data, we revealed regions for which functional processing has shifted to topologically divergent locations during evolution. We conclude that substantial evolution-driven functional reorganizations have occurred, not always consistent with cortical expansion processes. This framework for evaluating changes in functional architecture is crucial to building more accurate evolutionary models.

  19. A Statistical Model of Protein Sequence Similarity and Function Similarity Reveals Overly-Specific Function Predictions

    PubMed Central

    Kolker, Eugene

    2009-01-01

    Background Predicting protein function from primary sequence is an important open problem in modern biology. Not only are there many thousands of proteins of unknown function, current approaches for predicting function must be improved upon. One problem in particular is overly-specific function predictions which we address here with a new statistical model of the relationship between protein sequence similarity and protein function similarity. Methodology Our statistical model is based on sets of proteins with experimentally validated functions and numeric measures of function specificity and function similarity derived from the Gene Ontology. The model predicts the similarity of function between two proteins given their amino acid sequence similarity measured by statistics from the BLAST sequence alignment algorithm. A novel aspect of our model is that it predicts the degree of function similarity shared between two proteins over a continuous range of sequence similarity, facilitating prediction of function with an appropriate level of specificity. Significance Our model shows nearly exact function similarity for proteins with high sequence similarity (bit score >244.7, e-value >1e−62, non-redundant NCBI protein database (NRDB)) and only small likelihood of specific function match for proteins with low sequence similarity (bit score <54.6, e-value <1e−05, NRDB). For sequence similarity ranges in between our annotation model shows an increasing relationship between function similarity and sequence similarity, but with considerable variability. We applied the model to a large set of proteins of unknown function, and predicted functions for thousands of these proteins ranging from general to very specific. We also applied the model to a data set of proteins with previously assigned, specific functions that were electronically based. We show that, on average, these prior function predictions are more specific (quite possibly overly-specific) compared to

  20. Revealing Ozgur's Thoughts of a Quadratic Function with a Clinical Interview: Concepts and Their Underlying Reasons

    ERIC Educational Resources Information Center

    Ozaltun Celik, Aytug; Bukova Guzel, Esra

    2017-01-01

    The quadratic function is an important concept for calculus but the students at high school have many difficulties related to this concept. It is important that the teaching of the quadratic function is realized considering the students' thinking. In this context, the aim of this study conducted through a qualitative case study is to reveal the…

  1. Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality

    PubMed Central

    Wu, Nicholas C.; Olson, C. Anders; Du, Yushen; Le, Shuai; Tran, Kevin; Remenyi, Roland; Gong, Danyang; Al-Mawsawi, Laith Q.; Qi, Hangfei; Wu, Ting-Ting; Sun, Ren

    2015-01-01

    Viruses often encode proteins with multiple functions due to their compact genomes. Existing approaches to identify functional residues largely rely on sequence conservation analysis. Inferring functional residues from sequence conservation can produce false positives, in which the conserved residues are functionally silent, or false negatives, where functional residues are not identified since they are species-specific and therefore non-conserved. Furthermore, the tedious process of constructing and analyzing individual mutations limits the number of residues that can be examined in a single study. Here, we developed a systematic approach to identify the functional residues of a viral protein by coupling experimental fitness profiling with protein stability prediction using the influenza virus polymerase PA subunit as the target protein. We identified a significant number of functional residues that were influenza type-specific and were evolutionarily non-conserved among different influenza types. Our results indicate that type-specific functional residues are prevalent and may not otherwise be identified by sequence conservation analysis alone. More importantly, this technique can be adapted to any viral (and potentially non-viral) protein where structural information is available. PMID:26132554

  2. Preliminary X-ray characterization of a novel type of anchoring cohesin from the cellulosome of Ruminococcus flavefaciens

    SciTech Connect

    Alber, Orly; Noach, Ilit; Lamed, Raphael; Shimon, Linda J. W.; Bayer, Edward A.; Frolow, Felix

    2008-02-01

    The cloning, expression, purification, crystallization and preliminary X-ray characterization of a novel class of cohesin module (type III) from the R. flavefaciens ScaE anchoring scaffoldin are described. Ruminococcus flavefaciens is an anaerobic bacterium that resides in the gastrointestinal tract of ruminants. It produces a highly organized multi-enzyme cellulosome complex that plays a key role in the degradation of plant cell walls. ScaE is one of the critical structural components of its cellulosome that serves to anchor the complex to the cell wall. The seleno-l-methionine-labelled derivative of the ScaE cohesin module has been cloned, expressed, purified and crystallized. The crystals belong to space group C2, with unit-cell parameters a = 155.6, b = 69.3, c = 93.0 Å, β = 123.4°, and contain four molecules in the asymmetric unit. Diffraction data were phased to 1.95 Å using the anomalous signal from the Se atoms.

  3. Dynamic functional connectivity reveals altered variability in functional connectivity among patients with major depressive disorder

    PubMed Central

    Tornador, Cristian; Falcón, Carles; López‐Solà, Marina; Hernández‐Ribas, Rosa; Pujol, Jesús; Menchón, José M.; Ritter, Petra; Cardoner, Narcis; Soriano‐Mas, Carles; Deco, Gustavo

    2016-01-01

    Abstract Resting‐state fMRI (RS‐fMRI) has become a useful tool to investigate the connectivity structure of mental health disorders. In the case of major depressive disorder (MDD), recent studies regarding the RS‐fMRI have found abnormal connectivity in several regions of the brain, particularly in the default mode network (DMN). Thus, the relevance of the DMN to self‐referential thoughts and ruminations has made the use of the resting‐state approach particularly important for MDD. The majority of such research has relied on the grand averaged functional connectivity measures based on the temporal correlations between the BOLD time series of various brain regions. We, in our study, investigated the variations in the functional connectivity over time at global and local level using RS‐fMRI BOLD time series of 27 MDD patients and 27 healthy control subjects. We found that global synchronization and temporal stability were significantly increased in the MDD patients. Furthermore, the participants with MDD showed significantly increased overall average (static) functional connectivity (sFC) but decreased variability of functional connectivity (vFC) within specific networks. Static FC increased to predominance among the regions pertaining to the default mode network (DMN), while the decreased variability of FC was observed in the connections between the DMN and the frontoparietal network. Hum Brain Mapp 37:2918–2930, 2016. © 2016 Wiley Periodicals, Inc. PMID:27120982

  4. Dynamic functional connectivity reveals altered variability in functional connectivity among patients with major depressive disorder.

    PubMed

    Demirtaş, Murat; Tornador, Cristian; Falcón, Carles; López-Solà, Marina; Hernández-Ribas, Rosa; Pujol, Jesús; Menchón, José M; Ritter, Petra; Cardoner, Narcis; Soriano-Mas, Carles; Deco, Gustavo

    2016-08-01

    Resting-state fMRI (RS-fMRI) has become a useful tool to investigate the connectivity structure of mental health disorders. In the case of major depressive disorder (MDD), recent studies regarding the RS-fMRI have found abnormal connectivity in several regions of the brain, particularly in the default mode network (DMN). Thus, the relevance of the DMN to self-referential thoughts and ruminations has made the use of the resting-state approach particularly important for MDD. The majority of such research has relied on the grand averaged functional connectivity measures based on the temporal correlations between the BOLD time series of various brain regions. We, in our study, investigated the variations in the functional connectivity over time at global and local level using RS-fMRI BOLD time series of 27 MDD patients and 27 healthy control subjects. We found that global synchronization and temporal stability were significantly increased in the MDD patients. Furthermore, the participants with MDD showed significantly increased overall average (static) functional connectivity (sFC) but decreased variability of functional connectivity (vFC) within specific networks. Static FC increased to predominance among the regions pertaining to the default mode network (DMN), while the decreased variability of FC was observed in the connections between the DMN and the frontoparietal network. Hum Brain Mapp 37:2918-2930, 2016. © 2016 Wiley Periodicals, Inc.

  5. Scaling behavior in turbulent Rayleigh-Bénard convection revealed by conditional structure functions.

    PubMed

    Ching, Emily S C; Tsang, Yue-Kin; Fok, T N; He, Xiaozhou; Tong, Penger

    2013-01-01

    We show that the nature of the scaling behavior can be revealed by studying the conditional structure functions evaluated at given values of the locally averaged thermal dissipation rate. These conditional structure functions have power-law dependence on the value of the locally averaged thermal dissipation rate, and such dependence for the Bolgiano-Obukhov scaling is different from the other scaling behaviors. Our analysis of experimental measurements verifies the power-law dependence and reveals the Bolgiano-Obukhov scaling behavior at the center of the bottom plate of the convection cell.

  6. Inactivation of the budding yeast cohesin loader Scc2 alters gene expression both globally and in response to a single DNA double strand break.

    PubMed

    Lindgren, Emma; Hägg, Sara; Giordano, Fosco; Björkegren, Johan; Ström, Lena

    2014-01-01

    Genome integrity is fundamental for cell survival and cell cycle progression. Important mechanisms for keeping the genome intact are proper sister chromatid segregation, correct gene regulation and efficient repair of damaged DNA. Cohesin and its DNA loader, the Scc2/4 complex have been implicated in all these cellular actions. The gene regulation role has been described in several organisms. In yeast it has been suggested that the proteins in the cohesin network would effect transcription based on its role as insulator. More recently, data are emerging indicating direct roles for gene regulation also in yeast. Here we extend these studies by investigating whether the cohesin loader Scc2 is involved in regulation of gene expression. We performed global gene expression profiling in the absence and presence of DNA damage, in wild type and Scc2 deficient G2/M arrested cells, when it is known that Scc2 is important for DNA double strand break repair and formation of damage induced cohesion. We found that not only the DNA damage specific transcriptional response is distorted after inactivation of Scc2 but also the overall transcription profile. Interestingly, these alterations did not correlate with changes in cohesin binding.

  7. Functional Analysis of GLRX5 Mutants Reveals Distinct Functionalities of GLRX5 Protein.

    PubMed

    Liu, Gang; Wang, Yongwei; Anderson, Gregory J; Camaschella, Clara; Chang, Yanzhong; Nie, Guangjun

    2016-01-01

    Glutaredoxin 5 (GLRX5) is a 156 amino acid mitochondrial protein that plays an essential role in mitochondrial iron-sulfur cluster transfer. Mutations in this protein were reported to result in sideroblastic anemia and variant nonketotic hyperglycinemia in human. Recently, we have characterized a Chinese congenital sideroblastic anemia patient who has two compound heterozygous missense mutations (c. 301 A>C and c. 443 T>C) in his GLRX5 gene. Herein, we developed a GLRX5 knockout K562 cell line and studied the biochemical functions of the identified pathogenic mutations and other conserved amino acids with predicted essential functions. We observed that the K101Q mutation (due to c. 301 A>C mutation) may prevent the binding of [Fe-S] to GLRX5 protein, while L148S (due to c. 443 T>C mutation) may interfere with [Fe-S] transfer from GLRX5 to iron regulatory protein 1 (IRP1), mitochondrial aconitase (m-aconitase) and ferrochelatase. We also demonstrated that L148S is functionally complementary to the K51del mutant with respect to Fe/S-ferrochelatase, Fe/S-IRP1, Fe/S-succinate dehydrogenase, and Fe/S-m-aconitase biosynthesis and lipoylation of pyruvate dehydrogenase complex and α-ketoglutarate dehydrogenase complex. Furthermore, we demonstrated that the mutations of highly conserved amino acid residues in GLRX5 protein can have different effects on downstream Fe/S proteins. Collectively, our current work demonstrates that GLRX5 protein is multifunctional in [Fe-S] protein synthesis and maturation and defects of the different amino acids of the protein will lead to distinct effects on downstream Fe/S biosynthesis.

  8. Metagenomic analysis reveals significant changes of microbial compositions and protective functions during drinking water treatment.

    PubMed

    Chao, Yuanqing; Ma, Liping; Yang, Ying; Ju, Feng; Zhang, Xu-Xiang; Wu, Wei-Min; Zhang, Tong

    2013-12-19

    The metagenomic approach was applied to characterize variations of microbial structure and functions in raw (RW) and treated water (TW) in a drinking water treatment plant (DWTP) at Pearl River Delta, China. Microbial structure was significantly influenced by the treatment processes, shifting from Gammaproteobacteria and Betaproteobacteria in RW to Alphaproteobacteria in TW. Further functional analysis indicated the basic metabolic functions of microorganisms in TW did not vary considerably. However, protective functions, i.e. glutathione synthesis genes in 'oxidative stress' and 'detoxification' subsystems, significantly increased, revealing the surviving bacteria may have higher chlorine resistance. Similar results were also found in glutathione metabolism pathway, which identified the major reaction for glutathione synthesis and supported more genes for glutathione metabolism existed in TW. This metagenomic study largely enhanced our knowledge about the influences of treatment processes, especially chlorination, on bacterial community structure and protective functions (e.g. glutathione metabolism) in ecosystems of DWTPs.

  9. Dynamic functional network connectivity reveals unique and overlapping profiles of insula subdivisions.

    PubMed

    Nomi, Jason S; Farrant, Kristafor; Damaraju, Eswar; Rachakonda, Srinivas; Calhoun, Vince D; Uddin, Lucina Q

    2016-05-01

    The human insular cortex consists of functionally diverse subdivisions that engage during tasks ranging from interoception to cognitive control. The multiplicity of functions subserved by insular subdivisions calls for a nuanced investigation of their functional connectivity profiles. Four insula subdivisions (dorsal anterior, dAI; ventral, VI; posterior, PI; middle, MI) derived using a data-driven approach were subjected to static- and dynamic functional network connectivity (s-FNC and d-FNC) analyses. Static-FNC analyses replicated previous work demonstrating a cognition-emotion-interoception division of the insula, where the dAI is functionally connected to frontal areas, the VI to limbic areas, and the PI and MI to sensorimotor areas. Dynamic-FNC analyses consisted of k-means clustering of sliding windows to identify variable insula connectivity states. The d-FNC analysis revealed that the most frequently occurring dynamic state mirrored the cognition-emotion-interoception division observed from the s-FNC analysis, with less frequently occurring states showing overlapping and unique subdivision connectivity profiles. In two of the states, all subdivisions exhibited largely overlapping profiles, consisting of subcortical, sensory, motor, and frontal connections. Two other states showed the dAI exhibited a unique connectivity profile compared with other insula subdivisions. Additionally, the dAI exhibited the most variable functional connections across the s-FNC and d-FNC analyses, and was the only subdivision to exhibit dynamic functional connections with regions of the default mode network. These results highlight how a d-FNC approach can capture functional dynamics masked by s-FNC approaches, and reveal dynamic functional connections enabling the functional flexibility of the insula across time. Hum Brain Mapp 37:1770-1787, 2016. © 2016 Wiley Periodicals, Inc.

  10. Statistical method for revealing form-function relations in biological networks

    PubMed Central

    Mugler, Andrew; Grinshpun, Boris; Franks, Riley

    2011-01-01

    Over the past decade, a number of researchers in systems biology have sought to relate the function of biological systems to their network-level descriptions—lists of the most important players and the pairwise interactions between them. Both for large networks (in which statistical analysis is often framed in terms of the abundance of repeated small subgraphs) and for small networks which can be analyzed in greater detail (or even synthesized in vivo and subjected to experiment), revealing the relationship between the topology of small subgraphs and their biological function has been a central goal. We here seek to pose this revelation as a statistical task, illustrated using a particular setup which has been constructed experimentally and for which parameterized models of transcriptional regulation have been studied extensively. The question “how does function follow form” is here mathematized by identifying which topological attributes correlate with the diverse possible information-processing tasks which a transcriptional regulatory network can realize. The resulting method reveals one form-function relationship which had earlier been predicted based on analytic results, and reveals a second for which we can provide an analytic interpretation. Resulting source code is distributed via http://formfunction.sourceforge.net. PMID:21183719

  11. Large-Scale Meta-Analysis of Human Medial Frontal Cortex Reveals Tripartite Functional Organization

    PubMed Central

    Chang, Luke J.; Banich, Marie T.; Wager, Tor D.; Yarkoni, Tal

    2016-01-01

    The functional organization of human medial frontal cortex (MFC) is a subject of intense study. Using fMRI, the MFC has been associated with diverse psychological processes, including motor function, cognitive control, affect, and social cognition. However, there have been few large-scale efforts to comprehensively map specific psychological functions to subregions of medial frontal anatomy. Here we applied a meta-analytic data-driven approach to nearly 10,000 fMRI studies to identify putatively separable regions of MFC and determine which psychological states preferentially recruit their activation. We identified regions at several spatial scales on the basis of meta-analytic coactivation, revealing three broad functional zones along a rostrocaudal axis composed of 2–4 smaller subregions each. Multivariate classification analyses aimed at identifying the psychological functions most strongly predictive of activity in each region revealed a tripartite division within MFC, with each zone displaying a relatively distinct functional signature. The posterior zone was associated preferentially with motor function, the middle zone with cognitive control, pain, and affect, and the anterior with reward, social processing, and episodic memory. Within each zone, the more fine-grained subregions showed distinct, but subtler, variations in psychological function. These results provide hypotheses about the functional organization of medial prefrontal cortex that can be tested explicitly in future studies. SIGNIFICANCE STATEMENT Activation of medial frontal cortex in fMRI studies is associated with a wide range of psychological states ranging from cognitive control to pain. However, this high rate of activation makes it challenging to determine how these various processes are topologically organized across medial frontal anatomy. We conducted a meta-analysis across nearly 10,000 studies to comprehensively map psychological states to discrete subregions in medial frontal cortex

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

    SciTech Connect

    Merchant, Sabeeha S

    2007-04-09

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

  13. Thermodynamic and functional characteristics of deep-sea enzymes revealed by pressure effects.

    PubMed

    Ohmae, Eiji; Miyashita, Yurina; Kato, Chiaki

    2013-09-01

    Hydrostatic pressure analysis is an ideal approach for studying protein dynamics and hydration. The development of full ocean depth submersibles and high pressure biological techniques allows us to investigate enzymes from deep-sea organisms at the molecular level. The aim of this review was to overview the thermodynamic and functional characteristics of deep-sea enzymes as revealed by pressure axis analysis after giving a brief introduction to the thermodynamic principles underlying the effects of pressure on the structural stability and function of enzymes.

  14. A comprehensive excitatory input map of the striatum reveals novel functional organization

    PubMed Central

    Hunnicutt, Barbara J; Jongbloets, Bart C; Birdsong, William T; Gertz, Katrina J; Zhong, Haining; Mao, Tianyi

    2016-01-01

    The striatum integrates excitatory inputs from the cortex and the thalamus to control diverse functions. Although the striatum is thought to consist of sensorimotor, associative and limbic domains, their precise demarcations and whether additional functional subdivisions exist remain unclear. How striatal inputs are differentially segregated into each domain is also poorly understood. This study presents a comprehensive map of the excitatory inputs to the mouse striatum. The input patterns reveal boundaries between the known striatal domains. The most posterior striatum likely represents the 4th functional subdivision, and the dorsomedial striatum integrates highly heterogeneous, multimodal inputs. The complete thalamo-cortico-striatal loop is also presented, which reveals that the thalamic subregions innervated by the basal ganglia preferentially interconnect with motor-related cortical areas. Optogenetic experiments show the subregion-specific heterogeneity in the synaptic properties of striatal inputs from both the cortex and the thalamus. This projectome will guide functional studies investigating diverse striatal functions. DOI: http://dx.doi.org/10.7554/eLife.19103.001 PMID:27892854

  15. Systematic probing of the bacterial RNA structurome to reveal new functions.

    PubMed

    Ignatova, Zoya; Narberhaus, Franz

    2017-02-01

    RNA folds into intricate structures. Recent discoveries using next-generation sequencing (NGS) approaches have revealed unprecedented structural complexity with a pivotal role in regulating RNA function and stability. Here, we present new discoveries from the transcriptome-wide determination of RNA structuromes in bacteria and discuss emerging concepts in the role of mRNA structures in regulating transcription, translation and degradation. We also provide critical viewpoints on the use of NGS approaches for elucidating of RNA structuromes at the systems level.

  16. Functional Traits Reveal Processes Driving Natural Afforestation at Large Spatial Scales

    PubMed Central

    Mason, Norman W. H.; Wiser, Susan K.; Richardson, Sarah J.; Thorsen, Michael J.; Holdaway, Robert J.; Dray, Stéphane; Thomson, Fiona J.; Carswell, Fiona E.

    2013-01-01

    An understanding of the processes governing natural afforestation over large spatial scales is vital for enhancing forest carbon sequestration. Models of tree species occurrence probability in non-forest vegetation could potentially identify the primary variables determining natural afforestation. However, inferring processes governing afforestation using tree species occurrence is potentially problematic, since it is impossible to know whether observed occurrences are due to recruitment or persistence of existing trees following disturbance. Plant functional traits have the potential to reveal the processes by which key environmental and land cover variables influence afforestation. We used 10,061 survey plots to identify the primary environmental and land cover variables influencing tree occurrence probability in non-forest vegetation in New Zealand. We also examined how these variables influenced diversity of functional traits linked to plant ecological strategy and dispersal ability. Mean annual temperature was the most important environmental predictor of tree occurrence. Local woody cover and distance to forest were the most important land cover variables. Relationships between these variables and ecological strategy traits revealed a trade-off between ability to compete for light and colonize sites that were marginal for tree occurrence. Biotically dispersed species occurred less frequently with declining temperature and local woody cover, suggesting that abiotic stress limited their establishment and that biotic dispersal did not increase ability to colonize non-woody vegetation. Functional diversity for ecological strategy traits declined with declining temperature and woody cover and increasing distance to forest. Functional diversity for dispersal traits showed the opposite trend. This suggests that low temperatures and woody cover and high distance to forest may limit tree species establishment through filtering on ecological strategy traits, but not on

  17. Functional traits reveal processes driving natural afforestation at large spatial scales.

    PubMed

    Mason, Norman W H; Wiser, Susan K; Richardson, Sarah J; Thorsen, Michael J; Holdaway, Robert J; Dray, Stéphane; Thomson, Fiona J; Carswell, Fiona E

    2013-01-01

    An understanding of the processes governing natural afforestation over large spatial scales is vital for enhancing forest carbon sequestration. Models of tree species occurrence probability in non-forest vegetation could potentially identify the primary variables determining natural afforestation. However, inferring processes governing afforestation using tree species occurrence is potentially problematic, since it is impossible to know whether observed occurrences are due to recruitment or persistence of existing trees following disturbance. Plant functional traits have the potential to reveal the processes by which key environmental and land cover variables influence afforestation. We used 10,061 survey plots to identify the primary environmental and land cover variables influencing tree occurrence probability in non-forest vegetation in New Zealand. We also examined how these variables influenced diversity of functional traits linked to plant ecological strategy and dispersal ability. Mean annual temperature was the most important environmental predictor of tree occurrence. Local woody cover and distance to forest were the most important land cover variables. Relationships between these variables and ecological strategy traits revealed a trade-off between ability to compete for light and colonize sites that were marginal for tree occurrence. Biotically dispersed species occurred less frequently with declining temperature and local woody cover, suggesting that abiotic stress limited their establishment and that biotic dispersal did not increase ability to colonize non-woody vegetation. Functional diversity for ecological strategy traits declined with declining temperature and woody cover and increasing distance to forest. Functional diversity for dispersal traits showed the opposite trend. This suggests that low temperatures and woody cover and high distance to forest may limit tree species establishment through filtering on ecological strategy traits, but not on

  18. Genetic diversity of coastal bottlenose dolphins revealed by structurally and functionally diverse hemoglobins.

    PubMed

    Remington, Nicole; Stevens, Robert D; Wells, Randall S; Holn, Aleta; Dhungana, Suraj; Taboy, Celine H; Crumbliss, Alvin L; Henkens, Robert; Bonaventura, Celia

    2007-08-15

    Studies of structure-function relationships in the respiratory proteins of marine mammals revealed unexpected variations in the number and types of hemoglobins (Hbs) present in coastal bottlenose dolphins, Tursiops truncatus. We obtained blood samples from free-ranging coastal bottlenose dolphins as a component of capture-release studies. We found that the oxygen-binding functions of bottlenose dolphin blood are poised between effector-saturated and unsaturated levels, enabling exercise-dependent shifts in oxygen transfer functions. Isolated bottlenose dolphin Hbs showed elevated pH sensitivities (Bohr effects) and appreciably lower oxygen affinities than adult human Hb in the absence of allosteric effectors. These properties may be an adaptive modification that enhances oxygen delivery during diving episodes when oxygen tensions and effector levels are low. The Hbs of individual dolphins showed similar oxygen affinities, responses to effectors, and expression of heme-heme interaction in oxygen binding, but differed in their redox potentials and rates of autoxidation. The heterogeneity suggested by these functional variations in Hbs of individual dolphins was born out by variations in the molecular weights and numbers of their alpha and beta globin chains. Although coastal bottlenose dolphins were expected to have a single type of Hb, the mass differences observed revealed considerable genetic diversity. There were multiple Hb forms in some individuals and differences in Hb patterns among individuals within the same community.

  19. Modular organization of the white spruce (Picea glauca) transcriptome reveals functional organization and evolutionary signatures.

    PubMed

    Raherison, Elie S M; Giguère, Isabelle; Caron, Sébastien; Lamara, Mebarek; MacKay, John J

    2015-07-01

    Transcript profiling has shown the molecular bases of several biological processes in plants but few studies have developed an understanding of overall transcriptome variation. We investigated transcriptome structure in white spruce (Picea glauca), aiming to delineate its modular organization and associated functional and evolutionary attributes. Microarray analyses were used to: identify and functionally characterize groups of co-expressed genes; investigate expressional and functional diversity of vascular tissue preferential genes which were conserved among Picea species, and identify expression networks underlying wood formation. We classified 22 857 genes as variable (79%; 22 coexpression groups) or invariant (21%) by profiling across several vegetative tissues. Modular organization and complex transcriptome restructuring among vascular tissue preferential genes was revealed by their assignment to coexpression groups with partially overlapping profiles and partially distinct functions. Integrated analyses of tissue-based and temporally variable profiles identified secondary xylem gene networks, showed their remodelling over a growing season and identified PgNAC-7 (no apical meristerm (NAM), Arabidopsis transcription activation factor (ATAF) and cup-shaped cotyledon (CUC) transcription factor 007 in Picea glauca) as a major hub gene specific to earlywood formation. Reference profiling identified comprehensive, statistically robust coexpressed groups, revealing that modular organization underpins the evolutionary conservation of the transcriptome structure.

  20. Meta-Analytic Connectivity Modeling Reveals Differential Functional Connectivity of the Medial and Lateral Orbitofrontal Cortex

    PubMed Central

    Zald, David H.; McHugo, Maureen; Ray, Kimberly L.; Glahn, David C.; Eickhoff, Simon B.; Laird, Angela R.

    2014-01-01

    The orbitofrontal cortex (OFC) is implicated in a broad range of behaviors and neuropsychiatric disorders. Anatomical tracing studies in nonhuman primates reveal differences in connectivity across subregions of the OFC, but data on the connectivity of the human OFC remain limited. We applied meta-analytic connectivity modeling in order to examine which brain regions are most frequently coactivated with the medial and lateral portions of the OFC in published functional neuroimaging studies. The analysis revealed a clear divergence in the pattern of connectivity for the medial OFC (mOFC) and lateral OFC (lOFC) regions. The lOFC showed coactivations with a network of prefrontal regions and areas involved in cognitive functions including language and memory. In contrast, the mOFC showed connectivity with default mode, autonomic, and limbic regions. Convergent patterns of coactivations were observed in the amygdala, hippocampus, striatum, and thalamus. A small number of regions showed connectivity specific to the anterior or posterior sectors of the OFC. Task domains involving memory, semantic processing, face processing, and reward were additionally analyzed in order to identify the different patterns of OFC functional connectivity associated with specific cognitive and affective processes. These data provide a framework for understanding the human OFC's position within widespread functional networks. PMID:23042731

  1. Characterizing Thalamocortical Disturbances in Cervical Spondylotic Myelopathy: Revealed by Functional Connectivity under Two Slow Frequency Bands

    PubMed Central

    Zhou, Fuqing; Wu, Lin; Liu, Xiaojia; Gong, Honghan; Luk, Keith Dip-Kei; Hu, Yong

    2015-01-01

    Background and Purpose Recent advanced MRI studies on cervical spondylotic myelopathy (CSM) revealed alterations of sensorimotor cortex, but the disturbances of large-scale thalamocortical systems remains elusive. The purpose of this study was to characterizing the CSM-related thalamocortical disturbances, which were associated with spinal cord structural injury, and clinical measures. Methods A total of 17 patients with degenerative CSM and well-matched control subjects participated. Thalamocortical disturbances were quantified using thalamus seed-based functional connectivity in two distinct low frequencies bands (slow-5 and slow-4), with different neural manifestations. The clinical measures were evaluated by Japanese Orthopaedic Association (JOA) score system and Neck Disability Index (NDI) questionnaires. Results Decreased functional connectivity was found in the thalamo-motor, -somatosensory, and -temporal circuits in the slow-5 band, indicating impairment of thalamo-cortical circuit degeneration or axon/synaptic impairment. By contrast, increased functional connectivity between thalami and the bilateral primary motor (M1), primary and secondary somatosensory (S1/S2), premotor cortex (PMC), and right temporal cortex was detected in the slow-4 band, and were associated with higher fractional anisotropy values in the cervical cord, corresponding to mild spinal cord structural injury. Conclusions These thalamocortical disturbances revealed by two slow frequency bands inform basic understanding and vital clues about the sensorimotor dysfunction in CSM. Further work is needed to evaluate its contribution in central functional reorganization during spinal cord degeneration. PMID:26053316

  2. Yeast gain-of-function mutations reveal structure–function relationships conserved among different subfamilies of transient receptor potential channels

    PubMed Central

    Su, Zhenwei; Zhou, Xinliang; Haynes, W. John; Loukin, Stephen H.; Anishkin, Andriy; Saimi, Yoshiro; Kung, Ching

    2007-01-01

    Transient receptor potential (TRP) channels found in animals, protists, and fungi are primary chemo-, thermo-, or mechanosensors. Current research emphasizes the characteristics of individual channels in each animal TRP subfamily but not the mechanisms common across subfamilies. A forward genetic screen of the TrpY1, the yeast TRP channel, recovered gain-of-function (GOF) mutations with phenotype in vivo and in vitro. Single-channel patch-clamp analyses of these GOF-mutant channels show prominent aberrations in open probability and channel kinetics. These mutations revealed functionally important aromatic amino acid residues in four locations: at the intracellular end of the fifth transmembrane helix (TM5), at both ends of TM6, and at the immediate extension of TM6. These aromatics have counterparts in most TRP subfamilies. The one in TM5 (F380L) aligns precisely with an exceptional Drosophila mutant allele (F550I) that causes constitutive activity in the canonical TRP channel, resulting in rapid and severe retinal degeneration beyond mere loss of phototaxis. Thus, this phenylalanine maintains the balance of various functional states (conformations) of a channel for insect phototransduction as well as one for fungal mechanotransduction. This residue is among a small cluster of phenylalanines found in all known subfamilies of TRP channels. This unique case illustrates that GOF mutations can reveal structure–function principles that can be generalized across different TRP subfamilies. It appears that the conserved aromatics in the four locations have conserved functions in most TRP channels. The possible mechanistic roles of these aromatics and the further use of yeast genetics to dissect TRP channels are discussed. PMID:18042709

  3. Neurodegenerative disease mutations in TREM2 reveal a functional surface and distinct loss-of-function mechanisms

    SciTech Connect

    Kober, Daniel L.; Alexander-Brett, Jennifer M.; Karch, Celeste M.; Cruchaga, Carlos; Colonna, Marco; Holtzman, Michael J.; Brett, Thomas J.

    2016-12-20

    Genetic variations in the myeloid immune receptor TREM2 are linked to several neurodegenerative diseases. To determine how TREM2 variants contribute to these diseases, we performed structural and functional studies of wild-type and variant proteins. Our 3.1 Å TREM2 crystal structure revealed that mutations found in Nasu-Hakola disease are buried whereas Alzheimer’s disease risk variants are found on the surface, suggesting that these mutations have distinct effects on TREM2 function. Biophysical and cellular methods indicate that Nasu-Hakola mutations impact protein stability and decrease folded TREM2 surface expression, whereas Alzheimer’s risk variants impact binding to a TREM2 ligand. Additionally, the Alzheimer’s risk variants appear to epitope map a functional surface on TREM2 that is unique within the larger TREM family. These findings provide a guide to structural and functional differences among genetic variants of TREM2, indicating that therapies targeting the TREM2 pathway should be tailored to these genetic and functional differences with patient-specific medicine approaches for neurodegenerative disorders.

  4. Neurodegenerative disease mutations in TREM2 reveal a functional surface and distinct loss-of-function mechanisms

    PubMed Central

    Kober, Daniel L; Alexander-Brett, Jennifer M; Karch, Celeste M; Cruchaga, Carlos; Colonna, Marco; Holtzman, Michael J; Brett, Thomas J

    2016-01-01

    Genetic variations in the myeloid immune receptor TREM2 are linked to several neurodegenerative diseases. To determine how TREM2 variants contribute to these diseases, we performed structural and functional studies of wild-type and variant proteins. Our 3.1 Å TREM2 crystal structure revealed that mutations found in Nasu-Hakola disease are buried whereas Alzheimer’s disease risk variants are found on the surface, suggesting that these mutations have distinct effects on TREM2 function. Biophysical and cellular methods indicate that Nasu-Hakola mutations impact protein stability and decrease folded TREM2 surface expression, whereas Alzheimer’s risk variants impact binding to a TREM2 ligand. Additionally, the Alzheimer’s risk variants appear to epitope map a functional surface on TREM2 that is unique within the larger TREM family. These findings provide a guide to structural and functional differences among genetic variants of TREM2, indicating that therapies targeting the TREM2 pathway should be tailored to these genetic and functional differences with patient-specific medicine approaches for neurodegenerative disorders. DOI: http://dx.doi.org/10.7554/eLife.20391.001 PMID:27995897

  5. Dynamic functional connectivity analysis reveals transient states of dysconnectivity in schizophrenia.

    PubMed

    Damaraju, E; Allen, E A; Belger, A; Ford, J M; McEwen, S; Mathalon, D H; Mueller, B A; Pearlson, G D; Potkin, S G; Preda, A; Turner, J A; Vaidya, J G; van Erp, T G; Calhoun, V D

    2014-01-01

    Schizophrenia is a psychotic disorder characterized by functional dysconnectivity or abnormal integration between distant brain regions. Recent functional imaging studies have implicated large-scale thalamo-cortical connectivity as being disrupted in patients. However, observed connectivity differences in schizophrenia have been inconsistent between studies, with reports of hyperconnectivity and hypoconnectivity between the same brain regions. Using resting state eyes-closed functional imaging and independent component analysis on a multi-site data that included 151 schizophrenia patients and 163 age- and gender matched healthy controls, we decomposed the functional brain data into 100 components and identified 47 as functionally relevant intrinsic connectivity networks. We subsequently evaluated group differences in functional network connectivity, both in a static sense, computed as the pairwise Pearson correlations between the full network time courses (5.4 minutes in length), and a dynamic sense, computed using sliding windows (44 s in length) and k-means clustering to characterize five discrete functional connectivity states. Static connectivity analysis revealed that compared to healthy controls, patients show significantly stronger connectivity, i.e., hyperconnectivity, between the thalamus and sensory networks (auditory, motor and visual), as well as reduced connectivity (hypoconnectivity) between sensory networks from all modalities. Dynamic analysis suggests that (1), on average, schizophrenia patients spend much less time than healthy controls in states typified by strong, large-scale connectivity, and (2), that abnormal connectivity patterns are more pronounced during these connectivity states. In particular, states exhibiting cortical-subcortical antagonism (anti-correlations) and strong positive connectivity between sensory networks are those that show the group differences of thalamic hyperconnectivity and sensory hypoconnectivity. Group

  6. Individual-based analyses reveal limited functional overlap in a coral reef fish community.

    PubMed

    Brandl, Simon J; Bellwood, David R

    2014-05-01

    Detailed knowledge of a species' functional niche is crucial for the study of ecological communities and processes. The extent of niche overlap, functional redundancy and functional complementarity is of particular importance if we are to understand ecosystem processes and their vulnerability to disturbances. Coral reefs are among the most threatened marine systems, and anthropogenic activity is changing the functional composition of reefs. The loss of herbivorous fishes is particularly concerning as the removal of algae is crucial for the growth and survival of corals. Yet, the foraging patterns of the various herbivorous fish species are poorly understood. Using a multidimensional framework, we present novel individual-based analyses of species' realized functional niches, which we apply to a herbivorous coral reef fish community. In calculating niche volumes for 21 species, based on their microhabitat utilization patterns during foraging, and computing functional overlaps, we provide a measurement of functional redundancy or complementarity. Complementarity is the inverse of redundancy and is defined as less than 50% overlap in niche volumes. The analyses reveal extensive complementarity with an average functional overlap of just 15.2%. Furthermore, the analyses divide herbivorous reef fishes into two broad groups. The first group (predominantly surgeonfishes and parrotfishes) comprises species feeding on exposed surfaces and predominantly open reef matrix or sandy substrata, resulting in small niche volumes and extensive complementarity. In contrast, the second group consists of species (predominantly rabbitfishes) that feed over a wider range of microhabitats, penetrating the reef matrix to exploit concealed surfaces of various substratum types. These species show high variation among individuals, leading to large niche volumes, more overlap and less complementarity. These results may have crucial consequences for our understanding of herbivorous processes on

  7. A functional genomics screen in planarians reveals regulators of whole-brain regeneration

    PubMed Central

    Roberts-Galbraith, Rachel H; Brubacher, John L; Newmark, Phillip A

    2016-01-01

    Planarians regenerate all body parts after injury, including the central nervous system (CNS). We capitalized on this distinctive trait and completed a gene expression-guided functional screen to identify factors that regulate diverse aspects of neural regeneration in Schmidtea mediterranea. Our screen revealed molecules that influence neural cell fates, support the formation of a major connective hub, and promote reestablishment of chemosensory behavior. We also identified genes that encode signaling molecules with roles in head regeneration, including some that are produced in a previously uncharacterized parenchymal population of cells. Finally, we explored genes downregulated during planarian regeneration and characterized, for the first time, glial cells in the planarian CNS that respond to injury by repressing several transcripts. Collectively, our studies revealed diverse molecules and cell types that underlie an animal’s ability to regenerate its brain. DOI: http://dx.doi.org/10.7554/eLife.17002.001 PMID:27612384

  8. A functional genomics screen in planarians reveals regulators of whole-brain regeneration.

    PubMed

    Roberts-Galbraith, Rachel H; Brubacher, John L; Newmark, Phillip A

    2016-09-09

    Planarians regenerate all body parts after injury, including the central nervous system (CNS). We capitalized on this distinctive trait and completed a gene expression-guided functional screen to identify factors that regulate diverse aspects of neural regeneration in Schmidtea mediterranea. Our screen revealed molecules that influence neural cell fates, support the formation of a major connective hub, and promote reestablishment of chemosensory behavior. We also identified genes that encode signaling molecules with roles in head regeneration, including some that are produced in a previously uncharacterized parenchymal population of cells. Finally, we explored genes downregulated during planarian regeneration and characterized, for the first time, glial cells in the planarian CNS that respond to injury by repressing several transcripts. Collectively, our studies revealed diverse molecules and cell types that underlie an animal's ability to regenerate its brain.

  9. Combining functional and anatomical connectivity reveals brain networks for auditory language comprehension.

    PubMed

    Saur, Dorothee; Schelter, Björn; Schnell, Susanne; Kratochvil, David; Küpper, Hanna; Kellmeyer, Philipp; Kümmerer, Dorothee; Klöppel, Stefan; Glauche, Volkmar; Lange, Rüdiger; Mader, Wolfgang; Feess, David; Timmer, Jens; Weiller, Cornelius

    2010-02-15

    Cognitive functions are organized in distributed, overlapping, and interacting brain networks. Investigation of those large-scale brain networks is a major task in neuroimaging research. Here, we introduce a novel combination of functional and anatomical connectivity to study the network topology subserving a cognitive function of interest. (i) In a given network, direct interactions between network nodes are identified by analyzing functional MRI time series with the multivariate method of directed partial correlation (dPC). This method provides important improvements over shortcomings that are typical for ordinary (partial) correlation techniques. (ii) For directly interacting pairs of nodes, a region-to-region probabilistic fiber tracking on diffusion tensor imaging data is performed to identify the most probable anatomical white matter fiber tracts mediating the functional interactions. This combined approach is applied to the language domain to investigate the network topology of two levels of auditory comprehension: lower-level speech perception (i.e., phonological processing) and higher-level speech recognition (i.e., semantic processing). For both processing levels, dPC analyses revealed the functional network topology and identified central network nodes by the number of direct interactions with other nodes. Tractography showed that these interactions are mediated by distinct ventral (via the extreme capsule) and dorsal (via the arcuate/superior longitudinal fascicle fiber system) long- and short-distance association tracts as well as commissural fibers. Our findings demonstrate how both processing routines are segregated in the brain on a large-scale network level. Combining dPC with probabilistic tractography is a promising approach to unveil how cognitive functions emerge through interaction of functionally interacting and anatomically interconnected brain regions.

  10. Structural and functional analysis of amphioxus HIFα reveals ancient features of the HIFα family.

    PubMed

    Gao, Shan; Lu, Ling; Bai, Yan; Zhang, Peng; Song, Weibo; Duan, Cunming

    2014-04-01

    Hypoxia-inducible factors (HIFs) are master regulators of the transcriptional response to hypoxia. To gain insight into the structural and functional evolution of the HIF family, we characterized the HIFα gene from amphioxus, an invertebrate chordate, and identified several alternatively spliced HIFα isoforms. Whereas HIFα Ia, the full-length isoform, contained a complete oxygen-dependent degradation (ODD) domain, the isoforms Ib, Ic, and Id had 1 or 2 deletions in the ODD domain. When tagged with GFP and tested in mammalian cells, the amphioxus HIFα Ia protein level increased in response to hypoxia or CoCl2 treatment, whereas HIFα Ib, Ic, and Id showed reduced or no hypoxia regulation. Deletion of the ODD sequence in HIFα Ia up-regulated the HIFα Ia levels under normoxia. Gene expression analysis revealed HIFα Ic to be the predominant isoform in embryos and larvae, whereas isoform Ia was the most abundant form in the adult stage. The expression levels of Ib and Id were very low. Hypoxia treatment of adults had no effect on the mRNA levels of these HIFα isoforms. Functional analyses in mammalian cells showed all 4 HIFα isoforms capable of entering the nucleus and activating hypoxia response element-dependent reporter gene expression. The functional nuclear location signal (NLS) mapped to 3 clusters of basic residues. (775)KKARL functioned as the primary NLS, but (737)KRK and (754)KK also contributed to the nuclear localization. All amphioxus HIFα isoforms had 2 functional transactivation domains (TADs). Its C-terminal transactivation (C-TAD) shared high sequence identity with the human HIF-1α and HIF-2α C-TAD. This domain contained a conserved asparagine, and its mutation resulted in an increase in transcriptional activity. These findings reveal many ancient features of the HIFα family and provide novel insights into the evolution of the HIFα family.

  11. Acetylproteomic analysis reveals functional implications of lysine acetylation in human spermatozoa (sperm).

    PubMed

    Yu, Heguo; Diao, Hua; Wang, Chunmei; Lin, Yan; Yu, Fudong; Lu, Hui; Xu, Wei; Li, Zheng; Shi, Huijuan; Zhao, Shimin; Zhou, Yuchuan; Zhang, Yonglian

    2015-04-01

    Male infertility is a medical condition that has been on the rise globally. Lysine acetylation of human sperm, an essential posttranslational modification involved in the etiology of sperm abnormality, is not fully understood. Therefore, we first generated a qualified pan-anti-acetyllysine monoclonal antibody to characterize the global lysine acetylation of uncapacitated normal human sperm with a proteomics approach. With high enrichment ratios that were up to 31%, 973 lysine-acetylated sites that matched to 456 human sperm proteins, including 671 novel lysine acetylation sites and 205 novel lysine-acetylated proteins, were identified. These proteins exhibited conserved motifs XXXKYXXX, XXXKFXXX, and XXXKHXXX, were annotated to function in multiple metabolic processes, and were localized predominantly in the mitochondrion and cytoplasmic fractions. Between the uncapacitated and capacitated sperm, different acetylation profiles in regard to functional proteins involved in sperm capacitation, sperm-egg recognition, sperm-egg plasma fusion, and fertilization were observed, indicating that acetylation of functional proteins may be required during sperm capacitation. Bioinformatics analysis revealed association of acetylated proteins with diseases and drugs. Novel acetylation of voltage-dependent anion channel proteins was also found. With clinical sperm samples, we observed differed lysine acetyltransferases and lysine deacetylases expression between normal sperm and abnormal sperm of asthenospermia or necrospermia. Furthermore, with sperm samples impaired by epigallocatechin gallate to mimic asthenospermia, we observed that inhibition of sperm motility was partly through the blockade of voltage-dependent anion channel 2 Lys-74 acetylation combined with reduced ATP levels and mitochondrial membrane potential. Taken together, we obtained a qualified pan-anti-acetyllysine monoclonal antibody, analyzed the acetylproteome of uncapacitated human sperm, and revealed

  12. Genome-wide search for eliminylating domains reveals novel function for BLES03-like proteins.

    PubMed

    Khater, Shradha; Mohanty, Debasisa

    2014-07-24

    Bacterial phosphothreonine lyases catalyze a novel posttranslational modification involving formation of dehydrobutyrine/dehyroalanine by β elimination of the phosphate group of phosphothreonine or phosphoserine residues in their substrate proteins. Though there is experimental evidence for presence of dehydro amino acids in human proteins, no eukaryotic homologs of these lyases have been identified as of today. A comprehensive genome-wide search for identifying phosphothreonine lyase homologs in eukaryotes was carried out. Our fold-based search revealed structural and catalytic site similarity between bacterial phosphothreonine lyases and BLES03 (basophilic leukemia-expressed protein 03), a human protein with unknown function. Ligand induced conformational changes similar to bacterial phosphothreonine lyases, and movement of crucial arginines in the loop region to the catalytic pocket upon binding of phosphothreonine-containing peptides was seen during docking and molecular dynamics studies. Genome-wide search for BLES03 homologs using sensitive profile-based methods revealed their presence not only in eukaryotic classes such as chordata and fungi but also in bacterial and archaebacterial classes. The synteny of these archaebacterial BLES03-like proteins was remarkably similar to that of type IV lantibiotic synthetases which harbor LanL-like phosphothreonine lyase domains. Hence, context-based analysis reinforced our earlier sequence/structure-based prediction of phosphothreonine lyase catalytic function for BLES03. Our in silico analysis has revealed that BLES03-like proteins with previously unknown function are novel eukaryotic phosphothreonine lyases involved in biosynthesis of dehydro amino acids, whereas their bacterial and archaebacterial counterparts might be involved in biosynthesis of natural products similar to lantibiotics.

  13. Analyses of soil microbial community compositions and functional genes reveal potential consequences of natural forest succession

    NASA Astrophysics Data System (ADS)

    Cong, Jing; Yang, Yunfeng; Liu, Xueduan; Lu, Hui; Liu, Xiao; Zhou, Jizhong; Li, Diqiang; Yin, Huaqun; Ding, Junjun; Zhang, Yuguang

    2015-05-01

    The succession of microbial community structure and function is a central ecological topic, as microbes drive the Earth’s biogeochemical cycles. To elucidate the response and mechanistic underpinnings of soil microbial community structure and metabolic potential relevant to natural forest succession, we compared soil microbial communities from three adjacent natural forests: a coniferous forest (CF), a mixed broadleaf forest (MBF) and a deciduous broadleaf forest (DBF) on Shennongjia Mountain in central China. In contrary to plant communities, the microbial taxonomic diversity of the DBF was significantly (P < 0.05) higher than those of CF and MBF, rendering their microbial community compositions markedly different. Consistently, microbial functional diversity was also highest in the DBF. Furthermore, a network analysis of microbial carbon and nitrogen cycling genes showed the network for the DBF samples was relatively large and tight, revealing strong couplings between microbes. Soil temperature, reflective of climate regimes, was important in shaping microbial communities at both taxonomic and functional gene levels. As a first glimpse of both the taxonomic and functional compositions of soil microbial communities, our results suggest that microbial community structure and function potentials will be altered by future environmental changes, which have implications for forest succession.

  14. Analyses of soil microbial community compositions and functional genes reveal potential consequences of natural forest succession

    PubMed Central

    Cong, Jing; Yang, Yunfeng; Liu, Xueduan; Lu, Hui; Liu, Xiao; Zhou, Jizhong; Li, Diqiang; Yin, Huaqun; Ding, Junjun; Zhang, Yuguang

    2015-01-01

    The succession of microbial community structure and function is a central ecological topic, as microbes drive the Earth’s biogeochemical cycles. To elucidate the response and mechanistic underpinnings of soil microbial community structure and metabolic potential relevant to natural forest succession, we compared soil microbial communities from three adjacent natural forests: a coniferous forest (CF), a mixed broadleaf forest (MBF) and a deciduous broadleaf forest (DBF) on Shennongjia Mountain in central China. In contrary to plant communities, the microbial taxonomic diversity of the DBF was significantly (P < 0.05) higher than those of CF and MBF, rendering their microbial community compositions markedly different. Consistently, microbial functional diversity was also highest in the DBF. Furthermore, a network analysis of microbial carbon and nitrogen cycling genes showed the network for the DBF samples was relatively large and tight, revealing strong couplings between microbes. Soil temperature, reflective of climate regimes, was important in shaping microbial communities at both taxonomic and functional gene levels. As a first glimpse of both the taxonomic and functional compositions of soil microbial communities, our results suggest that microbial community structure and function potentials will be altered by future environmental changes, which have implications for forest succession. PMID:25943705

  15. Analyses of soil microbial community compositions and functional genes reveal potential consequences of natural forest succession.

    PubMed

    Cong, Jing; Yang, Yunfeng; Liu, Xueduan; Lu, Hui; Liu, Xiao; Zhou, Jizhong; Li, Diqiang; Yin, Huaqun; Ding, Junjun; Zhang, Yuguang

    2015-05-06

    The succession of microbial community structure and function is a central ecological topic, as microbes drive the Earth's biogeochemical cycles. To elucidate the response and mechanistic underpinnings of soil microbial community structure and metabolic potential relevant to natural forest succession, we compared soil microbial communities from three adjacent natural forests: a coniferous forest (CF), a mixed broadleaf forest (MBF) and a deciduous broadleaf forest (DBF) on Shennongjia Mountain in central China. In contrary to plant communities, the microbial taxonomic diversity of the DBF was significantly (P < 0.05) higher than those of CF and MBF, rendering their microbial community compositions markedly different. Consistently, microbial functional diversity was also highest in the DBF. Furthermore, a network analysis of microbial carbon and nitrogen cycling genes showed the network for the DBF samples was relatively large and tight, revealing strong couplings between microbes. Soil temperature, reflective of climate regimes, was important in shaping microbial communities at both taxonomic and functional gene levels. As a first glimpse of both the taxonomic and functional compositions of soil microbial communities, our results suggest that microbial community structure and function potentials will be altered by future environmental changes, which have implications for forest succession.

  16. Targeted capture and resequencing of 1040 genes reveal environmentally driven functional variation in grey wolves.

    PubMed

    Schweizer, Rena M; Robinson, Jacqueline; Harrigan, Ryan; Silva, Pedro; Galverni, Marco; Musiani, Marco; Green, Richard E; Novembre, John; Wayne, Robert K

    2016-01-01

    In an era of ever-increasing amounts of whole-genome sequence data for individuals and populations, the utility of traditional single nucleotide polymorphisms (SNPs) array-based genome scans is uncertain. We previously performed a SNP array-based genome scan to identify candidate genes under selection in six distinct grey wolf (Canis lupus) ecotypes. Using this information, we designed a targeted capture array for 1040 genes, including all exons and flanking regions, as well as 5000 1-kb nongenic neutral regions, and resequenced these regions in 107 wolves. Selection tests revealed striking patterns of variation within candidate genes relative to noncandidate regions and identified potentially functional variants related to local adaptation. We found 27% and 47% of candidate genes from the previous SNP array study had functional changes that were outliers in sweed and bayenv analyses, respectively. This result verifies the use of genomewide SNP surveys to tag genes that contain functional variants between populations. We highlight nonsynonymous variants in APOB, LIPG and USH2A that occur in functional domains of these proteins, and that demonstrate high correlation with precipitation seasonality and vegetation. We find Arctic and High Arctic wolf ecotypes have higher numbers of genes under selection, which highlight their conservation value and heightened threat due to climate change. This study demonstrates that combining genomewide genotyping arrays with large-scale resequencing and environmental data provides a powerful approach to discern candidate functional variants in natural populations.

  17. Quantitative protein localization signatures reveal an association between spatial and functional divergences of proteins.

    PubMed

    Loo, Lit-Hsin; Laksameethanasan, Danai; Tung, Yi-Ling

    2014-03-01

    Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST), an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast Saccharomyces cerevisiae as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to elucidate protein

  18. Functional splicing network reveals extensive regulatory potential of the core spliceosomal machinery.

    PubMed

    Papasaikas, Panagiotis; Tejedor, J Ramón; Vigevani, Luisa; Valcárcel, Juan

    2015-01-08

    Pre-mRNA splicing relies on the poorly understood dynamic interplay between >150 protein components of the spliceosome. The steps at which splicing can be regulated remain largely unknown. We systematically analyzed the effect of knocking down the components of the splicing machinery on alternative splicing events relevant for cell proliferation and apoptosis and used this information to reconstruct a network of functional interactions. The network accurately captures known physical and functional associations and identifies new ones, revealing remarkable regulatory potential of core spliceosomal components, related to the order and duration of their recruitment during spliceosome assembly. In contrast with standard models of regulation at early steps of splice site recognition, factors involved in catalytic activation of the spliceosome display regulatory properties. The network also sheds light on the antagonism between hnRNP C and U2AF, and on targets of antitumor drugs, and can be widely used to identify mechanisms of splicing regulation.

  19. Single-cell analysis reveals functionally distinct classes within the planarian stem cell compartment

    PubMed Central

    van Wolfswinkel, Josien C.; Wagner, Daniel E.; Reddien, Peter W.

    2014-01-01

    Planarians are flatworms capable of regenerating any missing body region. This capacity is mediated by neoblasts, a proliferative cell population that contains pluripotent stem cells. Although population-based studies have revealed many neoblast characteristics, whether functionally distinct classes exist within this population is unclear. Here, we used high-dimensional single-cell transcriptional profiling from over a thousand individual neoblasts to directly compare gene expression fingerprints during homeostasis and regeneration. We identified two prominent neoblast classes that we named ζ (zeta) and σ (sigma). Zeta-neoblasts encompass specified cells that give rise to an abundant postmitotic lineage including epidermal cells, and are not required for regeneration. By contrast, sigma-neoblasts proliferate in response to injury, possess broad lineage capacity, and can give rise to zeta-neoblasts. These findings present a new view of planarian neoblasts, in which the population is comprised of two major and functionally distinct cellular compartments. PMID:25017721

  20. Conservation and rewiring of functional modules revealed by an epistasis map in fission yeast.

    PubMed

    Roguev, Assen; Bandyopadhyay, Sourav; Zofall, Martin; Zhang, Ke; Fischer, Tamas; Collins, Sean R; Qu, Hongjing; Shales, Michael; Park, Han-Oh; Hayles, Jacqueline; Hoe, Kwang-Lae; Kim, Dong-Uk; Ideker, Trey; Grewal, Shiv I; Weissman, Jonathan S; Krogan, Nevan J

    2008-10-17

    An epistasis map (E-MAP) was constructed in the fission yeast, Schizosaccharomyces pombe, by systematically measuring the phenotypes associated with pairs of mutations. This high-density, quantitative genetic interaction map focused on various aspects of chromosome function, including transcription regulation and DNA repair/replication. The E-MAP uncovered a previously unidentified component of the RNA interference (RNAi) machinery (rsh1) and linked the RNAi pathway to several other biological processes. Comparison of the S. pombe E-MAP to an analogous genetic map from the budding yeast revealed that, whereas negative interactions were conserved between genes involved in similar biological processes, positive interactions and overall genetic profiles between pairs of genes coding for physically associated proteins were even more conserved. Hence, conservation occurs at the level of the functional module (protein complex), but the genetic cross talk between modules can differ substantially.

  1. Metagenomic sequencing reveals microbiota and its functional potential associated with periodontal disease

    PubMed Central

    Wang, Jinfeng; Qi, Ji; Zhao, Hui; He, Shu; Zhang, Yifei; Wei, Shicheng; Zhao, Fangqing

    2013-01-01

    Although attempts have been made to reveal the relationships between bacteria and human health, little is known about the species and function of the microbial community associated with oral diseases. In this study, we report the sequencing of 16 metagenomic samples collected from dental swabs and plaques representing four periodontal states. Insights into the microbial community structure and the metabolic variation associated with periodontal health and disease were obtained. We observed a strong correlation between community structure and disease status, and described a core disease-associated community. A number of functional genes and metabolic pathways including bacterial chemotaxis and glycan biosynthesis were over-represented in the microbiomes of periodontal disease. A significant amount of novel species and genes were identified in the metagenomic assemblies. Our study enriches the understanding of the oral microbiome and sheds light on the contribution of microorganisms to the formation and succession of dental plaques and oral diseases. PMID:23673380

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

    PubMed

    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.

  3. Quantification of Left Ventricular Function with Premature Ventricular Complexes Reveals Variable Hemodynamics

    PubMed Central

    Contijoch, Francisco; Rogers, Kelly; Rears, Hannah; Shahid, Mohammed; Kellman, Peter; Gorman, Joseph; Gorman, Robert C.; Yushkevich, Paul; Zado, Erica S.; Supple, Gregory E.; Marchlinski, Francis E.; Witschey, Walter R.T.; Han, Yuchi

    2016-01-01

    Background Premature ventricular complexes (PVC) are prevalent in the general population and are sometimes associated with reduced ventricular function. Current echocardiographic and cardiovascular magnetic resonance imaging (CMR) techniques do not adequately address the effect of PVCs on left ventricular function. Methods and Results Fifteen subjects with a history of frequent PVCs undergoing CMR had real-time slice volume quantification performed using a 2D real-time CMR imaging technique. Synchronization of 2D real-time imaging with patient ECG allowed for different beats to be categorized by the loading beat RR-duration and beat RR-duration. For each beat type, global volumes were quantified via summation over all slices covering the entire ventricle. Different patterns of ectopy including isolated PVCs, bigeminy, trigeminy, and interpolated PVCs were observed. Global functional measurement of the different beat types based on timing demonstrated differences in preload, stroke volume, and ejection fraction. An average of hemodynamic function was quantified for each subject depending on the frequency of each observed beat type. Conclusions Application of real-time CMR imaging in patients with PVCs revealed differential contribution of PVCs to hemodynamics. PMID:27009416

  4. Validation of skeletal muscle cis-regulatory module predictions reveals nucleotide composition bias in functional enhancers.

    PubMed

    Kwon, Andrew T; Chou, Alice Yi; Arenillas, David J; Wasserman, Wyeth W

    2011-12-01

    We performed a genome-wide scan for muscle-specific cis-regulatory modules (CRMs) using three computational prediction programs. Based on the predictions, 339 candidate CRMs were tested in cell culture with NIH3T3 fibroblasts and C2C12 myoblasts for capacity to direct selective reporter gene expression to differentiated C2C12 myotubes. A subset of 19 CRMs validated as functional in the assay. The rate of predictive success reveals striking limitations of computational regulatory sequence analysis methods for CRM discovery. Motif-based methods performed no better than predictions based only on sequence conservation. Analysis of the properties of the functional sequences relative to inactive sequences identifies nucleotide sequence composition can be an important characteristic to incorporate in future methods for improved predictive specificity. Muscle-related TFBSs predicted within the functional sequences display greater sequence conservation than non-TFBS flanking regions. Comparison with recent MyoD and histone modification ChIP-Seq data supports the validity of the functional regions.

  5. Cryptic biodiversity effects: importance of functional redundancy revealed through addition of food web complexity.

    PubMed

    Philpott, Stacy M; Pardee, Gabriella L; Gonthier, David J

    2012-05-01

    Interactions between predators and the degree of functional redundancy among multiple predator species may determine whether herbivores experience increased or decreased predation risk. Specialist parasites can modify predator behavior, yet rarely have cascading effects on multiple predator species and prey been evaluated. We examined influences of specialist phorid parasites (Pseudacteon spp.) on three predatory ant species and herbivores in a coffee agroecosystem. Specifically, we examined whether changes in ant richness affected fruit damage by the coffee berry borer (Hypothenemus hampei) and whether phorids altered multi-predator effects. Each ant species reduced borer damage, and without phorids, increasing predator richness did not further decrease borer damage. However, with phorids, activity of one ant species was reduced, indicating that the presence of multiple ant species was necessary to limit borer damage. In addition, phorid presence revealed synergistic effects of multiple ant species, not observed without the presence of this parasite. Thus, a trait-mediated cascade resulting from a parasite-induced predator behavioral change revealed the importance of functional redundancy, predator diversity, and food web complexity for control of this important pest.

  6. Predicting invasive species impacts: a community module functional response approach reveals context dependencies.

    PubMed

    Paterson, Rachel A; Dick, Jaimie T A; Pritchard, Daniel W; Ennis, Marilyn; Hatcher, Melanie J; Dunn, Alison M

    2015-03-01

    Predatory functional responses play integral roles in predator-prey dynamics, and their assessment promises greater understanding and prediction of the predatory impacts of invasive species. Other interspecific interactions, however, such as parasitism and higher-order predation, have the potential to modify predator-prey interactions and thus the predictive capability of the comparative functional response approach. We used a four-species community module (higher-order predator; focal native or invasive predators; parasites of focal predators; native prey) to compare the predatory functional responses of native Gammarus duebeni celticus and invasive Gammarus pulex amphipods towards three invertebrate prey species (Asellus aquaticus, Simulium spp., Baetis rhodani), thus, quantifying the context dependencies of parasitism and a higher-order fish predator on these functional responses. Our functional response experiments demonstrated that the invasive amphipod had a higher predatory impact (lower handling time) on two of three prey species, which reflects patterns of impact observed in the field. The community module also revealed that parasitism had context-dependent influences, for one prey species, with the potential to further reduce the predatory impact of the invasive amphipod or increase the predatory impact of the native amphipod in the presence of a higher-order fish predator. Partial consumption of prey was similar for both predators and occurred increasingly in the order A. aquaticus, Simulium spp. and B. rhodani. This was associated with increasing prey densities, but showed no context dependencies with parasitism or higher-order fish predator. This study supports the applicability of comparative functional responses as a tool to predict and assess invasive species impacts incorporating multiple context dependencies.

  7. Predicting invasive species impacts: a community module functional response approach reveals context dependencies

    PubMed Central

    Paterson, Rachel A; Dick, Jaimie T A; Pritchard, Daniel W; Ennis, Marilyn; Hatcher, Melanie J; Dunn, Alison M

    2015-01-01

    Summary Predatory functional responses play integral roles in predator–prey dynamics, and their assessment promises greater understanding and prediction of the predatory impacts of invasive species. Other interspecific interactions, however, such as parasitism and higher-order predation, have the potential to modify predator–prey interactions and thus the predictive capability of the comparative functional response approach. We used a four-species community module (higher-order predator; focal native or invasive predators; parasites of focal predators; native prey) to compare the predatory functional responses of native Gammarus duebeni celticus and invasive Gammarus pulex amphipods towards three invertebrate prey species (Asellus aquaticus, Simulium spp., Baetis rhodani), thus, quantifying the context dependencies of parasitism and a higher-order fish predator on these functional responses. Our functional response experiments demonstrated that the invasive amphipod had a higher predatory impact (lower handling time) on two of three prey species, which reflects patterns of impact observed in the field. The community module also revealed that parasitism had context-dependent influences, for one prey species, with the potential to further reduce the predatory impact of the invasive amphipod or increase the predatory impact of the native amphipod in the presence of a higher-order fish predator. Partial consumption of prey was similar for both predators and occurred increasingly in the order A. aquaticus, Simulium spp. and B. rhodani. This was associated with increasing prey densities, but showed no context dependencies with parasitism or higher-order fish predator. This study supports the applicability of comparative functional responses as a tool to predict and assess invasive species impacts incorporating multiple context dependencies. PMID:25265905

  8. Cluster analysis reveals a binary effect of storage on boar sperm motility function.

    PubMed

    Henning, Heiko; Petrunkina, Anna M; Harrison, Robin A P; Waberski, Dagmar

    2014-06-01

    Storage of liquid-preserved boar spermatozoa is associated with a loss of fertilising ability of the preserved spermatozoa, which standard semen parameters barely reflect. Monitoring responses to molecular effectors of sperm function (e.g. bicarbonate) has proven to be a more sensitive approach to investigating storage effects. Bicarbonate not only initiates capacitation in spermatozoa, but also induces motility activation. This occurs at ejaculation, but also happens throughout passage through the oviduct. In the present study we tested whether the specific response of boar sperm subpopulations to bicarbonate, as assessed by motility activation, is altered with the duration of storage in vitro. Three ejaculates from each of seven boars were diluted in Beltsville thawing solution and stored at 17°C. Only minor changes in the parameters of diluted semen were revealed over a period of 72h storage. For assessment of bicarbonate responses, subsamples of diluted spermatozoa were centrifuged through a discontinuous Percoll gradient after 12, 24 and 72h storage. Subsequently, spermatozoa were incubated in two Ca2+-free variants of Tyrode's medium either without (TyrControl) or with (TyrBic) 15mM bicarbonate, and computer-aided sperm analysis motility measurements were made. Cluster analysis of imaging data from motile spermatozoa revealed the presence of five major sperm subpopulations with distinct motility characteristics, differing between TyrBic and TyrControl at any given time (P<0.001). Although there was an increasing loss of motility function in both media, bicarbonate induced an increase in a 'fast linear' cohort of spermatozoa in TyrBic regardless of storage (66.4% at 12h and 63.9% at 72h). These results imply a binary pattern in response of sperm motility function descriptors to storage: although the quantitative descriptor (percentage of motile spermatozoa) declines in washed semen samples, the qualitative descriptor (percentage of spermatozoa stimulated into

  9. A fibrolytic potential in the human ileum mucosal microbiota revealed by functional metagenomic

    PubMed Central

    Patrascu, Orlane; Béguet-Crespel, Fabienne; Marinelli, Ludovica; Le Chatelier, Emmanuelle; Abraham, Anne-Laure; Leclerc, Marion; Klopp, Christophe; Terrapon, Nicolas; Henrissat, Bernard; Blottière, Hervé M.; Doré, Joël; Béra-Maillet, Christel

    2017-01-01

    The digestion of dietary fibers is a major function of the human intestinal microbiota. So far this function has been attributed to the microorganisms inhabiting the colon, and many studies have focused on this distal part of the gastrointestinal tract using easily accessible fecal material. However, microbial fermentations, supported by the presence of short-chain fatty acids, are suspected to occur in the upper small intestine, particularly in the ileum. Using a fosmid library from the human ileal mucosa, we screened 20,000 clones for their activities against carboxymethylcellulose and xylans chosen as models of the major plant cell wall (PCW) polysaccharides from dietary fibres. Eleven positive clones revealed a broad range of CAZyme encoding genes from Bacteroides and Clostridiales species, as well as Polysaccharide Utilization Loci (PULs). The functional glycoside hydrolase genes were identified, and oligosaccharide break-down products examined from different polysaccharides including mixed-linkage β-glucans. CAZymes and PULs were also examined for their prevalence in human gut microbiome. Several clusters of genes of low prevalence in fecal microbiome suggested they belong to unidentified strains rather specifically established upstream the colon, in the ileum. Thus, the ileal mucosa-associated microbiota encompasses the enzymatic potential for PCW polysaccharide degradation in the small intestine. PMID:28091525

  10. Conditional Degradation of Plasmodium Calcineurin Reveals Functions in Parasite Colonization of both Host and Vector

    PubMed Central

    Philip, Nisha; Waters, Andrew P.

    2015-01-01

    Summary Functional analysis of essential genes in the malarial parasite, Plasmodium, is hindered by lack of efficient strategies for conditional protein regulation. We report the development of a rapid, specific, and inducible chemical-genetic tool in the rodent malaria parasite, P. berghei, in which endogenous proteins engineered to contain the auxin-inducible degron (AID) are selectively degraded upon adding auxin. Application of AID to the calcium-regulated protein phosphatase, calcineurin, revealed functions in host and vector stages of parasite development. Whereas depletion of calcineurin in late-stage schizonts demonstrated its critical role in erythrocyte attachment and invasion in vivo, stage-specific depletion uncovered roles in gamete development, fertilization, and ookinete-to-oocyst and sporozoite-to-liver stage transitions. Furthermore, AID technology facilitated concurrent generation and phenotyping of transgenic lines, allowing multiple lines to be assessed simultaneously with significant reductions in animal use. This study highlights the broad applicability of AID for functional analysis of proteins across the Plasmodium life cycle. PMID:26118994

  11. Diurnal Changes in Mitochondrial Function Reveal Daily Optimization of Light and Dark Respiratory Metabolism in Arabidopsis*

    PubMed Central

    Lee, Chun Pong; Eubel, Holger; Millar, A. Harvey

    2010-01-01

    Biomass production by plants is often negatively correlated with respiratory rate, but the value of this rate changes dramatically during diurnal cycles, and hence, biomass is the cumulative result of complex environment-dependent metabolic processes. Mitochondria in photosynthetic plant tissues undertake substantially different metabolic roles during light and dark periods that are dictated by substrate availability and the functional capacity of mitochondria defined by their protein composition. We surveyed the heterogeneity of the mitochondrial proteome and its function during a typical night and day cycle in Arabidopsis shoots. This used a staged, quantitative analysis of the proteome across 10 time points covering 24 h of the life of 3-week-old Arabidopsis shoots grown under 12-h dark and 12-h light conditions. Detailed analysis of enzyme capacities and substrate-dependent respiratory processes of isolated mitochondria were also undertaken during the same time course. Together these data reveal a range of dynamic changes in mitochondrial capacity and uncover day- and night-enhanced protein components. Clear diurnal changes were evident in mitochondrial capacities to drive the TCA cycle and to undertake functions associated with nitrogen and sulfur metabolism, redox poise, and mitochondrial antioxidant defense. These data quantify the nature and nuances of a daily rhythm in Arabidopsis mitochondrial respiratory capacity. PMID:20601493

  12. Comparative proteomics reveal fundamental structural and functional differences between the two progeny phenotypes of a baculovirus.

    PubMed

    Hou, Dianhai; Zhang, Leike; Deng, Fei; Fang, Wei; Wang, Ranran; Liu, Xijia; Guo, Lin; Rayner, Simon; Chen, Xinwen; Wang, Hualin; Hu, Zhihong

    2013-01-01

    The replication of lepidopteran baculoviruses is characterized by the production of two progeny phenotypes: the occlusion-derived virus (ODV), which establishes infection in midgut cells, and the budded virus (BV), which disseminates infection to different tissues within a susceptible host. To understand the structural, and hence functional, differences between BV and ODV, we employed multiple proteomic methods to reveal the protein compositions and posttranslational modifications of the two phenotypes of Helicoverpa armigera nucleopolyhedrovirus. In addition, Western blotting and quantitative mass spectrometry were used to identify the localization of proteins in the envelope or nucleocapsid fractions. Comparative protein portfolios of BV and ODV showing the distribution of 54 proteins, encompassing the 21 proteins shared by BV and ODV, the 12 BV-specific proteins, and the 21 ODV-specific proteins, were obtained. Among the 11 ODV-specific envelope proteins, 8 either are essential for or contribute to oral infection. Twenty-three phosphorylated and 6 N-glycosylated viral proteins were also identified. While the proteins that are shared by the two phenotypes appear to be important for nucleocapsid assembly and trafficking, the structural and functional differences between the two phenotypes are evidently characterized by the envelope proteins and posttranslational modifications. This comparative proteomics study provides new insight into how BV and ODV are formed and why they function differently.

  13. Functional analysis reveals that RBM10 mutations contribute to lung adenocarcinoma pathogenesis by deregulating splicing

    PubMed Central

    Zhao, Jiawei; Sun, Yue; Huang, Yin; Song, Fan; Huang, Zengshu; Bao, Yufang; Zuo, Ji; Saffen, David; Shao, Zhen; Liu, Wen; Wang, Yongbo

    2017-01-01

    RBM10 is an RNA splicing regulator that is frequently mutated in lung adenocarcinoma (LUAD) and has recently been proposed to be a cancer gene. How RBM10 mutations observed in LUAD affect its normal functions, however, remains largely unknown. Here integrative analysis of RBM10 mutation and RNA expression data revealed that LUAD-associated RBM10 mutations exhibit a mutational spectrum similar to that of tumor suppressor genes. In addition, this analysis showed that RBM10 mutations identified in LUAD patients lacking canonical oncogenes are associated with significantly reduced RBM10 expression. To systematically investigate RBM10 mutations, we developed an experimental pipeline for elucidating their functional effects. Among six representative LUAD-associated RBM10 mutations, one nonsense and one frameshift mutation caused loss-of-function as expected, whereas four missense mutations differentially affected RBM10-mediated splicing. Importantly, changes in proliferation rates of LUAD-derived cells caused by these RBM10 missense mutants correlated with alterations in RNA splicing of RBM10 target genes. Together, our data implies that RBM10 mutations contribute to LUAD pathogenesis, at least in large part, by deregulating splicing. The methods described in this study should be useful for analyzing mutations in additional cancer-associated RNA splicing regulators. PMID:28091594

  14. Conditional Degradation of Plasmodium Calcineurin Reveals Functions in Parasite Colonization of both Host and Vector.

    PubMed

    Philip, Nisha; Waters, Andrew P

    2015-07-08

    Functional analysis of essential genes in the malarial parasite, Plasmodium, is hindered by lack of efficient strategies for conditional protein regulation. We report the development of a rapid, specific, and inducible chemical-genetic tool in the rodent malaria parasite, P. berghei, in which endogenous proteins engineered to contain the auxin-inducible degron (AID) are selectively degraded upon adding auxin. Application of AID to the calcium-regulated protein phosphatase, calcineurin, revealed functions in host and vector stages of parasite development. Whereas depletion of calcineurin in late-stage schizonts demonstrated its critical role in erythrocyte attachment and invasion in vivo, stage-specific depletion uncovered roles in gamete development, fertilization, and ookinete-to-oocyst and sporozoite-to-liver stage transitions. Furthermore, AID technology facilitated concurrent generation and phenotyping of transgenic lines, allowing multiple lines to be assessed simultaneously with significant reductions in animal use. This study highlights the broad applicability of AID for functional analysis of proteins across the Plasmodium life cycle.

  15. Comparative expression profiling reveals gene functions in female meiosis and gametophyte development in Arabidopsis.

    PubMed

    Zhao, Lihua; He, Jiangman; Cai, Hanyang; Lin, Haiyan; Li, Yanqiang; Liu, Renyi; Yang, Zhenbiao; Qin, Yuan

    2014-11-01

    Megasporogenesis is essential for female fertility, and requires the accomplishment of meiosis and the formation of functional megaspores. The inaccessibility and low abundance of female meiocytes make it particularly difficult to elucidate the molecular basis underlying megasporogenesis. We used high-throughput tag-sequencing analysis to identify genes expressed in female meiocytes (FMs) by comparing gene expression profiles from wild-type ovules undergoing megasporogenesis with those from the spl mutant ovules, which lack megasporogenesis. A total of 862 genes were identified as FMs, with levels that are consistently reduced in spl ovules in two biological replicates. Fluorescence-assisted cell sorting followed by RNA-seq analysis of DMC1:GFP-labeled female meiocytes confirmed that 90% of the FMs are indeed detected in the female meiocyte protoplast profiling. We performed reverse genetic analysis of 120 candidate genes and identified four FM genes with a function in female meiosis progression in Arabidopsis. We further revealed that KLU, a putative cytochrome P450 monooxygenase, is involved in chromosome pairing during female meiosis, most likely by affecting the normal expression pattern of DMC1 in ovules during female meiosis. Our studies provide valuable information for functional genomic analyses of plant germline development as well as insights into meiosis.

  16. Gas7-Deficient Mouse Reveals Roles in Motor Function and Muscle Fiber Composition during Aging

    PubMed Central

    Huang, Bo-Tsang; Chang, Pu-Yuan; Su, Ching-Hua; Chao, Chuck C.-K.; Lin-Chao, Sue

    2012-01-01

    Background Growth arrest-specific gene 7 (Gas7) has previously been shown to be involved in neurite outgrowth in vitro; however, its actual role has yet to be determined. To investigate the physiological function of Gas7 in vivo, here we generated a Gas7-deficient mouse strain with a labile Gas7 mutant protein whose functions are similar to wild-type Gas7. Methodology/Principal Findings Our data show that aged Gas7-deficient mice have motor activity defects due to decreases in the number of spinal motor neurons and in muscle strength, of which the latter may be caused by changes in muscle fiber composition as shown in the soleus. In cross sections of the soleus of Gas7-deficient mice, gross morphological features and levels of myosin heavy chain I (MHC I) and MHC II markers revealed significantly fewer fast fibers. In addition, we found that nerve terminal sprouting, which may be associated with slow and fast muscle fiber composition, was considerably reduced at neuromuscular junctions (NMJ) during aging. Conclusions/Significance These findings indicate that Gas7 is involved in motor neuron function associated with muscle strength maintenance. PMID:22662195

  17. Edge reconstruction in armchair phosphorene nanoribbons revealed by discontinuous Galerkin density functional theory.

    PubMed

    Hu, Wei; Lin, Lin; Yang, Chao

    2015-12-21

    With the help of our recently developed massively parallel DGDFT (Discontinuous Galerkin Density Functional Theory) methodology, we perform large-scale Kohn-Sham density functional theory calculations on phosphorene nanoribbons with armchair edges (ACPNRs) containing a few thousands to ten thousand atoms. The use of DGDFT allows us to systematically achieve a conventional plane wave basis set type of accuracy, but with a much smaller number (about 15) of adaptive local basis (ALB) functions per atom for this system. The relatively small number of degrees of freedom required to represent the Kohn-Sham Hamiltonian, together with the use of the pole expansion the selected inversion (PEXSI) technique that circumvents the need to diagonalize the Hamiltonian, results in a highly efficient and scalable computational scheme for analyzing the electronic structures of ACPNRs as well as their dynamics. The total wall clock time for calculating the electronic structures of large-scale ACPNRs containing 1080-10,800 atoms is only 10-25 s per self-consistent field (SCF) iteration, with accuracy fully comparable to that obtained from conventional planewave DFT calculations. For the ACPNR system, we observe that the DGDFT methodology can scale to 5000-50,000 processors. We use DGDFT based ab initio molecular dynamics (AIMD) calculations to study the thermodynamic stability of ACPNRs. Our calculations reveal that a 2 × 1 edge reconstruction appears in ACPNRs at room temperature.

  18. Structure of Prokaryotic Polyamine Deacetylase Reveals Evolutionary Functional Relationships with Eukaryotic Histone Deacetylases

    PubMed Central

    Lombardi, Patrick M.; Angell, Heather D.; Whittington, Douglas A.; Flynn, Erin F.; Rajashankar, Kanagalaghatta R.; Christianson, David W.

    2011-01-01

    Polyamines are a ubiquitous class of polycationic small molecules that can influence gene expression by binding to nucleic acids. Reversible polyamine acetylation regulates nucleic acid binding and is required for normal cell cycle progression and proliferation. Here, we report the structures of Mycoplana ramosa acetylpolyamine amidohydrolase (APAH) complexed with a transition state analogue and a hydroxamate inhibitor, and an inactive mutant complexed with two acetylpolyamine substrates. The structure of APAH is the first of a histone deacetylase-like oligomer and reveals that an 18-residue insert in the L2 loop promotes dimerization and the formation of an 18-Å long “L”-shaped active site tunnel at the dimer interface, accessible only to narrow and flexible substrates. The importance of dimerization for polyamine deacetylase function leads to the suggestion that a comparable dimeric or double-domain histone deacetylase could catalyze polyamine deacetylation reactions in eukaryotes. PMID:21268586

  19. Transcriptome analyses reveal molecular mechanisms underlying functional recovery after spinal cord injury

    PubMed Central

    Duan, Hongmei; Ge, Weihong; Zhang, Aifeng; Xi, Yue; Chen, Zhihua; Luo, Dandan; Cheng, Yin; Fan, Kevin S.; Horvath, Steve; Sofroniew, Michael V.; Cheng, Liming; Yang, Zhaoyang; Sun, Yi E.; Li, Xiaoguang

    2015-01-01

    Spinal cord injury (SCI) is considered incurable because axonal regeneration in the central nervous system (CNS) is extremely challenging, due to harsh CNS injury environment and weak intrinsic regeneration capability of CNS neurons. We discovered that neurotrophin-3 (NT3)-loaded chitosan provided an excellent microenvironment to facilitate nerve growth, new neurogenesis, and functional recovery of completely transected spinal cord in rats. To acquire mechanistic insight, we conducted a series of comprehensive transcriptome analyses of spinal cord segments at the lesion site, as well as regions immediately rostral and caudal to the lesion, over a period of 90 days after SCI. Using weighted gene coexpression network analysis (WGCNA), we established gene modules/programs corresponding to various pathological events at different times after SCI. These objective measures of gene module expression also revealed that enhanced new neurogenesis and angiogenesis, and reduced inflammatory responses were keys to conferring the effect of NT3-chitosan on regeneration. PMID:26460053

  20. Spatiotemporal analysis with a genetically encoded fluorescent RNA probe reveals TERRA function around telomeres

    PubMed Central

    Yamada, Toshimichi; Yoshimura, Hideaki; Shimada, Rintaro; Hattori, Mitsuru; Eguchi, Masatoshi; Fujiwara, Takahiro K.; Kusumi, Akihiro; Ozawa, Takeaki

    2016-01-01

    Telomeric repeat-containing RNA (TERRA) controls the structure and length of telomeres through interactions with numerous telomere-binding proteins. However, little is known about the mechanism by which TERRA regulates the accessibility of the proteins to telomeres, mainly because of the lack of spatiotemporal information of TERRA and its-interacting proteins. We developed a fluorescent probe to visualize endogenous TERRA to investigate its dynamics in living cells. Single-particle fluorescence imaging revealed that TERRA accumulated in a telomere-neighboring region and trapped diffusive heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), thereby inhibiting hnRNPA1 localization to the telomere. These results suggest that TERRA regulates binding of hnRNPA1 to the telomere in a region surrounding the telomere, leading to a deeper understanding of the mechanism of TERRA function. PMID:27958374

  1. Structure of Prokaryotic Polyamine Deacetylase Reveals Evolutionary Functional Relationships with Eukaryotic Histone Deacetylases

    SciTech Connect

    P Lombardi; H Angell; D Whittington; E Flynn; K Rajashankar; D Christianson

    2011-12-31

    Polyamines are a ubiquitous class of polycationic small molecules that can influence gene expression by binding to nucleic acids. Reversible polyamine acetylation regulates nucleic acid binding and is required for normal cell cycle progression and proliferation. Here, we report the structures of Mycoplana ramosa acetylpolyamine amidohydrolase (APAH) complexed with a transition state analogue and a hydroxamate inhibitor and an inactive mutant complexed with two acetylpolyamine substrates. The structure of APAH is the first of a histone deacetylase-like oligomer and reveals that an 18-residue insert in the L2 loop promotes dimerization and the formation of an 18 {angstrom} long 'L'-shaped active site tunnel at the dimer interface, accessible only to narrow and flexible substrates. The importance of dimerization for polyamine deacetylase function leads to the suggestion that a comparable dimeric or double-domain histone deacetylase could catalyze polyamine deacetylation reactions in eukaryotes.

  2. Transcriptome analyses reveal molecular mechanisms underlying functional recovery after spinal cord injury.

    PubMed

    Duan, Hongmei; Ge, Weihong; Zhang, Aifeng; Xi, Yue; Chen, Zhihua; Luo, Dandan; Cheng, Yin; Fan, Kevin S; Horvath, Steve; Sofroniew, Michael V; Cheng, Liming; Yang, Zhaoyang; Sun, Yi E; Li, Xiaoguang

    2015-10-27

    Spinal cord injury (SCI) is considered incurable because axonal regeneration in the central nervous system (CNS) is extremely challenging, due to harsh CNS injury environment and weak intrinsic regeneration capability of CNS neurons. We discovered that neurotrophin-3 (NT3)-loaded chitosan provided an excellent microenvironment to facilitate nerve growth, new neurogenesis, and functional recovery of completely transected spinal cord in rats. To acquire mechanistic insight, we conducted a series of comprehensive transcriptome analyses of spinal cord segments at the lesion site, as well as regions immediately rostral and caudal to the lesion, over a period of 90 days after SCI. Using weighted gene coexpression network analysis (WGCNA), we established gene modules/programs corresponding to various pathological events at different times after SCI. These objective measures of gene module expression also revealed that enhanced new neurogenesis and angiogenesis, and reduced inflammatory responses were keys to conferring the effect of NT3-chitosan on regeneration.

  3. Real-time Redox Measurements during Endoplasmic Reticulum Stress Reveal Interlinked Protein Folding Functions

    PubMed Central

    Merksamer, Philip I.; Trusina, Ala; Papa, Feroz R.

    2008-01-01

    SUMMARY Disruption of protein folding in the endoplasmic reticulum (ER) causes unfolded proteins to accumulate, triggering the unfolded protein response (UPR). UPR outputs in turn decrease ER unfolded proteins to close a negative feedback loop. However, because it is infeasible to directly measure the concentration of unfolded proteins in vivo, cells are generically described as experiencing “ER stress” whenever the UPR is active. Because ER redox potential is optimized for oxidative protein folding, we reasoned that measureable redox changes should accompany unfolded protein accumulation. To test this concept, we employed fluorescent protein reporters to dynamically measure ER redox status and UPR activity in single cells. Using these tools, we show that diverse stressors, both experimental and physiological, compromise ER protein oxidation when UPR-imposed homeostatic control is lost. Using genetic analysis we uncovered redox heterogeneities in isogenic cell populations, and revealed functional interlinks between ER protein folding, modification, and quality control systems. PMID:19026441

  4. Targeted mutagenesis of zebrafish antithrombin III triggers disseminated intravascular coagulation and thrombosis, revealing insight into function

    PubMed Central

    Liu, Yang; Kretz, Colin A.; Maeder, Morgan L.; Richter, Catherine E.; Tsao, Philip; Vo, Andy H.; Huarng, Michael C.; Rode, Thomas; Hu, Zhilian; Mehra, Rohit; Olson, Steven T.; Joung, J. Keith

    2014-01-01

    Pathologic blood clotting is a leading cause of morbidity and mortality in the developed world, underlying deep vein thrombosis, myocardial infarction, and stroke. Genetic predisposition to thrombosis is still poorly understood, and we hypothesize that there are many additional risk alleles and modifying factors remaining to be discovered. Mammalian models have contributed to our understanding of thrombosis, but are low throughput and costly. We have turned to the zebrafish, a tool for high-throughput genetic analysis. Using zinc finger nucleases, we show that disruption of the zebrafish antithrombin III (at3) locus results in spontaneous venous thrombosis in larvae. Although homozygous mutants survive into early adulthood, they eventually succumb to massive intracardiac thrombosis. Characterization of null fish revealed disseminated intravascular coagulation in larvae secondary to unopposed thrombin activity and fibrinogen consumption, which could be rescued by both human and zebrafish at3 complementary DNAs. Mutation of the human AT3-reactive center loop abolished the ability to rescue, but the heparin-binding site was dispensable. These results demonstrate overall conservation of AT3 function in zebrafish, but reveal developmental variances in the ability to tolerate excessive clot formation. The accessibility of early zebrafish development will provide unique methods for dissection of the underlying mechanisms of thrombosis. PMID:24782510

  5. Algebraic Topology of Multi-Brain Connectivity Networks Reveals Dissimilarity in Functional Patterns during Spoken Communications

    PubMed Central

    Tadić, Bosiljka; Andjelković, Miroslav; Boshkoska, Biljana Mileva; Levnajić, Zoran

    2016-01-01

    Human behaviour in various circumstances mirrors the corresponding brain connectivity patterns, which are suitably represented by functional brain networks. While the objective analysis of these networks by graph theory tools deepened our understanding of brain functions, the multi-brain structures and connections underlying human social behaviour remain largely unexplored. In this study, we analyse the aggregate graph that maps coordination of EEG signals previously recorded during spoken communications in two groups of six listeners and two speakers. Applying an innovative approach based on the algebraic topology of graphs, we analyse higher-order topological complexes consisting of mutually interwoven cliques of a high order to which the identified functional connections organise. Our results reveal that the topological quantifiers provide new suitable measures for differences in the brain activity patterns and inter-brain synchronisation between speakers and listeners. Moreover, the higher topological complexity correlates with the listener’s concentration to the story, confirmed by self-rating, and closeness to the speaker’s brain activity pattern, which is measured by network-to-network distance. The connectivity structures of the frontal and parietal lobe consistently constitute distinct clusters, which extend across the listener’s group. Formally, the topology quantifiers of the multi-brain communities exceed the sum of those of the participating individuals and also reflect the listener’s rated attributes of the speaker and the narrated subject. In the broader context, the presented study exposes the relevance of higher topological structures (besides standard graph measures) for characterising functional brain networks under different stimuli. PMID:27880802

  6. Sparse representation of HCP grayordinate data reveals novel functional architecture of cerebral cortex.

    PubMed

    Jiang, Xi; Li, Xiang; Lv, Jinglei; Zhang, Tuo; Zhang, Shu; Guo, Lei; Liu, Tianming

    2015-12-01

    The recently publicly released Human Connectome Project (HCP) grayordinate-based fMRI data not only has high spatial and temporal resolution, but also offers group-corresponding fMRI signals across a large population for the first time in the brain imaging field, thus significantly facilitating mapping the functional brain architecture with much higher resolution and in a group-wise fashion. In this article, we adopt the HCP grayordinate task-based fMRI (tfMRI) data to systematically identify and characterize task-based heterogeneous functional regions (THFRs) on cortical surface, i.e., the regions that are activated during multiple tasks conditions and contribute to multiple task-evoked systems during a specific task performance, and to assess the spatial patterns of identified THFRs on cortical gyri and sulci by applying a computational framework of sparse representations of grayordinate brain tfMRI signals. Experimental results demonstrate that both consistent task-evoked networks and intrinsic connectivity networks across all subjects and tasks in HCP grayordinate data are effectively and robustly reconstructed via the proposed sparse representation framework. Moreover, it is found that there are relatively consistent THFRs locating at bilateral parietal lobe, frontal lobe, and visual association cortices across all subjects and tasks. Particularly, those identified THFRs locate significantly more on gyral regions than on sulcal regions. These results based on sparse representation of HCP grayordinate data reveal novel functional architecture of cortical gyri and sulci, and might provide a foundation to better understand functional mechanisms of the human cerebral cortex in the future.

  7. Metagenomes from High-Temperature Chemotrophic Systems Reveal Geochemical Controls on Microbial Community Structure and Function

    PubMed Central

    Inskeep, William P.; Rusch, Douglas B.; Jay, Zackary J.; Herrgard, Markus J.; Kozubal, Mark A.; Richardson, Toby H.; Macur, Richard E.; Hamamura, Natsuko; Jennings, Ryan deM.; Fouke, Bruce W.; Reysenbach, Anna-Louise; Roberto, Frank; Young, Mark; Schwartz, Ariel; Boyd, Eric S.; Badger, Jonathan H.; Mathur, Eric J.; Ortmann, Alice C.; Bateson, Mary; Geesey, Gill; Frazier, Marvin

    2010-01-01

    The Yellowstone caldera contains the most numerous and diverse geothermal systems on Earth, yielding an extensive array of unique high-temperature environments that host a variety of deeply-rooted and understudied Archaea, Bacteria and Eukarya. The combination of extreme temperature and chemical conditions encountered in geothermal environments often results in considerably less microbial diversity than other terrestrial habitats and offers a tremendous opportunity for studying the structure and function of indigenous microbial communities and for establishing linkages between putative metabolisms and element cycling. Metagenome sequence (14–15,000 Sanger reads per site) was obtained for five high-temperature (>65°C) chemotrophic microbial communities sampled from geothermal springs (or pools) in Yellowstone National Park (YNP) that exhibit a wide range in geochemistry including pH, dissolved sulfide, dissolved oxygen and ferrous iron. Metagenome data revealed significant differences in the predominant phyla associated with each of these geochemical environments. Novel members of the Sulfolobales are dominant in low pH environments, while other Crenarchaeota including distantly-related Thermoproteales and Desulfurococcales populations dominate in suboxic sulfidic sediments. Several novel archaeal groups are well represented in an acidic (pH 3) Fe-oxyhydroxide mat, where a higher O2 influx is accompanied with an increase in archaeal diversity. The presence or absence of genes and pathways important in S oxidation-reduction, H2-oxidation, and aerobic respiration (terminal oxidation) provide insight regarding the metabolic strategies of indigenous organisms present in geothermal systems. Multiple-pathway and protein-specific functional analysis of metagenome sequence data corroborated results from phylogenetic analyses and clearly demonstrate major differences in metabolic potential across sites. The distribution of functional genes involved in electron transport

  8. Multivoxel patterns reveal functionally differentiated networks underlying auditory feedback processing of speech.

    PubMed

    Zheng, Zane Z; Vicente-Grabovetsky, Alejandro; MacDonald, Ewen N; Munhall, Kevin G; Cusack, Rhodri; Johnsrude, Ingrid S

    2013-03-06

    The everyday act of speaking involves the complex processes of speech motor control. An important component of control is monitoring, detection, and processing of errors when auditory feedback does not correspond to the intended motor gesture. Here we show, using fMRI and converging operations within a multivoxel pattern analysis framework, that this sensorimotor process is supported by functionally differentiated brain networks. During scanning, a real-time speech-tracking system was used to deliver two acoustically different types of distorted auditory feedback or unaltered feedback while human participants were vocalizing monosyllabic words, and to present the same auditory stimuli while participants were passively listening. Whole-brain analysis of neural-pattern similarity revealed three functional networks that were differentially sensitive to distorted auditory feedback during vocalization, compared with during passive listening. One network of regions appears to encode an "error signal" regardless of acoustic features of the error: this network, including right angular gyrus, right supplementary motor area, and bilateral cerebellum, yielded consistent neural patterns across acoustically different, distorted feedback types, only during articulation (not during passive listening). In contrast, a frontotemporal network appears sensitive to the speech features of auditory stimuli during passive listening; this preference for speech features was diminished when the same stimuli were presented as auditory concomitants of vocalization. A third network, showing a distinct functional pattern from the other two, appears to capture aspects of both neural response profiles. Together, our findings suggest that auditory feedback processing during speech motor control may rely on multiple, interactive, functionally differentiated neural systems.

  9. Metagenomes from high-temperature chemotrophic systems reveal geochemical controls on microbial community structure and function

    SciTech Connect

    Frank Roberto

    2010-03-01

    The Yellowstone caldera contains the most numerous and diverse geothermal systems on Earth, yielding an extensive array of unique high-temperature environments that host numerous deeply-rooted and understudied Archaea, Bacteria and Eukarya. The combination of extreme temperature and chemical conditions encountered in geothermal environments often results in considerably less microbial diversity than other terrestrial habitats and offers a tremendous opportunity for studying the structure and function of indigenous microbial communities and for establishing linkages between putative metabolisms and element cycling. Metagenome sequence (14-15,000 Sanger reads per site) was obtained for five high-temperature (> 65 oC) chemotrophic microbial communities sampled from geothermal springs (or pools) in Yellowstone National Park (YNP) that exhibit a wide range in geochemistry including pH, dissolved sulfide, dissolved O2 and ferrous Fe. Metagenome data revealed significant differences in the predominant phyla associated with each of these geochemical environments. Novel members of the Sulfolobales are dominant in low pH environments, while other Crenarchaeota including distantly-related Thermoproteales and Desulfurococcales populations dominate in suboxic sulfidic sediments. Several novel archaeal groups are well represented in an acidic (pH 3) Fe-oxyhydroxide mat, where a higher O2 influx is accompanied with an increase in archaeal diversity. The presence or absence of genes and pathways important in S oxidation-reduction, H2-oxidation, and aerobic respiration (terminal oxidation) provide insight regarding the metabolic strategies of indigenous organisms present in geothermal systems. Multiple-pathway and protein-specific functional analysis of metagenome sequence data corroborated results from phylogenetic analyses and clearly demonstrate major differences in metabolic potential across sites. The distribution of functional genes involved in electron transport is

  10. Metagenomes from high-temperature chemotrophic systems reveal geochemical controls on microbial community structure and function.

    PubMed

    Inskeep, William P; Rusch, Douglas B; Jay, Zackary J; Herrgard, Markus J; Kozubal, Mark A; Richardson, Toby H; Macur, Richard E; Hamamura, Natsuko; Jennings, Ryan deM; Fouke, Bruce W; Reysenbach, Anna-Louise; Roberto, Frank; Young, Mark; Schwartz, Ariel; Boyd, Eric S; Badger, Jonathan H; Mathur, Eric J; Ortmann, Alice C; Bateson, Mary; Geesey, Gill; Frazier, Marvin

    2010-03-19

    The Yellowstone caldera contains the most numerous and diverse geothermal systems on Earth, yielding an extensive array of unique high-temperature environments that host a variety of deeply-rooted and understudied Archaea, Bacteria and Eukarya. The combination of extreme temperature and chemical conditions encountered in geothermal environments often results in considerably less microbial diversity than other terrestrial habitats and offers a tremendous opportunity for studying the structure and function of indigenous microbial communities and for establishing linkages between putative metabolisms and element cycling. Metagenome sequence (14-15,000 Sanger reads per site) was obtained for five high-temperature (>65 degrees C) chemotrophic microbial communities sampled from geothermal springs (or pools) in Yellowstone National Park (YNP) that exhibit a wide range in geochemistry including pH, dissolved sulfide, dissolved oxygen and ferrous iron. Metagenome data revealed significant differences in the predominant phyla associated with each of these geochemical environments. Novel members of the Sulfolobales are dominant in low pH environments, while other Crenarchaeota including distantly-related Thermoproteales and Desulfurococcales populations dominate in suboxic sulfidic sediments. Several novel archaeal groups are well represented in an acidic (pH 3) Fe-oxyhydroxide mat, where a higher O2 influx is accompanied with an increase in archaeal diversity. The presence or absence of genes and pathways important in S oxidation-reduction, H2-oxidation, and aerobic respiration (terminal oxidation) provide insight regarding the metabolic strategies of indigenous organisms present in geothermal systems. Multiple-pathway and protein-specific functional analysis of metagenome sequence data corroborated results from phylogenetic analyses and clearly demonstrate major differences in metabolic potential across sites. The distribution of functional genes involved in electron

  11. The role of meiotic cohesin REC8 in chromosome segregation in {gamma} irradiation-induced endopolyploid tumour cells

    SciTech Connect

    Erenpreisa, Jekaterina; Cragg, Mark S.; Salmina, Kristine; Hausmann, Michael; Scherthan, Harry

    2009-09-10

    Escape from mitotic catastrophe and generation of endopolyploid tumour cells (ETCs) represents a potential survival strategy of tumour cells in response to genotoxic treatments. ETCs that resume the mitotic cell cycle have reduced ploidy and are often resistant to these treatments. In search for a mechanism for genome reduction, we previously observed that ETCs express meiotic proteins among which REC8 (a meiotic cohesin component) is of particular interest, since it favours reductional cell division in meiosis. In the present investigation, we induced endopolyploidy in p53-dysfunctional human tumour cell lines (Namalwa, WI-L2-NS, HeLa) by gamma irradiation, and analysed the sub-cellular localisation of REC8 in the resulting ETCs. We observed by RT-PCR and Western blot that REC8 is constitutively expressed in these tumour cells, along with SGOL1 and SGOL2, and that REC8 becomes modified after irradiation. REC8 localised to paired sister centromeres in ETCs, the former co-segregating to opposite poles. Furthermore, REC8 localised to the centrosome of interphase ETCs and to the astral poles in anaphase cells where it colocalised with the microtubule-associated protein NuMA. Altogether, our observations indicate that radiation-induced ETCs express features of meiotic cell divisions and that these may facilitate chromosome segregation and genome reduction.

  12. The acetyltransferase activity of San stabilizes the mitotic cohesin at the centromeres in a shugoshin-independent manner

    PubMed Central

    Hou, Fajian; Chu, Chih-Wen; Kong, Xiangduo; Yokomori, Kyoko; Zou, Hui

    2007-01-01

    Proper sister chromatid cohesion is critical for maintaining genetic stability. San is a putative acetyltransferase that is important for sister chromatid cohesion in Drosophila melanogaster, but not in budding yeast. We showed that San is critical for sister chromatid cohesion in HeLa cells, suggesting that this mechanism may be conserved in metazoans. Furthermore, although a small fraction of San interacts with the NatA complex, San appears to mediate cohesion independently. San exhibits acetyltransferase activity in vitro, and its activity is required for sister chromatid cohesion in vivo. In the absence of San, Sgo1 localizes correctly throughout the cell cycle. However, cohesin is no longer detected at the mitotic centromeres. Furthermore, San localizes to the cytoplasm in interphase cells; thus, it may not gain access to chromosomes until mitosis. Moreover, in San-depleted cells, further depletion of Plk1 rescues the cohesion along the chromosome arms, but not at the centromeres. Collectively, San may be specifically required for the maintenance of the centromeric cohesion in mitosis. PMID:17502424

  13. Haspin inhibitors reveal centromeric functions of Aurora B in chromosome segregation

    PubMed Central

    Wang, Fangwei; Ulyanova, Natalia P.; Daum, John R.; Patnaik, Debasis; Kateneva, Anna V.; Gorbsky, Gary J.

    2012-01-01

    Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), providing a docking site for the Aurora B complex at centromeres. Aurora B functions to correct improper kinetochore–microtubule attachments and alert the spindle checkpoint to the presence of misaligned chromosomes. We show that Haspin inhibitors decreased H3T3ph, resulting in loss of centromeric Aurora B and reduced phosphorylation of centromere and kinetochore Aurora B substrates. Consequently, metaphase chromosome alignment and spindle checkpoint signaling were compromised. These effects were phenocopied by microinjection of anti-H3T3ph antibodies. Retargeting Aurora B to centromeres partially restored checkpoint signaling and Aurora B–dependent phosphorylation at centromeres and kinetochores, bypassing the need for Haspin activity. Haspin inhibitors did not obviously affect phosphorylation of histone H3 at serine-10 (H3S10ph) by Aurora B on chromosome arms but, in Aurora B reactivation assays, recovery of H3S10ph was delayed. Haspin inhibitors did not block Aurora B localization to the spindle midzone in anaphase or Aurora B function in cytokinesis. Thus, Haspin inhibitors reveal centromeric roles of Aurora B in chromosome movement and spindle checkpoint signaling. PMID:23071152

  14. Functional proteomic analyses of Bothrops atrox venom reveals phenotypes associated with habitat variation in the Amazon.

    PubMed

    Sousa, Leijiane F; Portes-Junior, José A; Nicolau, Carolina A; Bernardoni, Juliana L; Nishiyama-Jr, Milton Y; Amazonas, Diana R; Freitas-de-Sousa, Luciana A; Mourão, Rosa Hv; Chalkidis, Hipócrates M; Valente, Richard H; Moura-da-Silva, Ana M

    2017-03-06

    Venom variability is commonly reported for venomous snakes including Bothrops atrox. Here, we compared the composition of venoms from B. atrox snakes collected at Amazonian conserved habitats (terra-firme upland forest and várzea) and human modified areas (pasture and degraded areas). Venom samples were submitted to shotgun proteomic analysis as a whole or compared after fractionation by reversed-phase chromatography. Whole venom proteomes revealed a similar composition among the venoms with predominance of SVMPs, CTLs, and SVSPs and intermediate amounts of PLA2s and LAAOs. However, when distribution of particular isoforms was analyzed by either method, the venom from várzea snakes showed a decrease in hemorrhagic SVMPs and an increase in SVSPs, and procoagulant SVMPs and PLA2s. These differences were validated by experimental approaches including both enzymatic and in vivo assays, and indicated restrictions in respect to antivenom efficacy to variable components. Thus, proteomic analysis at the isoform level combined to in silico prediction of functional properties may indicate venom biological activity. These results also suggest that the prevalence of functionally distinct isoforms contributes to the variability of the venoms and could reflect the adaptation of B. atrox to distinct prey communities in different Amazon habitats.

  15. Structure reveals function of the dual variable domain immunoglobulin (DVD-Ig™) molecule.

    PubMed

    Jakob, Clarissa G; Edalji, Rohinton; Judge, Russell A; DiGiammarino, Enrico; Li, Yingchun; Gu, Jijie; Ghayur, Tariq

    2013-01-01

    Several bispecific antibody-based formats have been developed over the past 25 years in an effort to produce a new generation of immunotherapeutics that target two or more disease mechanisms simultaneously. One such format, the dual-variable domain immunoglobulin (DVD-Ig™), combines the target binding domains of two monoclonal antibodies via flexible naturally occurring linkers, which yields a tetravalent IgG - like molecule. We report the structure of an interleukin (IL)12-IL18 DVD-Ig™ Fab (DFab) fragment with IL18 bound to the inner variable domain (VD) that reveals the remarkable flexibility of the DVD-Ig™ molecule and how the DVD-Ig™ format can function to bind four antigens simultaneously. An understanding of how the inner variable domain retains function is of critical importance for designing DVD-Ig™ molecules, and for better understanding of the flexibility of immunoglobulin variable domains and linkers, which may aid in the design of improved bi- and multi-specific biologics in general.

  16. Yeast mitochondrial protein-protein interactions reveal diverse complexes and disease-relevant functional relationships.

    PubMed

    Jin, Ke; Musso, Gabriel; Vlasblom, James; Jessulat, Matthew; Deineko, Viktor; Negroni, Jacopo; Mosca, Roberto; Malty, Ramy; Nguyen-Tran, Diem-Hang; Aoki, Hiroyuki; Minic, Zoran; Freywald, Tanya; Phanse, Sadhna; Xiang, Qian; Freywald, Andrew; Aloy, Patrick; Zhang, Zhaolei; Babu, Mohan

    2015-02-06

    Although detailed, focused, and mechanistic analyses of associations among mitochondrial proteins (MPs) have identified their importance in varied biological processes, a systematic understanding of how MPs function in concert both with one another and with extra-mitochondrial proteins remains incomplete. Consequently, many questions regarding the role of mitochondrial dysfunction in the development of human disease remain unanswered. To address this, we compiled all existing mitochondrial physical interaction data for over 1200 experimentally defined yeast MPs and, through bioinformatic analysis, identified hundreds of heteromeric MP complexes having extensive associations both within and outside the mitochondria. We provide support for these complexes through structure prediction analysis, morphological comparisons of deletion strains, and protein co-immunoprecipitation. The integration of these MP complexes with reported genetic interaction data reveals substantial crosstalk between MPs and non-MPs and identifies novel factors in endoplasmic reticulum-mitochondrial organization, membrane structure, and mitochondrial lipid homeostasis. More than one-third of these MP complexes are conserved in humans, with many containing members linked to clinical pathologies, enabling us to identify genes with putative disease function through guilt-by-association. Although still remaining incomplete, existing mitochondrial interaction data suggests that the relevant molecular machinery is modular, yet highly integrated with non-mitochondrial processes.

  17. Comparative analysis and functional mapping of SACS mutations reveal novel insights into sacsin repeated architecture.

    PubMed

    Romano, Alessandro; Tessa, Alessandra; Barca, Amilcare; Fattori, Fabiana; de Leva, Maria Fulvia; Terracciano, Alessandra; Storelli, Carlo; Santorelli, Filippo Maria; Verri, Tiziano

    2013-03-01

    Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurological disease with mutations in SACS, encoding sacsin, a multidomain protein of 4,579 amino acids. The large size of SACS and its translated protein has hindered biochemical analysis of ARSACS, and how mutant sacsins lead to disease remains largely unknown. Three repeated sequences, called sacsin repeating region (SRR) supradomains, have been recognized, which contribute to sacsin chaperone-like activity. We found that the three SRRs are much larger (≥1,100 residues) than previously described, and organized in discrete subrepeats. We named the large repeated regions Sacsin Internal RePeaTs (SIRPT1, SIRPT2, and SIRPT3) and the subrepeats sr1, sr2, sr3, and srX. Comparative analysis of vertebrate sacsins in combination with fine positional mapping of a set of human mutations revealed that sr1, sr2, sr3, and srX are functional. Notably, the position of the pathogenic mutations in sr1, sr2, sr3, and srX appeared to be related to the severity of the clinical phenotype, as assessed by defining a severity scoring system. Our results suggest that the relative position of mutations in subrepeats will variably influence sacsin dysfunction. The characterization of the specific role of each repeated region will help in developing a comprehensive and integrated pathophysiological model of function for sacsin.

  18. Functional proteomics reveal the effect of Salvia miltiorrhiza aqueous extract against vascular atherosclerotic lesions.

    PubMed

    Hung, Yu-Chiang; Wang, Pei-Wen; Pan, Tai-Long

    2010-06-01

    Salvia miltiorrhiza is a Chinese herb widely used for cardiovascular disorder regimens, yet little is known about the cellular mechanisms that contribute to attenuated growth of smooth muscle cells (SMCs) under oxidative stress such as homocysteine (Hcy) treatment. As anticipated, a low dose (0.015 mg/mL) of S.miltiorrhiza aqueous extract (SMAE) significantly inhibited (>60%) the growth of a rat smooth muscle cell line (A10) under Hcy stimulation and the intracellular reactive oxygen species (ROS) concentration obviously decreased after SMAE treatment in terms of reducing p47(phox) translocation and increasing catalase activity. Signaling profile suggests that SMAE inhibited Hcy-induced A10 cell growth via the PKC/MAPK-dependent pathway. Two-dimensional electrophoresis (2-DE) coupled with mass spectrometry revealed statistically significant changes in the intensity of 14 proteins in response to Hcy and Hcy/SMAE. Meanwhile, SMAE attenuated carbonyl-modification of specific cytoskeleton and chaperone proteins leading to cell type transformation. Moreover, a network analysis using MetaCore shed more light on the molecular basis associated with SMAE efficacy. SMAE exerts its protective effect through the scavenging of ROS and subsequent modulation of protein carbonylation to inhibit cell proliferation. These signature networks and functional proteomics highlighted herein may facilitate the evaluation of potential therapeutic targets and elucidate novel mechanisms through which protein functions can be regulated by the redox status.

  19. Comparative materials differences revealed in engineered bone as a function of cell-specific differentiation

    NASA Astrophysics Data System (ADS)

    Gentleman, Eileen; Swain, Robin J.; Evans, Nicholas D.; Boonrungsiman, Suwimon; Jell, Gavin; Ball, Michael D.; Shean, Tamaryn A. V.; Oyen, Michelle L.; Porter, Alexandra; Stevens, Molly M.

    2009-09-01

    An important aim of regenerative medicine is to restore tissue function with implantable, laboratory-grown constructs that contain tissue-specific cells that replicate the function of their counterparts in the healthy native tissue. It remains unclear, however, whether cells used in bone regeneration applications produce a material that mimics the structural and compositional complexity of native bone. By applying multivariate analysis techniques to micro-Raman spectra of mineralized nodules formed in vitro, we reveal cell-source-dependent differences in interactions between multiple bone-like mineral environments. Although osteoblasts and adult stem cells exhibited bone-specific biological activities and created a material with many of the hallmarks of native bone, the `bone nodules' formed from embryonic stem cells were an order of magnitude less stiff, and lacked the distinctive nanolevel architecture and complex biomolecular and mineral composition noted in the native tissue. Understanding the biological mechanisms of bone formation in vitro that contribute to cell-source-specific materials differences may facilitate the development of clinically successful engineered bone.

  20. Evolution-guided functional analyses reveal diverse antiviral specificities encoded by IFIT1 genes in mammals

    PubMed Central

    Daugherty, Matthew D; Schaller, Aaron M; Geballe, Adam P; Malik, Harmit S

    2016-01-01

    IFIT (interferon-induced with tetratricopeptide repeats) proteins are critical mediators of mammalian innate antiviral immunity. Mouse IFIT1 selectively inhibits viruses that lack 2'O-methylation of their mRNA 5' caps. Surprisingly, human IFIT1 does not share this antiviral specificity. Here, we resolve this discrepancy by demonstrating that human and mouse IFIT1 have evolved distinct functions using a combination of evolutionary, genetic and virological analyses. First, we show that human IFIT1 and mouse IFIT1 (renamed IFIT1B) are not orthologs, but are paralogs that diverged >100 mya. Second, using a yeast genetic assay, we show that IFIT1 and IFIT1B proteins differ in their ability to be suppressed by a cap 2'O-methyltransferase. Finally, we demonstrate that IFIT1 and IFIT1B have divergent antiviral specificities, including the discovery that only IFIT1 proteins inhibit a virus encoding a cap 2'O-methyltransferase. These functional data, combined with widespread turnover of mammalian IFIT genes, reveal dramatic species-specific differences in IFIT-mediated antiviral repertoires. DOI: http://dx.doi.org/10.7554/eLife.14228.001 PMID:27240734

  1. Splicing Functions and Global Dependency on Fission Yeast Slu7 Reveal Diversity in Spliceosome Assembly

    PubMed Central

    Banerjee, Shataparna; Khandelia, Piyush; Melangath, Geetha; Bashir, Samirul; Nagampalli, Vijaykrishna

    2013-01-01

    The multiple short introns in Schizosaccharomyces pombe genes with degenerate cis sequences and atypically positioned polypyrimidine tracts make an interesting model to investigate canonical and alternative roles for conserved splicing factors. Here we report functions and interactions of the S. pombe slu7+ (spslu7+) gene product, known from Saccharomyces cerevisiae and human in vitro reactions to assemble into spliceosomes after the first catalytic reaction and to dictate 3′ splice site choice during the second reaction. By using a missense mutant of this essential S. pombe factor, we detected a range of global splicing derangements that were validated in assays for the splicing status of diverse candidate introns. We ascribe widespread, intron-specific SpSlu7 functions and have deduced several features, including the branch nucleotide-to-3′ splice site distance, intron length, and the impact of its A/U content at the 5′ end on the intron's dependence on SpSlu7. The data imply dynamic substrate-splicing factor relationships in multiintron transcripts. Interestingly, the unexpected early splicing arrest in spslu7-2 revealed a role before catalysis. We detected a salt-stable association with U5 snRNP and observed genetic interactions with spprp1+, a homolog of human U5-102k factor. These observations together point to an altered recruitment and dependence on SpSlu7, suggesting its role in facilitating transitions that promote catalysis, and highlight the diversity in spliceosome assembly. PMID:23754748

  2. Metagenomics Reveals Microbial Community Composition And Function With Depth In Arctic Permafrost Cores

    NASA Astrophysics Data System (ADS)

    Jansson, J.; Tas, N.; Wu, Y.; Ulrich, C.; Kneafsey, T. J.; Torn, M. S.; Hubbard, S. S.; Chakraborty, R.; Graham, D. E.; Wullschleger, S. D.

    2013-12-01

    The Arctic is one of the most climatically sensitive regions on Earth and current surveys show that permafrost degradation is widespread in arctic soils. Biogeochemical feedbacks of permafrost thaw are expected to be dominated by the release of currently stored carbon back into the atmosphere as CO2 and CH4. Understanding the dynamics of C release from permafrost requires assessment of microbial functions from different soil compartments. To this end, as part of the Next Generation Ecosystem Experiment in the Arctic, we collected two replicate permafrost cores (1m and 3m deep) from a transitional polygon near Barrow, AK. At this location, permafrost starts from 0.5m in depth and is characterized by variable ice content and higher pH than surface soils. Prior to sectioning, the cores were CT-scanned to determine the physical heterogeneity throughout the cores. In addition to detailed geochemical characterization, we used Illumina MiSeq technology to sequence 16SrRNA genes throughout the depths of the cores at 1 cm intervals. Selected depths were also chosen for metagenome sequencing of total DNA (including phylogenetic and functional genes) using the Illumina HiSeq platform. The 16S rRNA gene sequence data revealed that the microbial community composition and diversity changed dramatically with depth. The microbial diversity decreased sharply below the first few centimeters of the permafrost and then gradually increased in deeper layers. Based on the metagenome sequence data, the permafrost microbial communities were found to contain members with a large metabolic potential for carbon processing, including pathways for fermentation and methanogenesis. The surface active layers had more representatives of Verrucomicrobia (potential methane oxidizers) whereas the deep permafrost layers were dominated by several different species of Actinobacteria. The latter are known to have a diverse metabolic capability and are able to adapt to stress by entering a dormant yet

  3. Prokaryotic Caspase Homologs: Phylogenetic Patterns and Functional Characteristics Reveal Considerable Diversity

    PubMed Central

    Asplund-Samuelsson, Johannes; Bergman, Birgitta; Larsson, John

    2012-01-01

    Caspases accomplish initiation and execution of apoptosis, a programmed cell death process specific to metazoans. The existence of prokaryotic caspase homologs, termed metacaspases, has been known for slightly more than a decade. Despite their potential connection to the evolution of programmed cell death in eukaryotes, the phylogenetic distribution and functions of these prokaryotic metacaspase sequences are largely uncharted, while a few experiments imply involvement in programmed cell death. Aiming at providing a more detailed picture of prokaryotic caspase homologs, we applied a computational approach based on Hidden Markov Model search profiles to identify and functionally characterize putative metacaspases in bacterial and archaeal genomes. Out of the total of 1463 analyzed genomes, merely 267 (18%) were identified to contain putative metacaspases, but their taxonomic distribution included most prokaryotic phyla and a few archaea (Euryarchaeota). Metacaspases were particularly abundant in Alphaproteobacteria, Deltaproteobacteria and Cyanobacteria, which harbor many morphologically and developmentally complex organisms, and a distinct correlation was found between abundance and phenotypic complexity in Cyanobacteria. Notably, Bacillus subtilis and Escherichia coli, known to undergo genetically regulated autolysis, lacked metacaspases. Pfam domain architecture analysis combined with operon identification revealed rich and varied configurations among the metacaspase sequences. These imply roles in programmed cell death, but also e.g. in signaling, various enzymatic activities and protein modification. Together our data show a wide and scattered distribution of caspase homologs in prokaryotes with structurally and functionally diverse sub-groups, and with a potentially intriguing evolutionary role. These features will help delineate future characterizations of death pathways in prokaryotes. PMID:23185476

  4. Revealing the functional neuroanatomy of intrinsic alertness using fMRI: methodological peculiarities.

    PubMed

    Clemens, Benjamin; Zvyagintsev, Mikhail; Sack, Alexander T; Sack, Alexander; Heinecke, Armin; Willmes, Klaus; Sturm, Walter

    2011-01-01

    Clinical observations and neuroimaging data revealed a right-hemisphere fronto-parietal-thalamic-brainstem network for intrinsic alertness, and additional left fronto-parietal activity during phasic alertness. The primary objective of this fMRI study was to map the functional neuroanatomy of intrinsic alertness as precisely as possible in healthy participants, using a novel assessment paradigm already employed in clinical settings. Both the paradigm and the experimental design were optimized to specifically assess intrinsic alertness, while at the same time controlling for sensory-motor processing. The present results suggest that the processing of intrinsic alertness is accompanied by increased activity within the brainstem, thalamus, anterior cingulate gyrus, right insula, and right parietal cortex. Additionally, we found increased activation in the left hemisphere around the middle frontal gyrus (BA 9), the insula, the supplementary motor area, and the cerebellum. Our results further suggest that rather minute aspects of the experimental design may induce aspects of phasic alertness, which in turn might lead to additional brain activation in left-frontal areas not normally involved in intrinsic alertness. Accordingly, left BA 9 activation may be related to co-activation of the phasic alertness network due to the switch between rest and task conditions functioning as an external warning cue triggering the phasic alertness network. Furthermore, activation of the intrinsic alertness network during fixation blocks due to enhanced expectancy shortly before the switch to the task block might, when subtracted from the task block, lead to diminished activation in the typical right hemisphere intrinsic alertness network. Thus, we cautiously suggest that--as a methodological artifact--left frontal activations might show up due to phasic alertness involvement and intrinsic alertness activations might be weakened due to contrasting with fixation blocks, when assessing the

  5. Transcriptome and Functional Genomics Reveal the Participation of Adenine Phosphoribosyltransferase in Trypanosoma cruzi Resistance to Benznidazole.

    PubMed

    García-Huertas, Paola; Mejía-Jaramillo, Ana María; González, Laura; Triana Chávez, Omar

    2017-03-09

    Currently, the only available treatments for Trypanosoma cruzi are benznidazole (Bz) and nifurtimox (Nfx). The mechanisms of action and resistance to these drugs in this parasite are not complete known. In order to identify differentially expressed transcripts between sensitive and resistant parasites, a massive pyrosequencing of the T. cruzi transcriptome was carried out. Additionally, the 2D gel electrophoresis profile of sensitive and resistant parasites was analyzed and the data were supported with functional genomics. The results showed 133 differentially expressed genes in resistant parasites. The transcriptome analysis revealed the regulation of different genes with several functions and metabolic pathways, which could suggest that resistance in T. cruzi is a multigenic process. Additionally, using transcriptomics, one gene, adenine phosphoribosyltransferase (APRT), was found to be down-regulated in the resistant parasites and its expression profile was confirmed by 2D electrophoresis analysis. The role of this gene in the resistance to Bz was confirmed overexpressing it in sensitive and resistant parasites. Interestingly, both parasites became more sensitive to Bz and H2 O2 . This is the first RNA-seq study to identify regulated genes in T. cruzi associated with Bz resistance and to show the role of APRT in T. cruzi resistance. Although T. cruzi regulation is mainly post-transcriptional, the transcriptome analysis, supported by 2D gel analysis and functional genomic, provides an overall idea of the expression profiles of genes under resistance conditions. These results contribute essential information to further the understanding of the mechanisms of action and resistance to Bz in T. cruzi. This article is protected by copyright. All rights reserved.

  6. GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function

    PubMed Central

    Porokuokka, Lauriina L.; Panhelainen, Anne; Kuure, Satu; Marshall, Pepin; Karalija, Nina; Härma, Mari-Anne; Vilenius, Carolina; Lilleväli, Kersti; Tekko, Triin; Mijatovic, Jelena; Pulkkinen, Nita; Jakobson, Madis; Jakobson, Maili; Ola, Roxana; Palm, Erik; Lindahl, Maria; Strömberg, Ingrid; Võikar, Vootele; Piepponen, T. Petteri; Saarma, Mart; Andressoo, Jaan-Olle

    2015-01-01

    Degeneration of nigrostriatal dopaminergic system is the principal lesion in Parkinson’s disease. Because glial cell line-derived neurotrophic factor (GDNF) promotes survival of dopamine neurons in vitro and in vivo, intracranial delivery of GDNF has been attempted for Parkinson’s disease treatment but with variable success. For improving GDNF-based therapies, knowledge on physiological role of endogenous GDNF at the sites of its expression is important. However, due to limitations of existing genetic model systems, such knowledge is scarce. Here, we report that prevention of transcription of Gdnf 3’UTR in Gdnf endogenous locus yields GDNF hypermorphic mice with increased, but spatially unchanged GDNF expression, enabling analysis of postnatal GDNF function. We found that increased level of GDNF in the central nervous system increases the number of adult dopamine neurons in the substantia nigra pars compacta and the number of dopaminergic terminals in the dorsal striatum. At the functional level, GDNF levels increased striatal tissue dopamine levels and augmented striatal dopamine release and re-uptake. In a proteasome inhibitor lactacystin-induced model of Parkinson’s disease GDNF hypermorphic mice were protected from the reduction in striatal dopamine and failure of dopaminergic system function. Importantly, adverse phenotypic effects associated with spatially unregulated GDNF applications were not observed. Enhanced GDNF levels up-regulated striatal dopamine transporter activity by at least five fold resulting in enhanced susceptibility to 6-OHDA, a toxin transported into dopamine neurons by DAT. Further, we report how GDNF levels regulate kidney development and identify microRNAs miR-9, miR-96, miR-133, and miR-146a as negative regulators of GDNF expression via interaction with Gdnf 3’UTR in vitro. Our results reveal the role of GDNF in nigrostriatal dopamine system postnatal development and adult function, and highlight the importance of correct

  7. Functional Connectivity Reveals Which Language the “Control Regions” Control during Bilingual Production

    PubMed Central

    Li, Le; Emmorey, Karen; Feng, Xiaoxia; Lu, Chunming; Ding, Guosheng

    2016-01-01

    Bilingual studies have revealed critical roles for the dorsal anterior cingulate cortex (dACC) and the left caudate nucleus (Lcaudate) in controlling language processing, but how these regions manage activation of a bilingual’s two languages remains an open question. We addressed this question by identifying the functional connectivity (FC) of these control regions during a picture-naming task by bimodal bilinguals who were fluent in both a spoken and a signed language. To quantify language control processes, we measured the FC of the dACC and Lcaudate with a region specific to each language modality: left superior temporal gyrus (LSTG) for speech and left pre/postcentral gyrus (LPCG) for sign. Picture-naming occurred in either a single- or dual-language context. The results showed that in a single-language context, the dACC exhibited increased FC with the target language region, but not with the non-target language region. During the dual-language context when both languages were alternately the target language, the dACC showed strong FC to the LPCG, the region specific to the less proficient (signed) language. By contrast, the Lcaudate revealed a strong connectivity to the LPCG in the single-language context and to the LSTG (the region specific to spoken language) in the dual-language context. Our findings suggest that the dACC monitors and supports the processing of the target language, and that the Lcaudate controls the selection of the less accessible language. The results support the hypothesis that language control processes adapt to task demands that vary due to different interactional contexts. PMID:27965563

  8. Functional magnetic resonance imaging adaptation reveals a noncategorical representation of hue in early visual cortex

    PubMed Central

    Persichetti, Andrew S.; Thompson-Schill, Sharon L.; Butt, Omar H.; Brainard, David H.; Aguirre, Geoffrey K.

    2015-01-01

    Color names divide the fine-grained gamut of color percepts into discrete categories. A categorical transition must occur somewhere between the initial encoding of the continuous spectrum of light by the cones and the verbal report of the name of a color stimulus. Here, we used a functional magnetic resonance imaging (fMRI) adaptation experiment to examine the representation of hue in the early visual cortex. Our stimuli varied in hue between blue and green. We found in the early visual areas (V1, V2/3, and hV4) a smoothly increasing recovery from adaptation with increasing hue distance between adjacent stimuli during both passive viewing (Experiment 1) and active categorization (Experiment 2). We examined the form of the adaptation effect and found no evidence that a categorical representation mediates the release from adaptation for stimuli that cross the blue–green color boundary. Examination of the direct effect of stimulus hue on the fMRI response did, however, reveal an enhanced response to stimuli near the blue–green category border. This was largest in hV4 and when subjects were engaged in active categorization of the stimulus hue. In contrast with a recent report from another laboratory (Bird, Berens, Horner, & Franklin, 2014), we found no evidence for a categorical representation of color in the middle frontal gyrus. A post hoc whole-brain analysis, however, revealed several regions in the frontal cortex with a categorical effect in the adaptation response. Overall, our results support the idea that the representation of color in the early visual cortex is primarily fine grained and does not reflect color categories. PMID:26024465

  9. Emotional regulatory function of Receptor Interacting Protein 140 revealed in the ventromedial hypothalamus

    PubMed Central

    Flaisher-Grinberg, S; Tsai, HC; Feng, X; Wei, LN

    2014-01-01

    Receptor-interacting protein (RIP140) is a transcription co-regulator highly expressed in macrophages to regulate inflammatory and metabolic processes. However, its implication in neurological, cognitive and emotional conditions, and the cellular systems relevant to its biological activity within the central nervous system are currently less clear. A transgenic mouse line with macrophage-specific knockdown of RIP140 was generated (MΦRIPKD mice) and brain-region specific RIP140 knockdown efficiency evaluated. Mice were subjected to a battery of tests, designed to evaluate multiple behavioral domains at naïve or following site-specific RIP140 re-expression. Gene expression analysis assessed TNF-α, IL-1β, TGF-1β, IL1-RA and Neuropeptide Y (NPY) expression, and in-vitro studies examined the effects of macrophage’s RIP140 on astrocytes’ NPY production. We found RIP140 expression was dramatically reduced in macrophages within the ventromedial hypothalamus (VMH) and the cingulate cortex of MΦRIPKD mice. These animals exhibited increased anxiety- and depressive-like behaviors. VMH-targeted RIP140 re-expression in MΦRIPKD mice reversed its depressive- but not its anxiety-like phenotype. Analysis of specific neurochemical changes revealed reduced astrocytic-NPY expression within the hypothalamus of MΦRIPKD mice, and in-vitro analysis confirmed that conditioned medium of RIP140-silnenced macrophage culture could no longer stimulate NPY production from astrocytes. The current study revealed an emotional regulatory function of macrophage-derived RIP140 in the VMH, and secondary dysregulation of NPY within hypothalamic astrocyte population, which might be associated with the observed behavioral phenotype of MΦRIPKD mice. This study highlights RIP140 as a novel target for the development of potential therapeutic and intervention strategies for emotional regulation disorders. PMID:24726835

  10. Metagenomics, metatranscriptomics and single cell genomics reveal functional response of active Oceanospirillales to Gulf oil spill

    SciTech Connect

    Mason, Olivia U.; Hazen, Terry C.; Borglin, Sharon; Chain, Patrick S. G.; Dubinsky, Eric A.; Fortney, Julian L.; Han, James; Holman, Hoi-Ying N.; Hultman, Jenni; Lamendella, Regina; Mackelprang, Rachel; Malfatti, Stephanie; Tom, Lauren M.; Tringe, Susannah G.; Woyke, Tanja; Zhou, Jizhong; Rubin, Edward M.; Jansson, Janet K.

    2012-06-12

    The Deepwater Horizon oil spill in the Gulf of Mexico resulted in a deep-sea hydrocarbon plume that caused a shift in the indigenous microbial community composition with unknown ecological consequences. Early in the spill history, a bloom of uncultured, thus uncharacterized, members of the Oceanospirillales was previously detected, but their role in oil disposition was unknown. Here our aim was to determine the functional role of the Oceanospirillales and other active members of the indigenous microbial community using deep sequencing of community DNA and RNA, as well as single-cell genomics. Shotgun metagenomic and metatranscriptomic sequencing revealed that genes for motility, chemotaxis and aliphatic hydrocarbon degradation were significantly enriched and expressed in the hydrocarbon plume samples compared with uncontaminated seawater collected from plume depth. In contrast, although genes coding for degradation of more recalcitrant compounds, such as benzene, toluene, ethylbenzene, total xylenes and polycyclic aromatic hydrocarbons, were identified in the metagenomes, they were expressed at low levels, or not at all based on analysis of the metatranscriptomes. Isolation and sequencing of two Oceanospirillales single cells revealed that both cells possessed genes coding for n-alkane and cycloalkane degradation. Specifically, the near-complete pathway for cyclohexane oxidation in the Oceanospirillales single cells was elucidated and supported by both metagenome and metatranscriptome data. The draft genome also included genes for chemotaxis, motility and nutrient acquisition strategies that were also identified in the metagenomes and metatranscriptomes. These data point towards a rapid response of members of the Oceanospirillales to aliphatic hydrocarbons in the deep sea.

  11. REVEALING PROBABLE UNIVERSAL FEATURES IN THE LOWER RED GIANT BRANCH LUMINOSITY FUNCTIONS OF GALACTIC GLOBULAR CLUSTERS

    SciTech Connect

    Kravtsov, V. V.

    2009-06-15

    This paper aims at demonstrating, for the first time, very probable universal peculiarities of the evolution of stars in the lower red giant branch (RGB) of Galactic globular clusters (GCs), reflected in two corresponding dips in the luminosity functions (LFs). By relying on the database of Hubble Space Telescope photometry of GCs, we analyze the lower RGB LFs of a sample of 18 GCs in a wide metallicity range, {delta}[Fe/H] {approx} 1.9 dex. We first show that in the F555W-(F439W-F555W) color-magnitude diagrams (CMDs), the lower RGB of GCs, except for the most metal-poor of them, frequently shows an apparent 'knee'. It reveals itself as a fairly abrupt change of the RGB slope. At the same luminosity level, the RGB LFs show a feature in the form of a more or less pronounced dip. We find that the magnitude difference between the RGB base and the given feature is, on average, around {delta} F555W{sup dip} {sub base}{approx} 1.4 mag. It shows a marginal variation with metallicity, if any, comparable to the error. At the same time, the magnitude difference between the dip and the RGB bump, {delta} F555W{sup bump} {sub dip}, decreases with increasing metallicity and falls within the range 0.8 {approx}< {delta} F555W{sup bump} {sub dip} {approx}< 1.7 mag. Generalized LFs (GLFs) have been obtained for three subsamples of GCs within limited metallicity ranges and with different horizontal branch (HB) morphology. They reproduce the 'knee-related' dip that is statistically significant in two of the GLFs. This feature turns out to be more pronounced in the GLFs of GCs with either the blue or red HB morphology than with the intermediate one. The same GLFs also reveal an additional probable universal dip. It shows up below the RGB bump at {delta} F555W slightly increasing from {approx}0.3 to {approx}0.5 mag with increasing metallicity. Also, the statistical significance of this 'prebump' dip increases, on average, toward higher metallicity. Except for the well known RGB bump, no

  12. Functional analysis of environmental DNA-derived type II polyketide synthases reveals structurally diverse secondary metabolites

    PubMed Central

    Feng, Zhiyang; Kallifidas, Dimitris; Brady, Sean F.

    2011-01-01

    A single gram of soil is predicted to contain thousands of unique bacterial species. The majority of these species remain recalcitrant to standard culture methods, prohibiting their use as sources of unique bioactive small molecules. The cloning and analysis of DNA extracted directly from environmental samples (environmental DNA, eDNA) provides a means of exploring the biosynthetic capacity of natural bacterial populations. Environmental DNA libraries contain large reservoirs of bacterial genetic diversity from which new secondary metabolite gene clusters can be systematically recovered and studied. The identification and heterologous expression of type II polyketide synthase-containing eDNA clones is reported here. Functional analysis of three soil DNA-derived polyketide synthase systems in Streptomyces albus revealed diverse metabolites belonging to well-known, rare, and previously uncharacterized structural families. The first of these systems is predicted to encode the production of the known antibiotic landomycin E. The second was found to encode the production of a metabolite with a previously uncharacterized pentacyclic ring system. The third was found to encode the production of unique KB-3346-5 derivatives, which show activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. These results, together with those of other small-molecule-directed metagenomic studies, suggest that culture-independent approaches are capable of accessing biosynthetic diversity that has not yet been extensively explored using culture-based methods. The large-scale functional screening of eDNA clones should be a productive strategy for generating structurally previously uncharacterized chemical entities for use in future drug development efforts. PMID:21768346

  13. Functional malignant cell heterogeneity in pancreatic neuroendocrine tumors revealed by targeting of PDGF-DD.

    PubMed

    Cortez, Eliane; Gladh, Hanna; Braun, Sebastian; Bocci, Matteo; Cordero, Eugenia; Björkström, Niklas K; Miyazaki, Hideki; Michael, Iacovos P; Eriksson, Ulf; Folestad, Erika; Pietras, Kristian

    2016-02-16

    Intratumoral heterogeneity is an inherent feature of most human cancers and has profound implications for cancer therapy. As a result, there is an emergent need to explore previously unmapped mechanisms regulating distinct subpopulations of tumor cells and to understand their contribution to tumor progression and treatment response. Aberrant platelet-derived growth factor receptor beta (PDGFRβ) signaling in cancer has motivated the development of several antagonists currently in clinical use, including imatinib, sunitinib, and sorafenib. The discovery of a novel ligand for PDGFRβ, platelet-derived growth factor (PDGF)-DD, opened the possibility of a previously unidentified signaling pathway involved in tumor development. However, the precise function of PDGF-DD in tumor growth and invasion remains elusive. Here, making use of a newly generated Pdgfd knockout mouse, we reveal a functionally important malignant cell heterogeneity modulated by PDGF-DD signaling in pancreatic neuroendocrine tumors (PanNET). Our analyses demonstrate that tumor growth was delayed in the absence of signaling by PDGF-DD. Surprisingly, ablation of PDGF-DD did not affect the vasculature or stroma of PanNET; instead, we found that PDGF-DD stimulated bulk tumor cell proliferation by induction of paracrine mitogenic signaling between heterogeneous malignant cell clones, some of which expressed PDGFRβ. The presence of a subclonal population of tumor cells characterized by PDGFRβ expression was further validated in a cohort of human PanNET. In conclusion, we demonstrate a previously unrecognized heterogeneity in PanNET characterized by signaling through the PDGF-DD/PDGFRβ axis.

  14. Transcriptomic analysis of toxoplasma development reveals many novel functions and structures specific to sporozoites and oocysts.

    PubMed

    Fritz, Heather M; Buchholz, Kerry R; Chen, Xiucui; Durbin-Johnson, Blythe; Rocke, David M; Conrad, Patricia A; Boothroyd, John C

    2012-01-01

    Sexual reproduction of Toxoplasma gondii occurs exclusively within enterocytes of the definitive felid host. The resulting immature oocysts are excreted into the environment during defecation, where in the days following, they undergo a complex developmental process. Within each oocyst, this culminates in the generation of two sporocysts, each containing 4 sporozoites. A single felid host is capable of shedding millions of oocysts, which can survive for years in the environment, are resistant to most methods of microbial inactivation during water-treatment and are capable of producing infection in warm-blooded hosts at doses as low as 1-10 ingested oocysts. Despite its extremely interesting developmental biology and crucial role in initiating an infection, almost nothing is known about the oocyst stage beyond morphological descriptions. Here, we present a complete transcriptomic analysis of the oocyst from beginning to end of its development. In addition, and to identify genes whose expression is unique to this developmental form, we compared the transcriptomes of developing oocysts with those of in vitro-derived tachyzoites and in vivo-derived bradyzoites. Our results reveal many genes whose expression is specifically up- or down-regulated in different developmental stages, including many genes that are likely critical to oocyst development, wall formation, resistance to environmental destruction and sporozoite infectivity. Of special note is the up-regulation of genes that appear "off" in tachyzoites and bradyzoites but that encode homologues of proteins known to serve key functions in those asexual stages, including a novel pairing of sporozoite-specific paralogues of AMA1 and RON2, two proteins that have recently been shown to form a crucial bridge during tachyzoite invasion of host cells. This work provides the first in-depth insight into the development and functioning of one of the most important but least studied stages in the Toxoplasma life cycle.

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

    PubMed Central

    Lewis, Wesley R.; Malarkey, Erik B.; Tritschler, Douglas; Bower, Raqual; Pasek, Raymond C.; Porath, Jonathan D.; Birket, Susan E.; Saunier, Sophie; Antignac, Corinne; Leigh, Margaret W.; Zariwala, Maimoona A.; Drummond, Iain A.; Parant, John M.; Hildebrandt, Friedhelm; Yoder, Bradley K.

    2016-01-01

    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

  16. Functional malignant cell heterogeneity in pancreatic neuroendocrine tumors revealed by targeting of PDGF-DD

    PubMed Central

    Cortez, Eliane; Gladh, Hanna; Braun, Sebastian; Bocci, Matteo; Cordero, Eugenia; Björkström, Niklas K.; Miyazaki, Hideki; Michael, Iacovos P.; Eriksson, Ulf; Folestad, Erika; Pietras, Kristian

    2016-01-01

    Intratumoral heterogeneity is an inherent feature of most human cancers and has profound implications for cancer therapy. As a result, there is an emergent need to explore previously unmapped mechanisms regulating distinct subpopulations of tumor cells and to understand their contribution to tumor progression and treatment response. Aberrant platelet-derived growth factor receptor beta (PDGFRβ) signaling in cancer has motivated the development of several antagonists currently in clinical use, including imatinib, sunitinib, and sorafenib. The discovery of a novel ligand for PDGFRβ, platelet-derived growth factor (PDGF)-DD, opened the possibility of a previously unidentified signaling pathway involved in tumor development. However, the precise function of PDGF-DD in tumor growth and invasion remains elusive. Here, making use of a newly generated Pdgfd knockout mouse, we reveal a functionally important malignant cell heterogeneity modulated by PDGF-DD signaling in pancreatic neuroendocrine tumors (PanNET). Our analyses demonstrate that tumor growth was delayed in the absence of signaling by PDGF-DD. Surprisingly, ablation of PDGF-DD did not affect the vasculature or stroma of PanNET; instead, we found that PDGF-DD stimulated bulk tumor cell proliferation by induction of paracrine mitogenic signaling between heterogeneous malignant cell clones, some of which expressed PDGFRβ. The presence of a subclonal population of tumor cells characterized by PDGFRβ expression was further validated in a cohort of human PanNET. In conclusion, we demonstrate a previously unrecognized heterogeneity in PanNET characterized by signaling through the PDGF-DD/PDGFRβ axis. PMID:26831065

  17. Structures of mesophilic and extremophilic citrate synthases reveal rigidity and flexibility for function.

    PubMed

    Wells, Stephen A; Crennell, Susan J; Danson, Michael J

    2014-10-01

    Citrate synthase (CS) catalyses the entry of carbon into the citric acid cycle and is highly-conserved structurally across the tree of life. Crystal structures of dimeric CSs are known in both "open" and "closed" forms, which differ by a substantial domain motion that closes the substrate-binding clefts. We explore both the static rigidity and the dynamic flexibility of CS structures from mesophilic and extremophilic organisms from all three evolutionary domains. The computational expense of this wide-ranging exploration is kept to a minimum by the use of rigidity analysis and rapid all-atom simulations of flexible motion, combining geometric simulation and elastic network modeling. CS structures from thermophiles display increased structural rigidity compared with the mesophilic enzyme. A CS structure from a psychrophile, stabilized by strong ionic interactions, appears to display likewise increased rigidity in conventional rigidity analysis; however, a novel modified analysis, taking into account the weakening of the hydrophobic effect at low temperatures, shows a more appropriate decreased rigidity. These rigidity variations do not, however, affect the character of the flexible dynamics, which are well conserved across all the structures studied. Simulation trajectories not only duplicate the crystallographically observed symmetric open-to-closed transitions, but also identify motions describing a previously unidentified antisymmetric functional motion. This antisymmetric motion would not be directly observed in crystallography but is revealed as an intrinsic property of the CS structure by modeling of flexible motion. This suggests that the functional motion closing the binding clefts in CS may be independent rather than symmetric and cooperative.

  18. Metagenomic analysis reveals that modern microbialites and polar microbial mats have similar taxonomic and functional potential

    PubMed Central

    White, Richard Allen; Power, Ian M.; Dipple, Gregory M.; Southam, Gordon; Suttle, Curtis A.

    2015-01-01

    Within the subarctic climate of Clinton Creek, Yukon, Canada, lies an abandoned and flooded open-pit asbestos mine that harbors rapidly growing microbialites. To understand their formation we completed a metagenomic community profile of the microbialites and their surrounding sediments. Assembled metagenomic data revealed that bacteria within the phylum Proteobacteria numerically dominated this system, although the relative abundances of taxa within the phylum varied among environments. Bacteria belonging to Alphaproteobacteria and Gammaproteobacteria were dominant in the microbialites and sediments, respectively. The microbialites were also home to many other groups associated with microbialite formation including filamentous cyanobacteria and dissimilatory sulfate-reducing Deltaproteobacteria, consistent with the idea of a shared global microbialite microbiome. Other members were present that are typically not associated with microbialites including Gemmatimonadetes and iron-oxidizing Betaproteobacteria, which participate in carbon metabolism and iron cycling. Compared to the sediments, the microbialite microbiome has significantly more genes associated with photosynthetic processes (e.g., photosystem II reaction centers, carotenoid, and chlorophyll biosynthesis) and carbon fixation (e.g., CO dehydrogenase). The Clinton Creek microbialite communities had strikingly similar functional potentials to non-lithifying microbial mats from the Canadian High Arctic and Antarctica, but are functionally distinct, from non-lithifying mats or biofilms from Yellowstone. Clinton Creek microbialites also share metabolic genes (R2 < 0.750) with freshwater microbial mats from Cuatro Ciénegas, Mexico, but are more similar to polar Arctic mats (R2 > 0.900). These metagenomic profiles from an anthropogenic microbialite-forming ecosystem provide context to microbialite formation on a human-relevant timescale. PMID:26441900

  19. Opsin switch reveals function of the ultraviolet cone in fish foraging

    PubMed Central

    Novales Flamarique, Iñigo

    2013-01-01

    Although several studies have shown that ultraviolet (UV) wavelengths are important in naturally occurring, visually guided behaviours of vertebrates, the function of the UV cone in such behaviours is unknown. Here, I used thyroid hormone to transform the UV cones of young rainbow trout into blue cones, a phenomenon that occurs naturally as the animal grows, to test whether the resulting loss of UV sensitivity affected the animal's foraging performance on Daphnia magna, a prey zooplankton. The distances and angles at which prey were located (variables that are known indicators of foraging performance) were significantly reduced for UV knock-out fish compared with controls. Optical measurements and photon-catch calculations revealed that the contrast of Daphnia was greater when perceived by the visual system of control versus that of thyroid-hormone-treated fish, demonstrating that the UV cone enhanced the foraging performance of young rainbow trout. Because most juvenile fishes have UV cones and feed on zooplankton, this finding has wide implications for understanding the visual ecology of fishes. The enhanced target contrast provided by UV cones could be used by other vertebrates in various behaviours, including foraging, mate selection and communication. PMID:23222448

  20. Structure–function characterization reveals new catalytic diversity in the galactose oxidase and glyoxal oxidase family

    PubMed Central

    Yin, DeLu (Tyler); Urresti, Saioa; Lafond, Mickael; Johnston, Esther M.; Derikvand, Fatemeh; Ciano, Luisa; Berrin, Jean-Guy; Henrissat, Bernard; Walton, Paul H.; Davies, Gideon J.; Brumer, Harry

    2015-01-01

    Alcohol oxidases, including carbohydrate oxidases, have a long history of research that has generated fundamental biological understanding and biotechnological applications. Despite a long history of study, the galactose 6-oxidase/glyoxal oxidase family of mononuclear copper-radical oxidases, Auxiliary Activity Family 5 (AA5), is currently represented by only very few characterized members. Here we report the recombinant production and detailed structure–function analyses of two homologues from the phytopathogenic fungi Colletotrichum graminicola and C. gloeosporioides, CgrAlcOx and CglAlcOx, respectively, to explore the wider biocatalytic potential in AA5. EPR spectroscopy and crystallographic analysis confirm a common active-site structure vis-à-vis the archetypal galactose 6-oxidase from Fusarium graminearum. Strikingly, however, CgrAlcOx and CglAlcOx are essentially incapable of oxidizing galactose and galactosides, but instead efficiently catalyse the oxidation of diverse aliphatic alcohols. The results highlight the significant potential of prospecting the evolutionary diversity of AA5 to reveal novel enzyme specificities, thereby informing both biology and applications. PMID:26680532

  1. Comprehensive profiling of lysine acetylproteome analysis reveals diverse functions of lysine acetylation in common wheat

    PubMed Central

    Zhang, Yumei; Song, Limin; Liang, Wenxing; Mu, Ping; Wang, Shu; Lin, Qi

    2016-01-01

    Lysine acetylation of proteins, a dynamic and reversible post-translational modification, plays a critical regulatory role in both eukaryotes and prokaryotes. Several researches have been carried out on acetylproteome in plants. However, until now, there have been no data on common wheat, the major cereal crop in the world. In this study, we performed a global acetylproteome analysis of common wheat variety (Triticum aestivum L.), Chinese Spring. In total, 416 lysine modification sites were identified on 277 proteins, which are involved in a wide variety of biological processes. Consistent with previous studies, a large proportion of the acetylated proteins are involved in metabolic process. Interestingly, according to the functional enrichment analysis, 26 acetylated proteins are involved in photosynthesis and Calvin cycle, suggesting an important role of lysine acetylation in these processes. Moreover, protein interaction network analysis reveals that diverse interactions are modulated by protein acetylation. These data represent the first report of acetylome in common wheat and serve as an important resource for exploring the physiological role of lysine acetylation in this organism and likely in all plants. PMID:26875666

  2. The structure of the Helicobacter pylori ferric uptake regulator Fur reveals three functional metal binding sites.

    PubMed

    Dian, Cyril; Vitale, Sylvia; Leonard, Gordon A; Bahlawane, Christelle; Fauquant, Caroline; Leduc, Damien; Muller, Cécile; de Reuse, Hilde; Michaud-Soret, Isabelle; Terradot, Laurent

    2011-03-01

    Fur, the ferric uptake regulator, is a transcription factor that controls iron metabolism in bacteria. Binding of ferrous iron to Fur triggers a conformational change that activates the protein for binding to specific DNA sequences named Fur boxes. In Helicobacter pylori, HpFur is involved in acid response and is important for gastric colonization in model animals. Here we present the crystal structure of a functionally active HpFur mutant (HpFur2M; C78S-C150S) bound to zinc. Although its fold is similar to that of other Fur and Fur-like proteins, the crystal structure of HpFur reveals a unique structured N-terminal extension and an unusual C-terminal helix. The structure also shows three metal binding sites: S1 the structural ZnS₄ site previously characterized biochemically in HpFur and the two zinc sites identified in other Fur proteins. Site-directed mutagenesis and spectroscopy analyses of purified wild-type HpFur and various mutants show that the two metal binding sites common to other Fur proteins can be also metallated by cobalt. DNA protection and circular dichroism experiments demonstrate that, while these two sites influence the affinity of HpFur for DNA, only one is absolutely required for DNA binding and could be responsible for the conformational changes of Fur upon metal binding while the other is a secondary site.

  3. Histone-DNA contacts in structure/function relationships of nucleosomes as revealed by crosslinking

    SciTech Connect

    Usachenko, S.I.; Bradbury, E.M. |

    1998-12-31

    The magnitude of the problem of understanding the structure/function relationships of eukaryotic chromosomes can be appreciated from the fact that the human diploid genome contains more than 2 meters of DNA packaged into 46 chromosomes, each at metaphase being several microns in length. Each chromatid of a chromosome contains a single DNA molecule several centimeters in length. In addition to the DNA, chromosomes contain an equal weight of histones and an equal weight of non-histone chromosomal proteins. These histones are the major chromosomal structural proteins. The non-histone chromosomal proteins are involved in the DNA processes of transcription and replication, in chromosome organization and in nuclear architecture. Polytene chromosomes with their bands and interbands and puffs of active genetic loci provide visual evidence for long range order as do the bands and interbands of mammalian metaphase chromosomes. The gentle removal of histones and all but the most tightly bound 2--3% of non-histone proteins from metaphase chromosomes revealed by electron microscopy a residual protein scaffold constraining a halo of DNA loops extending out from the scaffold.

  4. Functional magnetic resonance imaging reveals brain regions mediating the response to resistive expiratory loads in humans.

    PubMed Central

    Gozal, D; Omidvar, O; Kirlew, K A; Hathout, G M; Lufkin, R B; Harper, R M

    1996-01-01

    Obstructive lung disease is the most common form of respiratory disturbance. However, the location of brain structures underlying the ventilatory response to resistive expiratory loads is unknown in humans. To study this issue, midsagittal magnetic resonance images were acquired in eight healthy volunteers before and after application of a moderate resistive expiratory load (30 cmH2O/liter/s), using functional magnetic resonance imaging (fMRI) strategies (1.5-T magnetic resonance; repetition time: 72 ms; echo time: 45 ms; flip angle: 30 degrees; field of view: 26 cm; slice thickness: 5 mm; 128 x 256 x 1 number of excitations). Digital image subtractions and region of interest analyses revealed significant increases in fMRI signal intensity in discrete areas of the ventral medulla, ventral and dorsal pontomedullary structures, basal forebrain, and cerebellum. Upon load withdrawal, a rapid fMRI signal off-transient occurred in all activated sites. Application of an identical load immediately after recovery from the initial stimulus resulted in smaller signal increases (P < 0.02). Prolongation of load duration was associated with progressive fMRI signal decrease across activated regions. In three additional subjects, the threshold for significant MRI signal increases was established at expiratory loads > or = 15 cmH2O/liter/s and was dose dependent with increasing loads. We conclude that resistive expiratory loads > or = 15 cmH2O/liter/s elicit regional activation of discrete brain locations in humans. PMID:8550849

  5. The Proteomic Investigation of Chromatin Functional Domains Reveals Novel Synergisms among Distinct Heterochromatin Components*

    PubMed Central

    Soldi, Monica; Bonaldi, Tiziana

    2013-01-01

    Chromatin is a highly dynamic, well-structured nucleoprotein complex of DNA and proteins that controls virtually all DNA transactions. Chromatin dynamicity is regulated at specific loci by the presence of various associated proteins, histones, post-translational modifications, histone variants, and DNA methylation. Until now the characterization of the proteomic component of chromatin domains has been held back by the challenge of enriching distinguishable, homogeneous regions for subsequent mass spectrometry analysis. Here we describe a modified protocol for chromatin immunoprecipitation combined with quantitative proteomics based on stable isotope labeling by amino acids in cell culture to identify known and novel histone modifications, variants, and complexes that specifically associate with silent and active chromatin domains. Our chromatin proteomics strategy revealed unique functional interactions among various chromatin modifiers, suggesting new regulatory pathways, such as a heterochromatin-specific modulation of DNA damage response involving H2A.X and WICH, both enriched in silent domains. Chromatin proteomics expands the arsenal of tools for deciphering how all the distinct protein components act together to enforce a given region-specific chromatin status. PMID:23319141

  6. Comparative Functional Genomic Analysis of Two Vibrio Phages Reveals Complex Metabolic Interactions with the Host Cell

    PubMed Central

    Skliros, Dimitrios; Kalatzis, Panos G.; Katharios, Pantelis; Flemetakis, Emmanouil

    2016-01-01

    Sequencing and annotation was performed for two large double stranded DNA bacteriophages, φGrn1 and φSt2 of the Myoviridae family, considered to be of great interest for phage therapy against Vibrios in aquaculture live feeds. In addition, phage–host metabolic interactions and exploitation was studied by transcript profiling of selected viral and host genes. Comparative genomic analysis with other large Vibrio phages was also performed to establish the presence and location of homing endonucleases highlighting distinct features for both phages. Phylogenetic analysis revealed that they belong to the “schizoT4like” clade. Although many reports of newly sequenced viruses have provided a large set of information, basic research related to the shift of the bacterial metabolism during infection remains stagnant. The function of many viral protein products in the process of infection is still unknown. Genome annotation identified the presence of several viral open reading frames (ORFs) participating in metabolism, including a Sir2/cobB (sirtuin) protein and a number of genes involved in auxiliary NAD+ and nucleotide biosynthesis, necessary for phage DNA replication. Key genes were subsequently selected for detail study of their expression levels during infection. This work suggests a complex metabolic interaction and exploitation of the host metabolic pathways and biochemical processes, including a possible post-translational protein modification, by the virus during infection. PMID:27895630

  7. Cryo-EM reveals a novel octameric integrase structure for β-retroviral intasome function

    PubMed Central

    Ballandras-Colas, Allison; Brown, Monica; Cook, Nicola J.; Dewdney, Tamaria G.; Demeler, Borries; Cherepanov, Peter; Lyumkis, Dmitry; Engelman, Alan N.

    2016-01-01

    Retroviral integrase (IN) catalyzes the integration of viral DNA (vDNA) into host target (tDNA), which is an essential step in the lifecycle of all retroviruses1. Prior structural characterization of IN-vDNA complexes, or intasomes, from the spumavirus prototype foamy virus (PFV) revealed a functional IN tetramer2–5, and it is generally believed that intasomes derived from other retroviral genera will employ tetrameric IN6–9. However, the intasomes of orthoretroviruses, which include all known pathogenic species, have not been characterized structurally. Using single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography, we determine here an unexpected octameric IN architecture for the β-retrovirus mouse mammary tumor virus (MMTV) intasome. The structure is composed of two core IN dimers, which interact with the vDNA ends and structurally mimic the PFV IN tetramer, and two flanking IN dimers that engage the core structure via their IN C-terminal domains (CTDs). Contrary to the belief that tetrameric IN components are sufficient to catalyze integration, the flanking IN dimers were necessary for MMTV IN activity. The IN octamer solves a conundrum for the β- as well as α-retroviruses by providing critical CTDs to the intasome core that cannot be provided in cis due to evolutionarily restrictive catalytic core domain (CCD)-CTD linker regions. The octameric architecture of the MMTV intasome provides a new paradigm for the structural basis of retroviral DNA integration. PMID:26887496

  8. Structure of the Spt16 Middle Domain Reveals Functional Features of the Histone Chaperone FACT*

    PubMed Central

    Kemble, David J.; Whitby, Frank G.; Robinson, Howard; McCullough, Laura L.; Formosa, Tim; Hill, Christopher P.

    2013-01-01

    The histone chaperone FACT is an essential and abundant heterodimer found in all eukaryotes. Here we report a crystal structure of the middle domain of the large subunit of FACT (Spt16-M) to reveal a double pleckstrin homology architecture. This structure was found previously in the Pob3-M domain of the small subunit of FACT and in the related histone chaperone Rtt106, although Spt16-M is distinguished from these structures by the presence of an extended α-helix and a C-terminal addition. Consistent with our finding that the double pleckstrin homology structure is common to these three histone chaperones and reports that Pob3 and Rtt106 double pleckstrin homology domains bind histones H3-H4, we also find that Spt16-M binds H3-H4 with low micromolar affinity. Our structure provides a framework for interpreting a large body of genetic data regarding the physiological functions of FACT, including the identification of potential interaction surfaces for binding histones or other proteins. PMID:23417676

  9. Comparative Functional Genomic Analysis of Two Vibrio Phages Reveals Complex Metabolic Interactions with the Host Cell.

    PubMed

    Skliros, Dimitrios; Kalatzis, Panos G; Katharios, Pantelis; Flemetakis, Emmanouil

    2016-01-01

    Sequencing and annotation was performed for two large double stranded DNA bacteriophages, φGrn1 and φSt2 of the Myoviridae family, considered to be of great interest for phage therapy against Vibrios in aquaculture live feeds. In addition, phage-host metabolic interactions and exploitation was studied by transcript profiling of selected viral and host genes. Comparative genomic analysis with other large Vibrio phages was also performed to establish the presence and location of homing endonucleases highlighting distinct features for both phages. Phylogenetic analysis revealed that they belong to the "schizoT4like" clade. Although many reports of newly sequenced viruses have provided a large set of information, basic research related to the shift of the bacterial metabolism during infection remains stagnant. The function of many viral protein products in the process of infection is still unknown. Genome annotation identified the presence of several viral open reading frames (ORFs) participating in metabolism, including a Sir2/cobB (sirtuin) protein and a number of genes involved in auxiliary NAD(+) and nucleotide biosynthesis, necessary for phage DNA replication. Key genes were subsequently selected for detail study of their expression levels during infection. This work suggests a complex metabolic interaction and exploitation of the host metabolic pathways and biochemical processes, including a possible post-translational protein modification, by the virus during infection.

  10. Functional Heterogeneity of Embryonic Stem Cells Revealed through Translational Amplification of an Early Endodermal Transcript

    PubMed Central

    Canham, Maurice A.; Sharov, Alexei A.; Ko, Minoru S. H.; Brickman, Joshua M.

    2010-01-01

    ES cells are defined as self-renewing, pluripotent cell lines derived from early embryos. Cultures of ES cells are also characterized by the expression of certain markers thought to represent the pluripotent state. However, despite the widespread expression of key markers such as Oct4 and the appearance of a characteristic undifferentiated morphology, functional ES cells may represent only a small fraction of the cultures grown under self-renewing conditions. Thus phenotypically “undifferentiated” cells may consist of a heterogeneous population of functionally distinct cell types. Here we use a transgenic allele designed to detect low level transcription in the primitive endoderm lineage as a tool to identify an immediate early endoderm-like ES cell state. This reporter employs a tandem array of internal ribosomal entry sites to drive translation of an enhanced Yellow Fluorescent Protein (Venus) from the transcript that normally encodes for the early endodermal marker Hex. Expression of this Venus transgene reports on single cells with low Hex transcript levels and reveals the existence of distinct populations of Oct4 positive undifferentiated ES cells. One of these cells types, characterized by both the expression of the Venus transgene and the ES cells marker SSEA-1 (V+S+), appears to represent an early step in primitive endoderm specification. We show that the fraction of cells present within this state is influenced by factors that both promote and suppress primitive endoderm differentiation, but conditions that support ES cell self-renewal prevent their progression into differentiation and support an equilibrium between this state and at least one other that resembles the Nanog positive inner cell mass of the mammalian blastocysts. Interestingly, while these subpopulations are equivalently and clonally interconvertible under self-renewing conditions, when induced to differentiate both in vivo and in vitro they exhibit different behaviours. Most strikingly

  11. Egg colour mimicry in the common cuckoo Cuculus canorus as revealed by modelling host retinal function

    PubMed Central

    Avilés, Jesús M

    2008-01-01

    Some parasite cuckoo species lay eggs that, to the human eye, appear to mimic the appearance of the eggs of their favourite hosts, which hinders discrimination and removal of their eggs by host species. Hitherto, perception of cuckoo–host egg mimicry has been estimated based on human vision or spectrophotometry, which does not account for what the receivers' eye (i.e. hosts) actually discriminates. Using a discrimination model approach that reproduces host retinal functioning, and museum egg collections collected in the south of Finland, where at least six different races of the European cuckoo (Cuculus canorus) coexist, I first assess whether the colour design of cuckoo eggs of different races maximizes matching for two favourite avian hosts, viz. the redstart (Phoenicurus phoenicurus) and the pied wagtail (Motacilla alba). Second, I assess the role of nest luminosity on host perception of mimicry by the same two hosts. Phoenicurus-cuckoo eggs showed a better chromatic matching with the redstart-host eggs than other cuckoo races, and in most cases can not be discriminated. Sylvia-cuckoo eggs, however, showed better achromatic matching with redstart-host eggs than Phoenicurus-cuckoo eggs. Also, Motacilla-cuckoo eggs showed poorer chromatic and achromatic matching with pied wagtail-host eggs than Sylvia-cuckoo eggs. Nest luminosity affected chromatic and achromatic differences between cuckoo and host eggs, although only minimally affected the proportion of cuckoo eggs discriminated by chromatic signals. These results reveal that cuckoo races as assessed by humans do not entirely match with host perception of matching and that achromatic mechanisms could play a main role in the discrimination of cuckoo eggs at low-light levels. PMID:18595836

  12. Mouse model of CADASIL reveals novel insights into Notch3 function in adult hippocampal neurogenesis.

    PubMed

    Ehret, Fanny; Vogler, Steffen; Pojar, Sherin; Elliott, David A; Bradke, Frank; Steiner, Barbara; Kempermann, Gerd

    2015-03-01

    Could impaired adult hippocampal neurogenesis be a relevant mechanism underlying CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy)? Memory symptoms in CADASIL, the most common hereditary form of vascular dementia, are usually thought to be primarily due to vascular degeneration and white matter lacunes. Since adult hippocampal neurogenesis, a process essential for the integration of new spatial memory occurs in a highly vascularized niche, we considered dysregulation of adult neurogenesis as a potential mechanism for the manifestation of dementia in CADASIL. Analysis in aged mice overexpressing Notch3 with a CADASIL mutation, revealed vascular deficits in arteries of the hippocampal fissure but not in the niche of the dentate gyrus. At 12 months of age, cell proliferation and survival of newborn neurons were reduced not only in CADASIL mice but also in transgenic controls overexpressing wild type Notch3. At 6 months, hippocampal neurogenesis was altered in CADASIL mice independent of overt vascular abnormalities in the fissure. Further, we identified Notch3 expression in hippocampal precursor cells and maturing neurons in vivo as well as in cultured hippocampal precursor cells. Overexpression and knockdown experiments showed that Notch3 signaling negatively regulated precursor cell proliferation. Notch3 overexpression also led to deficits in KCl-induced precursor cell activation. This suggests a cell-autonomous effect of Notch3 signaling in the regulation of precursor proliferation and activation and a loss-of-function effect in CADASIL. Consequently, besides vascular damage, aberrant precursor cell proliferation and differentiation due to Notch3 dysfunction might be an additional independent mechanism for the development of hippocampal dysfunction in CADASIL.

  13. Egg colour mimicry in the common cuckoo Cuculus canorus as revealed by modelling host retinal function.

    PubMed

    Avilés, Jesús M

    2008-10-22

    Some parasite cuckoo species lay eggs that, to the human eye, appear to mimic the appearance of the eggs of their favourite hosts, which hinders discrimination and removal of their eggs by host species. Hitherto, perception of cuckoo-host egg mimicry has been estimated based on human vision or spectrophotometry, which does not account for what the receivers' eye (i.e. hosts) actually discriminates. Using a discrimination model approach that reproduces host retinal functioning, and museum egg collections collected in the south of Finland, where at least six different races of the European cuckoo (Cuculus canorus) coexist, I first assess whether the colour design of cuckoo eggs of different races maximizes matching for two favourite avian hosts, viz. the redstart (Phoenicurus phoenicurus) and the pied wagtail (Motacilla alba). Second, I assess the role of nest luminosity on host perception of mimicry by the same two hosts. Phoenicurus-cuckoo eggs showed a better chromatic matching with the redstart-host eggs than other cuckoo races, and in most cases can not be discriminated. Sylvia-cuckoo eggs, however, showed better achromatic matching with redstart-host eggs than Phoenicurus-cuckoo eggs. Also, Motacilla-cuckoo eggs showed poorer chromatic and achromatic matching with pied wagtail-host eggs than Sylvia-cuckoo eggs. Nest luminosity affected chromatic and achromatic differences between cuckoo and host eggs, although only minimally affected the proportion of cuckoo eggs discriminated by chromatic signals. These results reveal that cuckoo races as assessed by humans do not entirely match with host perception of matching and that achromatic mechanisms could play a main role in the discrimination of cuckoo eggs at low-light levels.

  14. Deep small RNA sequencing from the nematode Ascaris reveals conservation, functional diversification, and novel developmental profiles.

    PubMed

    Wang, Jianbin; Czech, Benjamin; Crunk, Amanda; Wallace, Adam; Mitreva, Makedonka; Hannon, Gregory J; Davis, Richard E

    2011-09-01

    Eukaryotic cells express several classes of small RNAs that regulate gene expression and ensure genome maintenance. Endogenous siRNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs) mainly control gene and transposon expression in the germline, while microRNAs (miRNAs) generally function in post-transcriptional gene silencing in both somatic and germline cells. To provide an evolutionary and developmental perspective on small RNA pathways in nematodes, we identified and characterized known and novel small RNA classes through gametogenesis and embryo development in the parasitic nematode Ascaris suum and compared them with known small RNAs of Caenorhabditis elegans. piRNAs, Piwi-clade Argonautes, and other proteins associated with the piRNA pathway have been lost in Ascaris. miRNAs are synthesized immediately after fertilization in utero, before pronuclear fusion, and before the first cleavage of the zygote. This is the earliest expression of small RNAs ever described at a developmental stage long thought to be transcriptionally quiescent. A comparison of the two classes of Ascaris endo-siRNAs, 22G-RNAs and 26G-RNAs, to those in C. elegans, suggests great diversification and plasticity in the use of small RNA pathways during spermatogenesis in different nematodes. Our data reveal conserved characteristics of nematode small RNAs as well as features unique to Ascaris that illustrate significant flexibility in the use of small RNAs pathways, some of which are likely an adaptation to Ascaris' life cycle and parasitism. The transcriptome assembly has been submitted to NCBI Transcriptome Shotgun Assembly Sequence Database(http://www.ncbi.nlm.nih.gov/genbank/TSA.html) under accession numbers JI163767–JI182837 and JI210738–JI257410.

  15. Ribosomal protein uS19 mutants reveal its role in coordinating ribosome structure and function

    PubMed Central

    Bowen, Alicia M; Musalgaonkar, Sharmishtha; Moomau, Christine A; Gulay, Suna P; Mirvis, Mary; Dinman, Jonathan D

    2015-01-01

    Prior studies identified allosteric information pathways connecting functional centers in the large ribosomal subunit to the decoding center in the small subunit through the B1a and B1b/c intersubunit bridges in yeast. In prokaryotes a single SSU protein, uS13, partners with H38 (the A-site finger) and uL5 to form the B1a and B1b/c bridges respectively. In eukaryotes, the SSU component was split into 2 separate proteins during the course of evolution. One, also known as uS13, participates in B1b/c bridge with uL5 in eukaryotes. The other, called uS19 is the SSU partner in the B1a bridge with H38. Here, polyalanine mutants of uS19 involved in the uS19/uS13 and the uS19/H38 interfaces were used to elucidate the important amino acid residues involved in these intersubunit communication pathways. Two key clusters of amino acids were identified: one located at the junction between uS19 and uS13, and a second that appears to interact with the distal tip of H38. Biochemical analyses reveal that these mutations shift the ribosomal rotational equilibrium toward the unrotated state, increasing ribosomal affinity for tRNAs in the P-site and for ternary complex in the A-site, and inhibit binding of the translocase, eEF2. These defects in turn affect specific aspects of translational fidelity. These findings suggest that uS19 plays a critical role as a conduit of information exchange between the large and small ribosomal subunits directly through the B1a, and indirectly through the B1b/c bridges. PMID:26824029

  16. Functional Coding Variation in Recombinant Inbred Mouse Lines Reveals Novel Serotonin Transporter-Associated Phenotypes

    SciTech Connect

    Carneiro, Ana; Airey, David; Thompson, Brent; Zhu, C; Rinchik, Eugene M; Lu, Lu; Chesler, Elissa J; Erikson, Keith; Blakely, Randy

    2009-01-01

    The human serotonin (5-hydroxytryptamine, 5-HT) transporter (hSERT, SLC6A4) figures prominently in the etiology or treatment of many prevalent neurobehavioral disorders including anxiety, alcoholism, depression, autism and obsessive-compulsive disorder (OCD). Here we utilize naturally occurring polymorphisms in recombinant inbred (RI) lines to identify novel phenotypes associated with altered SERT function. The widely used mouse strain C57BL/6J, harbors a SERT haplotype defined by two nonsynonymous coding variants (Gly39 and Lys152 (GK)). At these positions, many other mouse lines, including DBA/2J, encode Glu39 and Arg152 (ER haplotype), assignments found also in hSERT. Synaptosomal 5-HT transport studies revealed reduced uptake associated with the GK variant. Heterologous expression studies confirmed a reduced SERT turnover rate for the GK variant. Experimental and in silico approaches using RI lines (C57Bl/6J X DBA/2J=BXD) identifies multiple anatomical, biochemical and behavioral phenotypes specifically impacted by GK/ER variation. Among our findings are multiple traits associated with anxiety and alcohol consumption, as well as of the control of dopamine (DA) signaling. Further bioinformatic analysis of BXD phenotypes, combined with biochemical evaluation of SERT knockout mice, nominates SERT-dependent 5-HT signaling as a major determinant of midbrain iron homeostasis that, in turn, dictates ironregulated DA phenotypes. Our studies provide a novel example of the power of coordinated in vitro, in vivo and in silico approaches using murine RI lines to elucidate and quantify the system-level impact of gene variation.

  17. Comparative and functional triatomine genomics reveals reductions and expansions in insecticide resistance-related gene families

    PubMed Central

    Traverso, Lucila; Lavore, Andrés; Sierra, Ivana; Palacio, Victorio; Martinez-Barnetche, Jesús; Latorre-Estivalis, José Manuel; Mougabure-Cueto, Gaston; Francini, Flavio; Lorenzo, Marcelo G.; Rodríguez, Mario Henry; Ons, Sheila; Rivera-Pomar, Rolando V.

    2017-01-01

    Background Triatomine insects are vectors of Trypanosoma cruzi, a protozoan parasite that is the causative agent of Chagas’ disease. This is a neglected disease affecting approximately 8 million people in Latin America. The existence of diverse pyrethroid resistant populations of at least two species demonstrates the potential of triatomines to develop high levels of insecticide resistance. Therefore, the incorporation of strategies for resistance management is a main concern for vector control programs. Three enzymatic superfamilies are thought to mediate xenobiotic detoxification and resistance: Glutathione Transferases (GSTs), Cytochromes P450 (CYPs) and Carboxyl/Cholinesterases (CCEs). Improving our knowledge of key triatomine detoxification enzymes will strengthen our understanding of insecticide resistance processes in vectors of Chagas’ disease. Methods and findings The discovery and description of detoxification gene superfamilies in normalized transcriptomes of three triatomine species: Triatoma dimidiata, Triatoma infestans and Triatoma pallidipennis is presented. Furthermore, a comparative analysis of these superfamilies among the triatomine transcriptomes and the genome of Rhodnius prolixus, also a triatomine vector of Chagas’ disease, and other well-studied insect genomes was performed. The expression pattern of detoxification genes in R. prolixus transcriptomes from key organs was analyzed. The comparisons reveal gene expansions in Sigma class GSTs, CYP3 in CYP superfamily and clade E in CCE superfamily. Moreover, several CYP families identified in these triatomines have not yet been described in other insects. Conversely, several groups of insecticide resistance related enzymes within each enzyme superfamily are reduced or lacking in triatomines. Furthermore, our qRT-PCR results showed an increase in the expression of a CYP4 gene in a T. infestans population resistant to pyrethroids. These results could point to an involvement of metabolic

  18. Inherited cobalamin malabsorption. Mutations in three genes reveal functional and ethnic patterns

    PubMed Central

    2012-01-01

    Background Inherited malabsorption of cobalamin (Cbl) causes hematological and neurological abnormalities that can be fatal. Three genes have been implicated in Cbl malabsorption; yet, only about 10% of ~400-500 reported cases have been molecularly studied to date. Recessive mutations in CUBN or AMN cause Imerslund-Gräsbeck Syndrome (IGS), while recessive mutations in GIF cause Intrinsic Factor Deficiency (IFD). IGS and IFD differ in that IGS usually presents with proteinuria, which is not observed in IFD. The genetic heterogeneity and numerous differential diagnoses make clinical assessment difficult. Methods We present a large genetic screening study of 154 families or patients with suspected hereditary Cbl malabsorption. Patients and their families have been accrued over a period spanning >12 years. Systematic genetic testing of the three genes CUBN, AMN, and GIF was accomplished using a combination of single strand conformation polymorphism and DNA and RNA sequencing. In addition, six genes that were contenders for a role in inherited Cbl malabsorption were studied in a subset of these patients. Results Our results revealed population-specific mutations, mutational hotspots, and functionally distinct regions in the three causal genes. We identified mutations in 126/154 unrelated cases (82%). Fifty-three of 126 cases (42%) were mutated in CUBN, 45/126 (36%) were mutated in AMN, and 28/126 (22%) had mutations in GIF. We found 26 undescribed mutations in CUBN, 19 in AMN, and 7 in GIF for a total of 52 novel defects described herein. We excluded six other candidate genes as culprits and concluded that additional genes might be involved. Conclusions Cbl malabsorption is found worldwide and genetically complex. However, our results indicate that population-specific founder mutations are quite common. Consequently, targeted genetic testing has become feasible if ethnic ancestry is considered. These results will facilitate clinical and molecular genetic testing of

  19. Cornelia de Lange syndrome: further delineation of phenotype, cohesin biology and educational focus, 5th Biennial Scientific and Educational Symposium abstracts.

    PubMed

    Kline, Antonie D; Calof, Anne L; Schaaf, Cheri A; Krantz, Ian D; Jyonouchi, Soma; Yokomori, Kyoko; Gauze, Maria; Carrico, Cheri S; Woodman, Julie; Gerton, Jennifer L; Vega, Hugo; Levin, Alex V; Shirahige, Katsuhiko; Champion, Michele; Goodban, Marjorie T; O'Connor, Julia T; Pipan, Mary; Horsfield, Julia; Deardorff, Matthew A; Ishman, Stacey L; Dorsett, Dale

    2014-06-01

    Cornelia de Lange syndrome (CdLS) is the prototype for the cohesinopathy disorders that have mutations in genes associated with the cohesin subunit in all cells. Roberts syndrome is the next most common cohesinopathy. In addition to the developmental implications of cohesin biology, there is much translational and basic research, with progress towards potential treatment for these conditions. Clinically, there are many issues in CdLS faced by the individual, parents and caretakers, professionals, and schools. The following abstracts are presentations from the 5th Cornelia de Lange Syndrome Scientific and Educational Symposium on June 20-21, 2012, in conjunction with the Cornelia de Lange Syndrome Foundation National Meeting, Lincolnshire, IL. The research committee of the CdLS Foundation organizes the meeting, reviews and accepts abstracts and subsequently disseminates the information to the families. In addition to the basic science and clinical discussions, there were educationally-focused talks related to practical aspects of management at home and in school. AMA CME credits were provided by Greater Baltimore Medical Center, Baltimore, MD.

  20. Clinical, developmental and molecular update on Cornelia de Lange syndrome and the cohesin complex: abstracts from the 2014 Scientific and Educational Symposium.

    PubMed

    Kline, Antonie D; Calof, Anne L; Lander, Arthur D; Gerton, Jennifer L; Krantz, Ian D; Dorsett, Dale; Deardorff, Matthew A; Blagowidow, Natalie; Yokomori, Kyoko; Shirahige, Katsuhiko; Santos, Rosaysela; Woodman, Julie; Megee, Paul C; O'Connor, Julia T; Egense, Alena; Noon, Sarah; Belote, Maurice; Goodban, Marjorie T; Hansen, Blake D; Timmons, Jenni Glad; Musio, Antonio; Ishman, Stacey L; Bryan, Yvon; Wu, Yaning; Bettini, Laura R; Mehta, Devanshi; Zakari, Musinu; Mills, Jason A; Srivastava, Siddharth; Haaland, Richard E

    2015-06-01

    Cornelia de Lange Syndrome (CdLS) is the most common example of disorders of the cohesin complex, or cohesinopathies. There are a myriad of clinical issues facing individuals with CdLS, particularly in the neurodevelopmental system, which also have implications for the parents and caretakers, involved professionals, therapists, and schools. Basic research in developmental and cell biology on cohesin is showing significant progress, with improved understanding of the mechanisms and the possibility of potential therapeutics. The following abstracts are presentations from the 6th Cornelia de Lange Syndrome Scientific and Educational Symposium, which took place on June 25-26, 2014, in conjunction with the Cornelia de Lange Syndrome Foundation National Meeting in Costa Mesa, CA. The Research Committee of the CdLS Foundation organizes the meeting, reviews and accepts abstracts, and subsequently disseminates the information to the families through members of the Clinical Advisory Board. In addition to the scientific and clinical discussions, there were educationally focused talks related to practical aspects of behavior and development. AMA CME credits were provided by Greater Baltimore Medical Center, Baltimore, MD.

  1. Purification and Crystallization of a Multimodular Heterotrimeric Complex Containing Both Type I and Type II Cohesin-dockerin Interactions from the Cellulosome of Clostridium thermocellum

    SciTech Connect

    M Currie; J Adams; S Ali; S Smith; Z Jia

    2011-12-31

    The multimodular scaffoldin subunit CipA is the central component of the cellulosome, a multienzyme plant cell-wall-degrading complex, from Clostridium thermocellum. It captures secreted cellulases and hemicellulases and anchors the entire complex to the cell surface via high-affinity calcium-dependent interactions between cohesin and dockerin modules termed type I and type II interactions. The crystallization of a heterotrimeric complex comprising the type II cohesin module from the cell-surface protein SdbA, a trimodular C-terminal fragment of the scaffoldin CipA and the type I dockerin module from the CelD cellulase is reported. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 119.37, b = 186.31, c = 191.17 {angstrom}. The crystals diffracted to 2.7 {angstrom} resolution with four or eight molecules of the ternary protein complex in the asymmetric unit.

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

    USGS Publications Warehouse

    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

  3. Epigenomics reveals a functional genome anatomy and a new approach to common disease.

    PubMed

    Feinberg, Andrew P

    2010-10-01

    Epigenomics provides the context for understanding the function of genome sequence, analogous to the functional anatomy of the human body provided by Vesalius a half-millennium ago. Much of the seemingly inconclusive genetic data related to common diseases could therefore become meaningful in an epigenomic context.

  4. Evolutionary analysis reveals regulatory and functional landscape of coding and non-coding RNA editing.

    PubMed

    Zhang, Rui; Deng, Patricia; Jacobson, Dionna; Li, Jin Billy

    2017-02-01

    Adenosine-to-inosine RNA editing diversifies the transcriptome and promotes functional diversity, particularly in the brain. A plethora of editing sites has been recently identified; however, how they are selected and regulated and which are functionally important are largely unknown. Here we show the cis-regulation and stepwise selection of RNA editing during Drosophila evolution and pinpoint a large number of functional editing sites. We found that the establishment of editing and variation in editing levels across Drosophila species are largely explained and predicted by cis-regulatory elements. Furthermore, editing events that arose early in the species tree tend to be more highly edited in clusters and enriched in slowly-evolved neuronal genes, thus suggesting that the main role of RNA editing is for fine-tuning neurological functions. While nonsynonymous editing events have been long recognized as playing a functional role, in addition to nonsynonymous editing sites, a large fraction of 3'UTR editing sites is evolutionarily constrained, highly edited, and thus likely functional. We find that these 3'UTR editing events can alter mRNA stability and affect miRNA binding and thus highlight the functional roles of noncoding RNA editing. Our work, through evolutionary analyses of RNA editing in Drosophila, uncovers novel insights of RNA editing regulation as well as its functions in both coding and non-coding regions.

  5. Evolutionary analysis reveals regulatory and functional landscape of coding and non-coding RNA editing

    PubMed Central

    Jacobson, Dionna

    2017-01-01

    Adenosine-to-inosine RNA editing diversifies the transcriptome and promotes functional diversity, particularly in the brain. A plethora of editing sites has been recently identified; however, how they are selected and regulated and which are functionally important are largely unknown. Here we show the cis-regulation and stepwise selection of RNA editing during Drosophila evolution and pinpoint a large number of functional editing sites. We found that the establishment of editing and variation in editing levels across Drosophila species are largely explained and predicted by cis-regulatory elements. Furthermore, editing events that arose early in the species tree tend to be more highly edited in clusters and enriched in slowly-evolved neuronal genes, thus suggesting that the main role of RNA editing is for fine-tuning neurological functions. While nonsynonymous editing events have been long recognized as playing a functional role, in addition to nonsynonymous editing sites, a large fraction of 3’UTR editing sites is evolutionarily constrained, highly edited, and thus likely functional. We find that these 3’UTR editing events can alter mRNA stability and affect miRNA binding and thus highlight the functional roles of noncoding RNA editing. Our work, through evolutionary analyses of RNA editing in Drosophila, uncovers novel insights of RNA editing regulation as well as its functions in both coding and non-coding regions. PMID:28166241

  6. Functional brain imaging in 14 patients with dissociative amnesia reveals right inferolateral prefrontal hypometabolism.

    PubMed

    Brand, Matthias; Eggers, Carsten; Reinhold, Nadine; Fujiwara, Esther; Kessler, Josef; Heiss, Wolf-Dieter; Markowitsch, Hans J

    2009-10-30

    Dissociative amnesia is a condition usually characterized by severely impaired retrograde memory functioning in the absence of structural brain damage. Recent case studies nevertheless found functional brain changes in patients suffering from autobiographical-episodic memory loss in the cause of dissociative amnesia. Functional changes were demonstrated in both resting state and memory retrieval conditions. In addition, some but not all cases also showed other neuropsychological impairments beyond retrograde memory deficits. However, there is no group study available that examined potential functional brain abnormalities and accompanying neuropsychological deteriorations in larger samples of patients with dissociative retrograde amnesia. We report functional imaging and neuropsychological data acquired in 14 patients with dissociative amnesia following stressful or traumatic events. All patients suffered from autobiographical memory loss. In addition, approximately half of the patients had deficits in anterograde memory and executive functioning. Accompanying functional brain changes were measured by [18F]fluorodeoxyglucose positron emission tomography (FDG-PET). Regional glucose utilization of the patients was compared with that of 19 healthy subjects, matched for age and gender. We found significantly decreased glucose utilization in the right inferolateral prefrontal cortex in the patients. Hypometabolism in this brain region, known to be involved in retrieval of autobiographical memories and self-referential processing, may be a functional brain correlate of dissociative amnesia.

  7. Structural and Functional Dissection of the Abp1 ADFH Actin-binding Domain Reveals Versatile In Vivo Adapter Functions

    SciTech Connect

    Quintero-Monzon,O.; Rodal, A.; Strokopytov, B.; Almo, S.; Goode, B.

    2005-01-01

    Abp1 is a multidomain protein that regulates the Arp2/3 complex and links proteins involved in endocytosis to the actin cytoskeleton. All of the proposed cellular functions of Abp1 involve actin filament binding, yet the actin binding site(s) on Abp1 have not been identified, nor has the importance of actin binding for Abp1 localization and function in vivo been tested. Here, we report the crystal structure of the Saccharomyces cerevisiae Abp1 actin-binding actin depolymerizing factor homology (ADFH) domain and dissect its activities by mutagenesis. Abp1-ADFH domain and ADF/cofilin structures are similar, and they use conserved surfaces to bind actin; however, there are also key differences that help explain their differential effects on actin dynamics. Using point mutations, we demonstrate that actin binding is required for localization of Abp1 in vivo, the lethality caused by Abp1 overexpression, and the ability of Abp1 to activate Arp2/3 complex. Furthermore, we genetically uncouple ABP1 functions that overlap with SAC6, SLA1, and SLA2, showing they require distinct combinations of activities and interactions. Together, our data provide the first structural and functional view of the Abp1-actin interaction and show that Abp1 has distinct cellular roles as an adapter, linking different sets of ligands for each function.

  8. Structure Function Studies of Vaccinia Virus Host Range Protein K1 Reveal a Novel Functional Surface for Ankyrin Repeat Proteins

    SciTech Connect

    Li, Yongchao; Meng, Xiangzhi; Xiang, Yan; Deng, Junpeng

    2010-06-15

    Poxvirus host tropism at the cellular level is regulated by virus-encoded host range proteins acting downstream of virus entry. The functioning mechanisms of most host range proteins are unclear, but many contain multiple ankyrin (ANK) repeats, a motif that is known for ligand interaction through a concave surface. We report here the crystal structure of one of the ANK repeat-containing host range proteins, the vaccinia virus K1 protein. The structure, at a resolution of 2.3 {angstrom}, showed that K1 consists entirely of ANK repeats, including seven complete ones and two incomplete ones, one each at the N and C terminus. Interestingly, Phe82 and Ser83, which were previously shown to be critical for K1's function, are solvent exposed and located on a convex surface, opposite the consensus ANK interaction surface. The importance of this convex surface was further supported by our additional mutagenesis studies. We found that K1's host range function was negatively affected by substitution of either Asn51 or Cys47 and completely abolished by substitution of both residues. Cys47 and Asn51 are also exposed on the convex surface, spatially adjacent to Phe82 and Ser83. Altogether, our data showed that K1 residues on a continuous convex ANK repeat surface are critical for the host range function, suggesting that K1 functions through ligand interaction and does so with a novel ANK interaction surface.

  9. Aberrant regional neural fluctuations and functional connectivity in generalized anxiety disorder revealed by resting-state functional magnetic resonance imaging.

    PubMed

    Wang, Wei; Hou, Jingming; Qian, Shaowen; Liu, Kai; Li, Bo; Li, Min; Peng, Zhaohui; Xin, Kuolin; Sun, Gang

    2016-06-15

    The purpose of this study was to investigate the neural activity and functional connectivity in generalized anxiety disorder (GAD) during resting state, and how these alterations correlate to patients' symptoms. Twenty-eight GAD patients and 28 matched healthy controls underwent resting-state functional magnetic resonance (fMRI) scans. Amplitude of low-frequency fluctuation (ALFF) and seed-based resting-state functional connectivity (RSFC) were computed to explore regional activity and functional integration, and were compared between the two groups using the voxel-based two-sample t test. Pearson's correlation analyses were performed to examine the neural relationships with demographics and clinical symptoms scores. Compared to controls, GAD patients showed functional abnormalities: higher ALFF in the bilateral dorsomedial prefrontal cortex, bilateral dorsolateral prefrontal cortex and left precuneus/posterior cingulate cortex; lower connectivity in prefrontal gyrus; lower in prefrontal-limbic and cingulate RSFC and higher prefrontal-hippocampus RSFC were correlated with clinical symptoms severity, but these associations were unable to withstand correction for multiple testing. These findings may help facilitate further understanding of the potential neural substrate of GAD.

  10. An atlas of the thioredoxin fold class reveals the complexity of function-enabling adaptations.

    PubMed

    Atkinson, Holly J; Babbitt, Patricia C

    2009-10-01

    The group of proteins that contain a thioredoxin (Trx) fold is huge and diverse. Assessment of the variation in catalytic machinery of Trx fold proteins is essential in providing a foundation for understanding their functional diversity and predicting the function of the many uncharacterized members of the class. The proteins of the Trx fold class retain common features-including variations on a dithiol CxxC active site motif-that lead to delivery of function. We use protein similarity networks to guide an analysis of how structural and sequence motifs track with catalytic function and taxonomic categories for 4,082 representative sequences spanning the known superfamilies of the Trx fold. Domain structure in the fold class is varied and modular, with 2.8% of sequences containing more than one Trx fold domain. Most member proteins are bacterial. The fold class exhibits many modifications to the CxxC active site motif-only 56.8% of proteins have both cysteines, and no functional groupings have absolute conservation of the expected catalytic motif. Only a small fraction of Trx fold sequences have been functionally characterized. This work provides a global view of the complex distribution of domains and catalytic machinery throughout the fold class, showing that each superfamily contains remnants of the CxxC active site. The unifying context provided by this work can guide the comparison of members of different Trx fold superfamilies to gain insight about their structure-function relationships, illustrated here with the thioredoxins and peroxiredoxins.

  11. Resting-state functional connectivity in the human brain revealed with diffuse optical tomography

    PubMed Central

    White, Brian R.; Snyder, Abraham Z.; Cohen, Alexander L.; Petersen, Steven E.; Raich-le, Marcus E.; Schlaggar, Bradley L.; Culver, Joseph P.

    2009-01-01

    Mapping resting-state networks allows insight into the brain's functional architecture and physiology and has rapidly become important in contemporary neuroscience research. Diffuse optical tomography (DOT) is an emerging functional neuroimaging technique with the advantages, relative to functional magnetic resonance imaging (fMRI), of portability and the ability to simultaneously measure both oxy- and deoxy-hemoglobin. Previous optical studies have evaluated the temporal features of spontaneous resting brain signals. Herein, we develop techniques for spatially mapping functional connectivity with DOT (fc-DOT). Simultaneous imaging over the motor and visual cortices yielded robust correlation maps reproducing the expected functional neural architecture. The localization of the maps was confirmed with task-response studies and with subject-matched fc-MRI. These fc-DOT methods provide a task-less approach to mapping brain function in populations that were previously difficult to research. Our advances may permit new studies of early childhood development and of unconscious patients. In addition, the comprehensive hemoglobin contrasts of fc-DOT enable innovative studies of the biophysical origin of the functional connectivity signal. PMID:19344773

  12. Distribution of neurons in functional areas of the mouse cerebral cortex reveals quantitatively different cortical zones.

    PubMed

    Herculano-Houzel, Suzana; Watson, Charles; Paxinos, George

    2013-01-01

    How are neurons distributed along the cortical surface and across functional areas? Here we use the isotropic fractionator (Herculano-Houzel and Lent, 2005) to analyze the distribution of neurons across the entire isocortex of the mouse, divided into 18 functional areas defined anatomically. We find that the number of neurons underneath a surface area (the N/A ratio) varies 4.5-fold across functional areas and neuronal density varies 3.2-fold. The face area of S1 contains the most neurons, followed by motor cortex and the primary visual cortex. Remarkably, while the distribution of neurons across functional areas does not accompany the distribution of surface area, it mirrors closely the distribution of cortical volumes-with the exception of the visual areas, which hold more neurons than expected for their volume. Across the non-visual cortex, the volume of individual functional areas is a shared linear function of their number of neurons, while in the visual areas, neuronal densities are much higher than in all other areas. In contrast, the 18 functional areas cluster into three different zones according to the relationship between the N/A ratio and cortical thickness and neuronal density: these three clusters can be called visual, sensory, and, possibly, associative. These findings are remarkably similar to those in the human cerebral cortex (Ribeiro et al., 2013) and suggest that, like the human cerebral cortex, the mouse cerebral cortex comprises two zones that differ in how neurons form the cortical volume, and three zones that differ in how neurons are distributed underneath the cortical surface, possibly in relation to local differences in connectivity through the white matter. Our results suggest that beyond the developmental divide into visual and non-visual cortex, functional areas initially share a common distribution of neurons along the parenchyma that become delimited into functional areas according to the pattern of connectivity established later.

  13. Network asymmetry of motor areas revealed by resting-state functional magnetic resonance imaging.

    PubMed

    Yan, Li-Rong; Wu, Yi-Bo; Hu, De-Wen; Qin, Shang-Zhen; Xu, Guo-Zheng; Zeng, Xiao-Hua; Song, Hua

    2012-02-01

    There are ample functional magnetic resonance imaging (fMRI) studies on functional brain asymmetries, and the asymmetry of cerebral network in the resting state may be crucial to brain function organization. In this paper, a unified schema of voxel-wise functional connectivity and asymmetry analysis was presented and the network asymmetry of motor areas was studied. Twelve healthy male subjects with mean age 29.8 ± 6.4 were studied. Functional network in the resting state was described by using functional connectivity magnetic resonance imaging (fcMRI) analysis. Motor areas were selected as regions of interest (ROIs). Network asymmetry, including intra- and inter-network asymmetries, was formulated and analyzed. The intra-network asymmetry was defined as the difference between the left and right part of a particular functional network. The inter-network asymmetry was defined as the difference between the networks for a specific ROI in the left hemisphere and its homotopic ROI in the right hemisphere. Primary motor area (M1), primary sensory area (S1) and premotor area (PMA) exhibited higher functional correlation with the right parietal-temporal-occipital circuit and the middle frontal gyrus than they did with the left hemisphere. Right S1 and right PMA exhibited higher functional correlation with the ipsilateral precentral and supramarginal areas. There exist the large-scale hierarchical network asymmetries of the motor areas in the resting state. These asymmetries imply the right hemisphere dominance for predictive motor coding based on spatial attention and higher sensory processing load for the motor performance of non-dominant hemisphere.

  14. Genotype Correlation Analysis Reveals Pathway-Based Functional Disequilibrium and Potential Epistasis in the Human Interactome

    PubMed Central

    Bush, William S.; Haines, Jonathan L.

    2016-01-01

    Epistasis is thought to be a pervasive part of complex phenotypes due to the dynamics and complexity of biological systems, and a further understanding of epistasis in the context of biological pathways may provide insight into the etiology of complex disease. In this study, we use genotype data from the International HapMap Project to characterize the functional dependencies between alleles in the human interactome as defined by KEGG pathways. We performed chi-square tests to identify non-independence between functionally-related SNP pairs within parental Caucasian and Yoruba samples. We further refine this list by testing for skewed transmission of pseudo-haplotypes to offspring using a haplotype-based TDT test. From these analyses, we identify pathways enriched for functional disequilibrium, and a set of 863 SNP pairs (representing 453 gene pairs) showing consistent non-independence and transmission distortion. These results represent gene pairs with strong evidence of epistasis within the context of a biological function.

  15. Spectral imaging reveals microvessel physiology and function from anastomoses to thromboses

    NASA Astrophysics Data System (ADS)

    Wankhede, Mamta; Agarwal, Nikita; Fraga-Silva, Rodrigo A.; Dedeugd, Casey; Raizada, Mohan K.; Oh, S. Paul; Sorg, Brian S.

    2010-01-01

    Abnormal microvascular physiology and function is common in many diseases. Numerous pathologies include hypervascularity, aberrant angiogenesis, or abnormal vascular remodeling among the characteristic features of the disease, and quantitative imaging and measurement of microvessel function can be important to increase understanding of these diseases. Several optical techniques are useful for direct imaging of microvascular function. Spectral imaging is one such technique that can be used to assess microvascular oxygen transport function with high spatial and temporal resolution in microvessel networks through measurements of hemoglobin saturation. We highlight novel observation made with our intravital microscopy spectral imaging system employed with mouse dorsal skin-fold window chambers for imaging hemoglobin saturation in microvessel networks. Specifically, we image acute oxygenation fluctuations in a tumor microvessel network, the development of arteriovenous malformations in a mouse model of hereditary hemorrhagic telangiectasia, and the formation of spontaneous and induced microvascular thromboses and occlusions.

  16. Annotation of Protein Domains Reveals Remarkable Conservation in the Functional Make up of Proteomes Across Superkingdoms.

    PubMed

    Nasir, Arshan; Naeem, Aisha; Khan, Muhammad Jawad; Nicora, Horacio D Lopez; Caetano-Anollés, Gustavo

    2011-11-08

    The functional repertoire of a cell is largely embodied in its proteome, the collection of proteins encoded in the genome of an organism. The molecular functions of proteins are the direct consequence of their structure and structure can be inferred from sequence using hidden Markov models of structural recognition. Here we analyze the functional annotation of protein domain structures in almost a thousand sequenced genomes, exploring the functional and structural diversity of proteomes. We find there is a remarkable conservation in the distribution of domains with respect to the molecular functions they perform in the three superkingdoms of life. In general, most of the protein repertoire is spent in functions related to metabolic processes but there are significant differences in the usage of domains for regulatory and extra-cellular processes both within and between superkingdoms. Our results support the hypotheses that the proteomes of superkingdom Eukarya evolved via genome expansion mechanisms that were directed towards innovating new domain architectures for regulatory and extra/intracellular process functions needed for example to maintain the integrity of multicellular structure or to interact with environmental biotic and abiotic factors (e.g., cell signaling and adhesion, immune responses, and toxin production). Proteomes of microbial superkingdoms Archaea and Bacteria retained fewer numbers of domains and maintained simple and smaller protein repertoires. Viruses appear to play an important role in the evolution of superkingdoms. We finally identify few genomic outliers that deviate significantly from the conserved functional design. These include Nanoarchaeum equitans, proteobacterial symbionts of insects with extremely reduced genomes, Tenericutes and Guillardia theta. These organisms spend most of their domains on information functions, including translation and transcription, rather than on metabolism and harbor a domain repertoire characteristic of

  17. Annotation of Protein Domains Reveals Remarkable Conservation in the Functional Make up of Proteomes Across Superkingdoms

    PubMed Central

    Nasir, Arshan; Naeem, Aisha; Khan, Muhammad Jawad; Lopez-Nicora, Horacio D.; Caetano-Anollés, Gustavo

    2011-01-01

    The functional repertoire of a cell is largely embodied in its proteome, the collection of proteins encoded in the genome of an organism. The molecular functions of proteins are the direct consequence of their structure and structure can be inferred from sequence using hidden Markov models of structural recognition. Here we analyze the functional annotation of protein domain structures in almost a thousand sequenced genomes, exploring the functional and structural diversity of proteomes. We find there is a remarkable conservation in the distribution of domains with respect to the molecular functions they perform in the three superkingdoms of life. In general, most of the protein repertoire is spent in functions related to metabolic processes but there are significant differences in the usage of domains for regulatory and extra-cellular processes both within and between superkingdoms. Our results support the hypotheses that the proteomes of superkingdom Eukarya evolved via genome expansion mechanisms that were directed towards innovating new domain architectures for regulatory and extra/intracellular process functions needed for example to maintain the integrity of multicellular structure or to interact with environmental biotic and abiotic factors (e.g., cell signaling and adhesion, immune responses, and toxin production). Proteomes of microbial superkingdoms Archaea and Bacteria retained fewer numbers of domains and maintained simple and smaller protein repertoires. Viruses appear to play an important role in the evolution of superkingdoms. We finally identify few genomic outliers that deviate significantly from the conserved functional design. These include Nanoarchaeum equitans, proteobacterial symbionts of insects with extremely reduced genomes, Tenericutes and Guillardia theta. These organisms spend most of their domains on information functions, including translation and transcription, rather than on metabolism and harbor a domain repertoire characteristic of

  18. Disrupted Brain Functional Organization in Epilepsy Revealed by Graph Theory Analysis.

    PubMed

    Song, Jie; Nair, Veena A; Gaggl, Wolfgang; Prabhakaran, Vivek

    2015-06-01

    The human brain is a complex and dynamic system that can be modeled as a large-scale brain network to better understand the reorganizational changes secondary to epilepsy. In this study, we developed a brain functional network model using graph theory methods applied to resting-state fMRI data acquired from a group of epilepsy patients and age- and gender-matched healthy controls. A brain functional network model was constructed based on resting-state functional connectivity. A minimum spanning tree combined with proportional thresholding approach was used to obtain sparse connectivity matrices for each subject, which formed the basis of brain networks. We examined the brain reorganizational changes in epilepsy thoroughly at the level of the whole brain, the functional network, and individual brain regions. At the whole-brain level, local efficiency was significantly decreased in epilepsy patients compared with the healthy controls. However, global efficiency was significantly increased in epilepsy due to increased number of functional connections between networks (although weakly connected). At the functional network level, there were significant proportions of newly formed connections between the default mode network and other networks and between the subcortical network and other networks. There was a significant proportion of decreasing connections between the cingulo-opercular task control network and other networks. Individual brain regions from different functional networks, however, showed a distinct pattern of reorganizational changes in epilepsy. These findings suggest that epilepsy alters brain efficiency in a consistent pattern at the whole-brain level, yet alters brain functional networks and individual brain regions differently.

  19. Functional genomics identifies a requirement of pre-mRNA splicing factors for sister chromatid cohesion.

    PubMed

    Sundaramoorthy, Sriramkumar; Vázquez-Novelle, María Dolores; Lekomtsev, Sergey; Howell, Michael; Petronczki, Mark

    2014-11-18

    Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation during cell division. Using functional genomic screening, we identify a set of 26 pre-mRNA splicing factors that are required for sister chromatid cohesion in human cells. Loss of spliceosome subunits increases the dissociation rate of cohesin from chromatin and abrogates cohesion after DNA replication, ultimately causing mitotic catastrophe. Depletion of splicing factors causes defective processing of the pre-mRNA encoding sororin, a factor required for the stable association of cohesin with chromatin, and an associated reduction of sororin protein level. Expression of an intronless version of sororin and depletion of the cohesin release protein WAPL suppress the cohesion defect in cells lacking splicing factors. We propose that spliceosome components contribute to sister chromatid cohesion and mitotic chromosome segregation through splicing of sororin pre-mRNA. Our results highlight the loss of cohesion as an early cellular consequence of compromised splicing. This may have clinical implications because SF3B1, a splicing factor that we identify to be essential for cohesion, is recurrently mutated in chronic lymphocytic leukaemia.

  20. Comparative transcriptome analyses of seven anurans reveal functions and adaptations of amphibian skin

    PubMed Central

    Huang, Li; Li, Jun; Anboukaria, Housseni; Luo, Zhenhua; Zhao, Mian; Wu, Hua

    2016-01-01

    Animal skin, which is the tissue that directly contacts the external surroundings, has evolved diverse functions to adapt to various environments. Amphibians represent the transitional taxon from aquatic to terrestrial life. Exploring the molecular basis of their skin function and adaptation is important to understand the survival and evolutionary mechanisms of vertebrates. However, comprehensive studies on the molecular mechanisms of skin functions in amphibians are scarce. In this study, we sequenced the skin transcriptomes of seven anurans belonging to three families and compared the similarities and differences in expressed genes and proteins. Unigenes and pathways related to basic biological processes and special functions, such as defense, immunity, and respiration, were enriched in functional annotations. A total of 108 antimicrobial peptides were identified. The highly expressed genes were similar in species of the same family but were different among families. Additionally, the positively selected orthologous groups were involved in biosynthesis, metabolism, immunity, and defense processes. This study is the first to generate extensive transcriptome data for the skin of seven anurans and provides unigenes and pathway candidates for further studies on amphibian skin function and adaptation. PMID:27040083

  1. In actio optophysiological analyses reveal functional diversification of dopaminergic neurons in the nematode C. elegans

    NASA Astrophysics Data System (ADS)

    Tanimoto, Yuki; Zheng, Ying Grace; Fei, Xianfeng; Fujie, Yukako; Hashimoto, Koichi; Kimura, Koutarou D.

    2016-05-01

    Many neuronal groups such as dopamine-releasing (dopaminergic) neurons are functionally divergent, although the details of such divergence are not well understood. Dopamine in the nematode Caenorhabditis elegans modulates various neural functions and is released from four left-right pairs of neurons. The terminal identities of these dopaminergic neurons are regulated by the same genetic program, and previous studies have suggested that they are functionally redundant. In this study, however, we show functional divergence within the dopaminergic neurons of C. elegans. Because dopaminergic neurons of the animals were supposedly activated by mechanical stimulus upon entry into a lawn of their food bacteria, we developed a novel integrated microscope system that can auto-track a freely-moving (in actio) C. elegans to individually monitor and stimulate the neuronal activities of multiple neurons. We found that only head-dorsal pair of dopaminergic neurons (CEPD), but not head-ventral or posterior pairs, were preferentially activated upon food entry. In addition, the optogenetic activation of CEPD neurons alone exhibited effects similar to those observed upon food entry. Thus, our results demonstrated functional divergence in the genetically similar dopaminergic neurons, which may provide a new entry point toward understanding functional diversity of neurons beyond genetic terminal identification.

  2. An integrative architecture for general intelligence and executive function revealed by lesion mapping

    PubMed Central

    Colom, Roberto; Solomon, Jeffrey; Krueger, Frank; Forbes, Chad; Grafman, Jordan

    2012-01-01

    Although cognitive neuroscience has made remarkable progress in understanding the involvement of the prefrontal cortex in executive control, the broader functional networks that support high-level cognition and give rise to general intelligence remain to be well characterized. Here, we investigated the neural substrates of the general factor of intelligence (g) and executive function in 182 patients with focal brain damage using voxel-based lesion–symptom mapping. The Wechsler Adult Intelligence Scale and Delis–Kaplan Executive Function System were used to derive measures of g and executive function, respectively. Impaired performance on these measures was associated with damage to a distributed network of left lateralized brain areas, including regions of frontal and parietal cortex and white matter association tracts, which bind these areas into a coordinated system. The observed findings support an integrative framework for understanding the architecture of general intelligence and executive function, supporting their reliance upon a shared fronto-parietal network for the integration and control of cognitive representations and making specific recommendations for the application of the Wechsler Adult Intelligence Scale and Delis–Kaplan Executive Function System to the study of high-level cognition in health and disease. PMID:22396393

  3. Functional diversity of the p24γ homologue Erp reveals physiological differences between two filamentous fungi.

    PubMed

    Wang, Fangzhong; Liang, Ya; Wang, Mingyu; Yang, Hui; Liu, Kuimei; Zhao, Qiushuang; Fang, Xu

    2013-12-01

    The protein hyper-secreting filamentous fungi impact their surrounding environments by secreting cellulases and digesting plant cell wall via microbe-plant interspecies interaction. This process is of paramount importance in biofuel production from the renewable lignocellulosic biomass, because cellulase production is the key factor in cost determination. Despite the importance of protein secretion, p24 protein, a key factor in eukaryotic protein maturation and secretion, was never investigated in filamentous fungi. The erp genes encoding p24γ homologues were identified in Trichoderma reesei and Penicillium decumbens. The roles of Erp and their participated cellular pathways were investigated via disruption of erp, revealing significant differences: sporulation was hampered in T. reesei Δerp but not in P. decumbens Δerp; in both species Erp maintains membrane integrity; Erp is likely involved in hyphae polarity maintenance in T. reesei. Protein- and transcription-level investigations of Erp participation in cellulase production revealed distinct regulatory mechanisms. In T. reesei, cellulase encoding genes were repressed under secretion stress. In contrast, activation of the same genes under the same stress was identified in P. decumbens. These observations revealed a novel cellulase gene regulation mechanism, clearly suggested the different physiological roles of Erp, and further demonstrated the different physiology of T. reesei and P. decumbens, despite above 75% sequence identity between the proteins and the close evolutionary relationship between the two species.

  4. Simultaneous PET-MRI reveals brain function in activated and resting state on metabolic, hemodynamic and multiple temporal scales.

    PubMed

    Wehrl, Hans F; Hossain, Mosaddek; Lankes, Konrad; Liu, Chih-Chieh; Bezrukov, Ilja; Martirosian, Petros; Schick, Fritz; Reischl, Gerald; Pichler, Bernd J

    2013-09-01

    Combined positron emission tomography (PET) and magnetic resonance imaging (MRI) is a new tool to study functional processes in the brain. Here we study brain function in response to a barrel-field stimulus simultaneously using PET, which traces changes in glucose metabolism on a slow time scale, and functional MRI (fMRI), which assesses fast vascular and oxygenation changes during activation. We found spatial and quantitative discrepancies between the PET and the fMRI activation data. The functional connectivity of the rat brain was assessed by both modalities: the fMRI approach determined a total of nine known neural networks, whereas the PET method identified seven glucose metabolism-related networks. These results demonstrate the feasibility of combined PET-MRI for the simultaneous study of the brain at activation and rest, revealing comprehensive and complementary information to further decode brain function and brain networks.

  5. Pull-down combined with proteomic strategy reveals functional diversity of synaptotagmin I.

    PubMed

    Guo, Tianyao; Duan, Zhigui; Chen, Jia; Xie, Chunliang; Wang, Ying; Chen, Ping; Wang, Xianchun

    2017-01-01

    Synaptotagmin I (Syt I) is most abundant in the brain and is involved in multiple cellular processes. Its two C2 domains, C2A and C2B, are the main functional regions. Our present study employed a pull-down combined with proteomic strategy to identify the C2 domain-interacting proteins to comprehensively understand the biological roles of the C2 domains and thus the functional diversity of Syt I. A total of 135 non-redundant proteins interacting with the C2 domains of Syt I were identified. Out of them, 32 and 64 proteins only bound to C2A or C2B domains, respectively, and 39 proteins bound to both of them. Compared with C2A, C2B could bind to many more proteins particularly those involved in synaptic transmission and metabolic regulation. Functional analysis indicated that Syt I may exert impacts by interacting with other proteins on multiple cellular processes, including vesicular membrane trafficking, synaptic transmission, metabolic regulation, catalysis, transmembrane transport and structure formation, etc. These results demonstrate that the functional diversity of Syt I is higher than previously expected, that its two domains may mediate the same and different cellular processes cooperatively or independently, and that C2B domain may play even more important roles than C2A in the functioning of Syt I. This work not only further deepened our understanding of the functional diversity of Syt I and the functional differences between its two C2 domains, but also provided important clues for the further related researches.

  6. Pull-down combined with proteomic strategy reveals functional diversity of synaptotagmin I

    PubMed Central

    Guo, Tianyao; Duan, Zhigui; Chen, Jia; Xie, Chunliang; Wang, Ying; Chen, Ping

    2017-01-01

    Synaptotagmin I (Syt I) is most abundant in the brain and is involved in multiple cellular processes. Its two C2 domains, C2A and C2B, are the main functional regions. Our present study employed a pull-down combined with proteomic strategy to identify the C2 domain-interacting proteins to comprehensively understand the biological roles of the C2 domains and thus the functional diversity of Syt I. A total of 135 non-redundant proteins interacting with the C2 domains of Syt I were identified. Out of them, 32 and 64 proteins only bound to C2A or C2B domains, respectively, and 39 proteins bound to both of them. Compared with C2A, C2B could bind to many more proteins particularly those involved in synaptic transmission and metabolic regulation. Functional analysis indicated that Syt I may exert impacts by interacting with other proteins on multiple cellular processes, including vesicular membrane trafficking, synaptic transmission, metabolic regulation, catalysis, transmembrane transport and structure formation, etc. These results demonstrate that the functional diversity of Syt I is higher than previously expected, that its two domains may mediate the same and different cellular processes cooperatively or independently, and that C2B domain may play even more important roles than C2A in the functioning of Syt I. This work not only further deepened our understanding of the functional diversity of Syt I and the functional differences between its two C2 domains, but also provided important clues for the further related researches. PMID:28194317

  7. Functional assays and metagenomic analyses reveals differences between the microbial communities inhabiting the soil horizons of a Norway spruce plantation.

    PubMed

    Uroz, Stéphane; Ioannidis, Panos; Lengelle, Juliette; Cébron, Aurélie; Morin, Emmanuelle; Buée, Marc; Martin, Francis

    2013-01-01

    In temperate ecosystems, acidic forest soils are among the most nutrient-poor terrestrial environments. In this context, the long-term differentiation of the forest soils into horizons may impact the assembly and the functions of the soil microbial communities. To gain a more comprehensive understanding of the ecology and functional potentials of these microbial communities, a suite of analyses including comparative metagenomics was applied on independent soil samples from a spruce plantation (Breuil-Chenue, France). The objectives were to assess whether the decreasing nutrient bioavailability and pH variations that naturally occurs between the organic and mineral horizons affects the soil microbial functional biodiversity. The 14 Gbp of pyrosequencing and Illumina sequences generated in this study revealed complex microbial communities dominated by bacteria. Detailed analyses showed that the organic soil horizon was significantly enriched in sequences related to Bacteria, Chordata, Arthropoda and Ascomycota. On the contrary the mineral horizon was significantly enriched in sequences related to Archaea. Our analyses also highlighted that the microbial communities inhabiting the two soil horizons differed significantly in their functional potentials according to functional assays and MG-RAST analyses, suggesting a functional specialisation of these microbial communities. Consistent with this specialisation, our shotgun metagenomic approach revealed a significant increase in the relative abundance of sequences related glycoside hydrolases in the organic horizon compared to the mineral horizon that was significantly enriched in glycoside transferases. This functional stratification according to the soil horizon was also confirmed by a significant correlation between the functional assays performed in this study and the functional metagenomic analyses. Together, our results suggest that the soil stratification and particularly the soil resource availability impact the

  8. The structure of BVU2987 from Bacteroides vulgatus reveals a superfamily of bacterial periplasmic proteins with possible inhibitory function

    PubMed Central

    Das, Debanu; Finn, Robert D.; Carlton, Dennis; Miller, Mitchell D.; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Bakolitsa, Constantina; Chen, Connie; Chiu, Hsiu-Ju; Chiu, Michelle; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Ellrott, Kyle; Ernst, Dustin; Farr, Carol L.; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Marciano, David; McMullan, Daniel; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Puckett, Christina; Reyes, Ron; Rife, Christopher L.; Sefcovic, Natasha; Tien, Henry J.; Trame, Christine B.; van den Bedem, Henry; Weekes, Dana; Wooten, Tiffany; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2010-01-01

    Proteins that contain the DUF2874 domain constitute a new Pfam family PF11396. Members of this family have predominantly been identified in microbes found in the human gut and oral cavity. The crystal structure of one member of this family, BVU2987 from Bacteroides vulgatus, has been determined, revealing a β-lactamase inhibitor protein-like structure with a tandem repeat of domains. Sequence analysis and structural comparisons reveal that BVU2987 and other DUF2874 proteins are related to β-lactamase inhibitor protein, PepSY and SmpA_OmlA proteins and hence are likely to function as inhibitory proteins. PMID:20944221

  9. Intraoperative mapping during repeat awake craniotomy reveals the functional plasticity of adult cortex.

    PubMed

    Southwell, Derek G; Hervey-Jumper, Shawn L; Perry, David W; Berger, Mitchel S

    2016-05-01

    OBJECT To avoid iatrogenic injury during the removal of intrinsic cerebral neoplasms such as gliomas, direct electrical stimulation (DES) is used to identify cortical and subcortical white matter pathways critical for language, motor, and sensory function. When a patient undergoes more than 1 brain tumor resection as in the case of tumor recurrence, the use of DES provides an unusual opportunity to examine brain plasticity in the setting of neurological disease. METHODS The authors examined 561 consecutive cases in which patients underwent DES mapping during surgery forglioma resection. "Positive" and "negative" sites-discrete cortical regions where electrical stimulation did (positive) or did not (negative) produce transient sensory, motor, or language disturbance-were identified prior to tumor resection and documented by intraoperative photography for categorization into functional maps. In this group of 561 patients, 18 were identified who underwent repeat surgery in which 1 or more stimulation sites overlapped with those tested during the initial surgery. The authors compared intraoperative sensory, motor, or language mapping results between initial and repeat surgeries, and evaluated the clinical outcomes for these patients. RESULTS A total of 117 sites were tested for sensory (7 sites, 6.0%), motor (9 sites, 7.7%), or language (101 sites, 86.3%) function during both initial and repeat surgeries. The mean interval between surgical procedures was 4.1 years. During initial surgeries, 95 (81.2%) of 117 sites were found to be negative and 22 (18.8%) of 117 sites were found to be positive. During repeat surgeries, 103 (88.0%) of 117 sites were negative and 14 (12.0%) of 117 were positive. Of the 95 sites that were negative at the initial surgery, 94 (98.9%) were also negative at the repeat surgery, while 1 (1.1%) site was found to be positive. Of the 22 sites that were initially positive, 13 (59.1%) remained positive at repeat surgery, while 9 (40.9%) had become

  10. Positron Emission Tomography Reveals Abnormal Topological Organization in Functional Brain Network in Diabetic Patients

    PubMed Central

    Qiu, Xiangzhe; Zhang, Yanjun; Feng, Hongbo; Jiang, Donglang

    2016-01-01

    Recent studies have demonstrated alterations in the topological organization of structural brain networks in diabetes mellitus (DM). However, the DM-related changes in the topological properties in functional brain networks are unexplored so far. We therefore used fluoro-D-glucose positron emission tomography (FDG-PET) data to construct functional brain networks of 73 DM patients and 91 sex- and age-matched normal controls (NCs), followed by a graph theoretical analysis. We found that both DM patients and NCs had a small-world topology in functional brain network. In comparison to the NC group, the DM group was found to have significantly lower small-world index, lower normalized clustering coefficients and higher normalized characteristic path length. Moreover, for diabetic patients, the nodal centrality was significantly reduced in the right rectus, the right cuneus, the left middle occipital gyrus, and the left postcentral gyrus, and it was significantly increased in the orbitofrontal region of the left middle frontal gyrus, the left olfactory region, and the right paracentral lobule. Our results demonstrated that the diabetic brain was associated with disrupted topological organization in the functional PET network, thus providing functional evidence for the abnormalities of brain networks in DM. PMID:27303259

  11. Protein similarity networks reveal relationships among sequence, structure, and function within the Cupin superfamily.

    PubMed

    Uberto, Richard; Moomaw, Ellen W

    2013-01-01

    The cupin superfamily is extremely diverse and includes catalytically inactive seed storage proteins, sugar-binding metal-independent epimerases, and metal-dependent enzymes possessing dioxygenase, decarboxylase, and other activities. Although numerous proteins of this superfamily have been structurally characterized, the functions of many of them have not been experimentally determined. We report the first use of protein similarity networks (PSNs) to visualize trends of sequence and structure in order to make functional inferences in this remarkably diverse superfamily. PSNs provide a way to visualize relatedness of structure and sequence among a given set of proteins. Structure- and sequence-based clustering of cupin members reflects functional clustering. Networks based only on cupin domains and networks based on the whole proteins provide complementary information. Domain-clustering supports phylogenetic conclusions that the N- and C-terminal domains of bicupin proteins evolved independently. Interestingly, although many functionally similar enzymatic cupin members bind the same active site metal ion, the structure and sequence clustering does not correlate with the identity of the bound metal. It is anticipated that the application of PSNs to this superfamily will inform experimental work and influence the functional annotation of databases.

  12. Metaproteogenomics Reveals Taxonomic and Functional Changes between Cecal and Fecal Microbiota in Mouse.

    PubMed

    Tanca, Alessandro; Manghina, Valeria; Fraumene, Cristina; Palomba, Antonio; Abbondio, Marcello; Deligios, Massimo; Silverman, Michael; Uzzau, Sergio

    2017-01-01

    Previous studies on mouse models report that cecal and fecal microbial communities may differ in the taxonomic structure, but little is known about their respective functional activities. Here, we employed a metaproteogenomic approach, including 16S rRNA gene sequencing, shotgun metagenomics and shotgun metaproteomics, to analyze the microbiota of paired mouse cecal contents (CCs) and feces, with the aim of identifying changes in taxon-specific functions. As a result, Gram-positive anaerobes were observed as considerably higher in CCs, while several key enzymes, involved in oxalate degradation, glutamate/glutamine metabolism, and redox homeostasis, and most actively expressed by Bacteroidetes, were clearly more represented in feces. On the whole, taxon and function abundance appeared to vary consistently with environmental changes expected to occur throughout the transit from the cecum to outside the intestine, especially when considering metaproteomic data. The results of this study indicate that functional and metabolic differences exist between CC and stool samples, paving the way to further metaproteogenomic investigations aimed at elucidating the functional dynamics of the intestinal microbiota.

  13. A reporter assay in lamprey embryos reveals both functional conservation and elaboration of vertebrate enhancers.

    PubMed

    Parker, Hugo J; Sauka-Spengler, Tatjana; Bronner, Marianne; Elgar, Greg

    2014-01-01

    The sea lamprey is an important model organism for investigating the evolutionary origins of vertebrates. As more vertebrate genome sequences are obtained, evolutionary developmental biologists are becoming increasingly able to identify putative gene regulatory elements across the breadth of the vertebrate taxa. The identification of these regions makes it possible to address how changes at the genomic level have led to changes in developmental gene regulatory networks and ultimately to the evolution of morphological diversity. Comparative genomics approaches using sea lamprey have already predicted a number of such regulatory elements in the lamprey genome. Functional characterisation of these sequences and other similar elements requires efficient reporter assays in lamprey. In this report, we describe the development of a transient transgenesis method for lamprey embryos. Focusing on conserved non-coding elements (CNEs), we use this method to investigate their functional conservation across the vertebrate subphylum. We find instances of both functional conservation and lineage-specific functional evolution of CNEs across vertebrates, emphasising the utility of functionally testing homologous CNEs in their host species.

  14. Fundamental gaps with approximate density functionals: the derivative discontinuity revealed from ensemble considerations.

    PubMed

    Kraisler, Eli; Kronik, Leeor

    2014-05-14

    The fundamental gap is a central quantity in the electronic structure of matter. Unfortunately, the fundamental gap is not generally equal to the Kohn-Sham gap of density functional theory (DFT), even in principle. The two gaps differ precisely by the derivative discontinuity, namely, an abrupt change in slope of the exchange-correlation energy as a function of electron number, expected across an integer-electron point. Popular approximate functionals are thought to be devoid of a derivative discontinuity, strongly compromising their performance for prediction of spectroscopic properties. Here we show that, in fact, all exchange-correlation functionals possess a derivative discontinuity, which arises naturally from the application of ensemble considerations within DFT, without any empiricism. This derivative discontinuity can be expressed in closed form using only quantities obtained in the course of a standard DFT calculation of the neutral system. For small, finite systems, addition of this derivative discontinuity indeed results in a greatly improved prediction for the fundamental gap, even when based on the most simple approximate exchange-correlation density functional--the local density approximation (LDA). For solids, the same scheme is exact in principle, but when applied to LDA it results in a vanishing derivative discontinuity correction. This failure is shown to be directly related to the failure of LDA in predicting fundamental gaps from total energy differences in extended systems.

  15. Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex

    NASA Astrophysics Data System (ADS)

    Ohki, Kenichi; Chung, Sooyoung; Ch'ng, Yeang H.; Kara, Prakash; Reid, R. Clay

    2005-02-01

    Neurons in the cerebral cortex are organized into anatomical columns, with ensembles of cells arranged from the surface to the white matter. Within a column, neurons often share functional properties, such as selectivity for stimulus orientation; columns with distinct properties, such as different preferred orientations, tile the cortical surface in orderly patterns. This functional architecture was discovered with the relatively sparse sampling of microelectrode recordings. Optical imaging of membrane voltage or metabolic activity elucidated the overall geometry of functional maps, but is averaged over many cells (resolution >100µm). Consequently, the purity of functional domains and the precision of the borders between them could not be resolved. Here, we labelled thousands of neurons of the visual cortex with a calcium-sensitive indicator in vivo. We then imaged the activity of neuronal populations at single-cell resolution with two-photon microscopy up to a depth of 400µm. In rat primary visual cortex, neurons had robust orientation selectivity but there was no discernible local structure; neighbouring neurons often responded to different orientations. In area 18 of cat visual cortex, functional maps were organized at a fine scale. Neurons with opposite preferences for stimulus direction were segregated with extraordinary spatial precision in three dimensions, with columnar borders one to two cells wide. These results indicate that cortical maps can be built with single-cell precision.

  16. Fundamental gaps with approximate density functionals: The derivative discontinuity revealed from ensemble considerations

    SciTech Connect

    Kraisler, Eli; Kronik, Leeor

    2014-05-14

    The fundamental gap is a central quantity in the electronic structure of matter. Unfortunately, the fundamental gap is not generally equal to the Kohn-Sham gap of density functional theory (DFT), even in principle. The two gaps differ precisely by the derivative discontinuity, namely, an abrupt change in slope of the exchange-correlation energy as a function of electron number, expected across an integer-electron point. Popular approximate functionals are thought to be devoid of a derivative discontinuity, strongly compromising their performance for prediction of spectroscopic properties. Here we show that, in fact, all exchange-correlation functionals possess a derivative discontinuity, which arises naturally from the application of ensemble considerations within DFT, without any empiricism. This derivative discontinuity can be expressed in closed form using only quantities obtained in the course of a standard DFT calculation of the neutral system. For small, finite systems, addition of this derivative discontinuity indeed results in a greatly improved prediction for the fundamental gap, even when based on the most simple approximate exchange-correlation density functional – the local density approximation (LDA). For solids, the same scheme is exact in principle, but when applied to LDA it results in a vanishing derivative discontinuity correction. This failure is shown to be directly related to the failure of LDA in predicting fundamental gaps from total energy differences in extended systems.

  17. Metaproteogenomics Reveals Taxonomic and Functional Changes between Cecal and Fecal Microbiota in Mouse

    PubMed Central

    Tanca, Alessandro; Manghina, Valeria; Fraumene, Cristina; Palomba, Antonio; Abbondio, Marcello; Deligios, Massimo; Silverman, Michael; Uzzau, Sergio

    2017-01-01

    Previous studies on mouse models report that cecal and fecal microbial communities may differ in the taxonomic structure, but little is known about their respective functional activities. Here, we employed a metaproteogenomic approach, including 16S rRNA gene sequencing, shotgun metagenomics and shotgun metaproteomics, to analyze the microbiota of paired mouse cecal contents (CCs) and feces, with the aim of identifying changes in taxon-specific functions. As a result, Gram-positive anaerobes were observed as considerably higher in CCs, while several key enzymes, involved in oxalate degradation, glutamate/glutamine metabolism, and redox homeostasis, and most actively expressed by Bacteroidetes, were clearly more represented in feces. On the whole, taxon and function abundance appeared to vary consistently with environmental changes expected to occur throughout the transit from the cecum to outside the intestine, especially when considering metaproteomic data. The results of this study indicate that functional and metabolic differences exist between CC and stool samples, paving the way to further metaproteogenomic investigations aimed at elucidating the functional dynamics of the intestinal microbiota. PMID:28352255

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

    PubMed

    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.

  19. The scaling behavior of hand motions reveals self-organization during an executive function task

    NASA Astrophysics Data System (ADS)

    Anastas, Jason R.; Stephen, Damian G.; Dixon, James A.

    2011-05-01

    Recent approaches to cognition explain cognitive phenomena in terms of interaction-dominant dynamics. In the current experiment, we extend this approach to executive function, a construct used to describe flexible, goal-oriented behavior. Participants were asked to perform a widely used executive function task, card sorting, under two conditions. In one condition, participants were given a rule with which to sort the cards. In the other condition, participants had to induce the rule from experimenter feedback. The motion of each participant’s hand was tracked during the sorting task. Detrended fluctuation analysis was performed on the inter-point time series using a windowing strategy to capture changes over each trial. For participants in the induction condition, the Hurst exponent sharply increased and then decreased. The Hurst exponents for the explicit condition did not show this pattern. Our results suggest that executive function may be understood in terms of changes in stability that arise from interaction-dominant dynamics.

  20. The functional micro-organization of grid cells revealed by cellular-resolution imaging.

    PubMed

    Heys, James G; Rangarajan, Krsna V; Dombeck, Daniel A

    2014-12-03

    Establishing how grid cells are anatomically arranged, on a microscopic scale, in relation to their firing patterns in the environment would facilitate a greater microcircuit-level understanding of the brain's representation of space. However, all previous grid cell recordings used electrode techniques that provide limited descriptions of fine-scale organization. We therefore developed a technique for cellular-resolution functional imaging of medial entorhinal cortex (MEC) neurons in mice navigating a virtual linear track, enabling a new experimental approach to study MEC. Using these methods, we show that grid cells are physically clustered in MEC compared to nongrid cells. Additionally, we demonstrate that grid cells are functionally micro-organized: the similarity between the environment firing locations of grid cell pairs varies as a function of the distance between them according to a "Mexican hat"-shaped profile. This suggests that, on average, nearby grid cells have more similar spatial firing phases than those further apart.

  1. Integrating abundance and functional traits reveals new global hotspots of fish diversity.

    PubMed

    Stuart-Smith, Rick D; Bates, Amanda E; Lefcheck, Jonathan S; Duffy, J Emmett; Baker, Susan C; Thomson, Russell J; Stuart-Smith, Jemina F; Hill, Nicole A; Kininmonth, Stuart J; Airoldi, Laura; Becerro, Mikel A; Campbell, Stuart J; Dawson, Terence P; Navarrete, Sergio A; Soler, German A; Strain, Elisabeth M A; Willis, Trevor J; Edgar, Graham J

    2013-09-26

    Species richness has dominated our view of global biodiversity patterns for centuries. The dominance of this paradigm is reflected in the focus by ecologists and conservation managers on richness and associated occurrence-based measures for understanding drivers of broad-scale diversity patterns and as a biological basis for management. However, this is changing rapidly, as it is now recognized that not only the number of species but the species present, their phenotypes and the number of individuals of each species are critical in determining the nature and strength of the relationships between species diversity and a range of ecological functions (such as biomass production and nutrient cycling). Integrating these measures should provide a more relevant representation of global biodiversity patterns in terms of ecological functions than that provided by simple species counts. Here we provide comparisons of a traditional global biodiversity distribution measure based on richness with metrics that incorporate species abundances and functional traits. We use data from standardized quantitative surveys of 2,473 marine reef fish species at 1,844 sites, spanning 133 degrees of latitude from all ocean basins, to identify new diversity hotspots in some temperate regions and the tropical eastern Pacific Ocean. These relate to high diversity of functional traits amongst individuals in the community (calculated using Rao's Q), and differ from previously reported patterns in functional diversity and richness for terrestrial animals, which emphasize species-rich tropical regions only. There is a global trend for greater evenness in the number of individuals of each species, across the reef fish species observed at sites ('community evenness'), at higher latitudes. This contributes to the distribution of functional diversity hotspots and contrasts with well-known latitudinal gradients in richness. Our findings suggest that the contribution of species diversity to a range of

  2. Resting state functional MRI reveals abnormal network connectivity in orthostatic tremor.

    PubMed

    Benito-León, Julián; Louis, Elan D; Manzanedo, Eva; Hernández-Tamames, Juan Antonio; Álvarez-Linera, Juan; Molina-Arjona, José Antonio; Matarazzo, Michele; Romero, Juan Pablo; Domínguez-González, Cristina; Domingo-Santos, Ángela; Sánchez-Ferro, Álvaro

    2016-07-01

    Very little is known about the pathogenesis of orthostatic tremor (OT). We have observed that OT patients might have deficits in specific aspects of neuropsychological function, particularly those thought to rely on the integrity of the prefrontal cortex, which suggests a possible involvement of frontocerebellar circuits. We examined whether resting-state functional magnetic resonance imaging (fMRI) might provide further insights into the pathogenesis on OT. Resting-state fMRI data in 13 OT patients (11 women and 2 men) and 13 matched healthy controls were analyzed using independent component analysis, in combination with a "dual-regression" technique, to identify group differences in several resting-state networks (RSNs). All participants also underwent neuropsychological testing during the same session. Relative to healthy controls, OT patients showed increased connectivity in RSNs involved in cognitive processes (default mode network [DMN] and frontoparietal networks), and decreased connectivity in the cerebellum and sensorimotor networks. Changes in network integrity were associated not only with duration (DMN and medial visual network), but also with cognitive function. Moreover, in at least 2 networks (DMN and medial visual network), increased connectivity was associated with worse performance on different cognitive domains (attention, executive function, visuospatial ability, visual memory, and language). In this exploratory study, we observed selective impairments of RSNs in OT patients. This and other future resting-state fMRI studies might provide a novel method to understand the pathophysiological mechanisms of motor and nonmotor features of OT.

  3. Muscle contraction phenotypic analysis enabled by optogenetics reveals functional relationships of sarcomere components in Caenorhabditis elegans

    NASA Astrophysics Data System (ADS)

    Hwang, Hyundoo; Barnes, Dawn E.; Matsunaga, Yohei; Benian, Guy M.; Ono, Shoichiro; Lu, Hang

    2016-01-01

    The sarcomere, the fundamental unit of muscle contraction, is a highly-ordered complex of hundreds of proteins. Despite decades of genetics work, the functional relationships and the roles of those sarcomeric proteins in animal behaviors remain unclear. In this paper, we demonstrate that optogenetic activation of the motor neurons that induce muscle contraction can facilitate quantitative studies of muscle kinetics in C. elegans. To increase the throughput of the study, we trapped multiple worms in parallel in a microfluidic device and illuminated for photoactivation of channelrhodopsin-2 to induce contractions in body wall muscles. Using image processing, the change in body size was quantified over time. A total of five parameters including rate constants for contraction and relaxation were extracted from the optogenetic assay as descriptors of sarcomere functions. To potentially relate the genes encoding the sarcomeric proteins functionally, a hierarchical clustering analysis was conducted on the basis of those parameters. Because it assesses physiological output different from conventional assays, this method provides a complement to the phenotypic analysis of C. elegans muscle mutants currently performed in many labs; the clusters may provide new insights and drive new hypotheses for functional relationships among the many sarcomere components.

  4. Diversity and functions of bacterial community in drinking water biofilms revealed by high-throughput sequencing.

    PubMed

    Chao, Yuanqing; Mao, Yanping; Wang, Zhiping; Zhang, Tong

    2015-06-12

    The development of biofilms in drinking water (DW) systems may cause various problems to water quality. To investigate the community structure of biofilms on different pipe materials and the global/specific metabolic functions of DW biofilms, PCR-based 454 pyrosequencing data for 16S rRNA genes and Illumina metagenomic data were generated and analysed. Considerable differences in bacterial diversity and taxonomic structure were identified between biofilms formed on stainless steel and biofilms formed on plastics, indicating that the metallic materials facilitate the formation of higher diversity biofilms. Moreover, variations in several dominant genera were observed during biofilm formation. Based on PCA analysis, the global functions in the DW biofilms were similar to other DW metagenomes. Beyond the global functions, the occurrences and abundances of specific protective genes involved in the glutathione metabolism, the SoxRS system, the OxyR system, RpoS regulated genes, and the production/degradation of extracellular polymeric substances were also evaluated. A near-complete and low-contamination draft genome was constructed from the metagenome of the DW biofilm, based on the coverage and tetranucleotide frequencies, and identified as a Bradyrhizobiaceae-like bacterium according to a phylogenetic analysis. Our findings provide new insight into DW biofilms, especially in terms of their metabolic functions.

  5. Analysis of functional variants reveals new candidate genes associated with alexithymia.

    PubMed

    Mezzavilla, Massimo; Ulivi, Sheila; Bianca, Martina La; Carlino, Davide; Gasparini, Paolo; Robino, Antonietta

    2015-06-30

    In this study we explored the possible association between 36,915 functional variants and alexithymia, a personality trait characterized by the inability to identify and describe emotions and feelings. From our analysis, variants in the genes ABCB4, TP53AIP1, ARHGAP32 and TMEM88B were identified linked to the alexithymia phenotype.

  6. Site-directed mutants of human RECQ1 reveal functional importance of the zinc binding domain.

    PubMed

    Sami, Furqan; Gary, Ronald K; Fang, Yayin; Sharma, Sudha

    2016-08-01

    RecQ helicases are a highly conserved family of ATP-dependent DNA-unwinding enzymes with key roles in DNA replication and repair in all kingdoms of life. The RECQ1 gene encodes the most abundant RecQ homolog in humans. We engineered full-length RECQ1 harboring point mutations in the zinc-binding motif (amino acids 419-480) within the conserved RecQ-specific-C-terminal (RQC) domain known to be critical for diverse biochemical and cellular functions of RecQ helicases. Wild-type RECQ1 contains a zinc ion. Substitution of three of the four conserved cysteine residues that coordinate zinc severely impaired the ATPase and DNA unwinding activities but retained DNA binding and single strand DNA annealing activities. Furthermore, alteration of these residues attenuated zinc binding and significantly changed the overall conformation of full-length RECQ1 protein. In contrast, substitution of cysteine residue at position 471 resulted in a wild-type like RECQ1 protein. Differential contribution of the conserved cysteine residues to the structure and functions of the RECQ1 protein is also inferred by homology modeling. Overall, our results indicate that the zinc binding motif in the RQC domain of RECQ1 is a key structural element that is essential for the structure-functions of RECQ1. Given the recent association of RECQ1 mutations with breast cancer, these results will contribute to understanding the molecular basis of RECQ1 functions in cancer etiology.

  7. Metaproteomics reveals functional shifts in microbial and human proteins during a preterm infant gut colonization case.

    PubMed

    Young, Jacque C; Pan, Chongle; Adams, Rachel M; Brooks, Brandon; Banfield, Jillian F; Morowitz, Michael J; Hettich, Robert L

    2015-10-01

    Microbial colonization of the human gastrointestinal tract plays an important role in establishing health and homeostasis. However, the time-dependent functional signatures of microbial and human proteins during early colonization of the gut have yet to be determined. To this end, we employed shotgun proteomics to simultaneously monitor microbial and human proteins in fecal samples from a preterm infant during the first month of life. Microbial community complexity increased over time, with compositional changes that were consistent with previous metagenomic and rRNA gene data. More specifically, the function of the microbial community initially involved biomass growth, protein production, and lipid metabolism, and then switched to more complex metabolic functions, such as carbohydrate metabolism, once the community stabilized and matured. Human proteins detected included those responsible for epithelial barrier function and antimicrobial activity. Some neutrophil-derived proteins increased in abundance early in the study period, suggesting activation of the innate immune system. Likewise, abundances of cytoskeletal and mucin proteins increased later in the time course, suggestive of subsequent adjustment to the increased microbial load. This study provides the first snapshot of coordinated human and microbial protein expression in a preterm infant's gut during early development.

  8. Metaproteomics Reveals Functional Shifts in Microbial and Human Proteins During a Preterm Infant Gut Colonization Case

    PubMed Central

    Young, Jacque C.; Pan, Chongle; Adams, Rachel; Brooks, Brandon; Banfield, Jillian F.; Morowitz, Michael J.; Hettich, Robert L.

    2015-01-01

    Microbial colonization of the human gastrointestinal tract plays an important role in establishing health and homeostasis. However, the time-dependent functional signatures of microbial and human proteins during early colonization of the gut have yet to be determined. To this end, we employed shotgun proteomics to simultaneously monitor microbial and human proteins in fecal samples from a preterm infant during the first month of life. Microbial community complexity increased over time, with compositional changes that were consistent with previous metagenomic and rRNA gene data. More specifically, the function of the microbial community initially involved biomass growth, protein production, and lipid metabolism, and then switched to more complex metabolic functions, such as carbohydrate metabolism, once the community stabilized and matured. Human proteins detected included those responsible for epithelial barrier function and antimicrobial activity. Some neutrophil-derived proteins increased in abundance early in the study period, suggesting activation of the innate immune system. Likewise, abundances of cytoskeletal and mucin proteins increased later in the time course, suggestive of subsequent adjustment to the increased microbial load. This study provides the first snapshot of coordinated human and microbial protein expression in a preterm infant’s gut during early development. PMID:26077811

  9. Comprehensive analysis of cellular galectin-3 reveals no consistent oncogenic function in pancreatic cancer cells.

    PubMed

    Hann, Alexander; Gruner, Anja; Chen, Ying; Gress, Thomas M; Buchholz, Malte

    2011-01-01

    Galectin-3 (Gal-3), a 31 kDa member of the family of beta-galactoside-binding proteins, has been implicated in the progression of different human cancers. However, the proposed roles differ widely, ranging from tumor-promoting cellular functions and negative impact on patient prognosis to tumor-suppressive properties and positive prognostic impact. We and others have previously identified Gal-3 as overexpressed in pancreatic cancer as compared to chronic pancreatitis and normal pancreatic tissue. The purpose of this study was thus the comprehensive analysis of putative cellular functions of Gal-3 by transient as well as stable silencing or overexpression of Gal-3 in a panel of 6 well-established pancreatic cancer cell lines. Our results confirm that galectin-3 is upregulated at the mRNA level in pancreatic cancer and strongly expressed in the majority of pancreatic cancer cell lines. In individual cell lines, transient knockdown of Gal-3 expression resulted in moderate inhibitory effects on proliferation, migration or anchorage-independent growth of the cells, but these effects were not consistent across the spectrum of analyzed cell lines. Moreover, functional effects of the modulation of Gal-3 expression were not observed in stable knockdown or overexpression approaches in vitro and did not alter the growth characteristics of nude mouse xenograft tumors in vivo. Our data thus do not support a direct functional role of Gal-3 in the malignant transformation of pancreatic epithelial cells, although paracrine or systemic effects of Gal-3 expression are not excluded.

  10. Diversity and functions of bacterial community in drinking water biofilms revealed by high-throughput sequencing

    PubMed Central

    Chao, Yuanqing; Mao, Yanping; Wang, Zhiping; Zhang, Tong

    2015-01-01

    The development of biofilms in drinking water (DW) systems may cause various problems to water quality. To investigate the community structure of biofilms on different pipe materials and the global/specific metabolic functions of DW biofilms, PCR-based 454 pyrosequencing data for 16S rRNA genes and Illumina metagenomic data were generated and analysed. Considerable differences in bacterial diversity and taxonomic structure were identified between biofilms formed on stainless steel and biofilms formed on plastics, indicating that the metallic materials facilitate the formation of higher diversity biofilms. Moreover, variations in several dominant genera were observed during biofilm formation. Based on PCA analysis, the global functions in the DW biofilms were similar to other DW metagenomes. Beyond the global functions, the occurrences and abundances of specific protective genes involved in the glutathione metabolism, the SoxRS system, the OxyR system, RpoS regulated genes, and the production/degradation of extracellular polymeric substances were also evaluated. A near-complete and low-contamination draft genome was constructed from the metagenome of the DW biofilm, based on the coverage and tetranucleotide frequencies, and identified as a Bradyrhizobiaceae-like bacterium according to a phylogenetic analysis. Our findings provide new insight into DW biofilms, especially in terms of their metabolic functions. PMID:26067561

  11. TURKEY FECAL MICROBIAL COMMUNITY STRUCTURE AND ECOLOGICAL FUNCTIONS REVEALED BY 16S RDNA AND METAGENOME SEQUENCES

    EPA Science Inventory

    Turkey feces are an important source of fecal waste in the United States. With the exception of isolated studies on bacterial pathogens, little is known about the type of bacteria inhabiting the turkey gut. In order to understand the microbial diversity and functional genes assoc...

  12. Cluster Analysis of p53 Binding Site Sequences Reveals Subsets with Different Functions

    PubMed Central

    Lim, Ji-Hyun; Latysheva, Natasha S.; Iggo, Richard D.; Barker, Daniel

    2016-01-01

    p53 is an important regulator of cell cycle arrest, senescence, apoptosis and metabolism, and is frequently mutated in tumors. It functions as a tetramer, where each component dimer binds to a decameric DNA region known as a response element. We identify p53 binding site subtypes and examine the functional and evolutionary properties of these subtypes. We start with over 1700 known binding sites and, with no prior labeling, identify two sets of response elements by unsupervised clustering. When combined, they give rise to three types of p53 binding sites. We find that probabilistic and alignment-based assessments of cross-species conservation show no strong evidence of differential conservation between types of binding sites. In contrast, functional analysis of the genes most proximal to the binding sites provides strong bioinformatic evidence of functional differentiation between the three types of binding sites. Our results are consistent with recent structural data identifying two conformations of the L1 loop in the DNA binding domain, suggesting that they reflect biologically meaningful groups imposed by the p53 protein structure. PMID:27812278

  13. Resting state functional MRI reveals abnormal network connectivity in orthostatic tremor

    PubMed Central

    Benito-León, Julián; Louis, Elan D.; Manzanedo, Eva; Hernández-Tamames, Juan Antonio; Álvarez-Linera, Juan; Molina-Arjona, José Antonio; Matarazzo, Michele; Romero, Juan Pablo; Domínguez-González, Cristina; Domingo-Santos, Ángela; Sánchez-Ferro, Álvaro

    2016-01-01

    Abstract Very little is known about the pathogenesis of orthostatic tremor (OT). We have observed that OT patients might have deficits in specific aspects of neuropsychological function, particularly those thought to rely on the integrity of the prefrontal cortex, which suggests a possible involvement of frontocerebellar circuits. We examined whether resting-state functional magnetic resonance imaging (fMRI) might provide further insights into the pathogenesis on OT. Resting-state fMRI data in 13 OT patients (11 women and 2 men) and 13 matched healthy controls were analyzed using independent component analysis, in combination with a “dual-regression” technique, to identify group differences in several resting-state networks (RSNs). All participants also underwent neuropsychological testing during the same session. Relative to healthy controls, OT patients showed increased connectivity in RSNs involved in cognitive processes (default mode network [DMN] and frontoparietal networks), and decreased connectivity in the cerebellum and sensorimotor networks. Changes in network integrity were associated not only with duration (DMN and medial visual network), but also with cognitive function. Moreover, in at least 2 networks (DMN and medial visual network), increased connectivity was associated with worse performance on different cognitive domains (attention, executive function, visuospatial ability, visual memory, and language). In this exploratory study, we observed selective impairments of RSNs in OT patients. This and other future resting-state fMRI studies might provide a novel method to understand the pathophysiological mechanisms of motor and nonmotor features of OT. PMID:27442678

  14. Resting-State Time-Varying Analysis Reveals Aberrant Variations of Functional Connectivity in Autism

    PubMed Central

    Yao, Zhijun; Hu, Bin; Xie, Yuanwei; Zheng, Fang; Liu, Guangyao; Chen, Xuejiao; Zheng, Weihao

    2016-01-01

    Recently, studies based on time-varying functional connectivity have unveiled brain states diversity in some neuropsychiatric disorders, such as schizophrenia and major depressive disorder. However, time-varying functional connectivity analysis of resting-state functional Magnetic Resonance Imaging (fMRI) have been rarely performed on the Autism Spectrum Disorder (ASD). Hence, we performed time-varying connectivity analysis on resting-state fMRI data to investigate brain states mutation in ASD children. ASD showed an imbalance of connectivity state and aberrant ratio of connectivity with different strengths in the whole brain network, and decreased connectivity associated precuneus/posterior cingulate gyrus with medial prefrontal gyrus in default mode network. As compared to typical development children, weak relevance condition (the strength of a large number of connectivities in the state was less than means minus standard deviation of all connection strength) was maintained for a longer time between brain areas of ASD children, and ratios of weak connectivity in brain states varied dramatically in the ASD. In the ASD, the abnormal brain state might be related to repetitive behaviors and stereotypical interests, and macroscopically reflect disruption of gamma-aminobutyric acid at the cellular level. The detection of brain states based on time-varying functional connectivity analysis of resting-state fMRI might be conducive for diagnosis and early intervention of ASD before obvious clinical symptoms. PMID:27695408

  15. Independent component analysis of localized resting-state functional magnetic resonance imaging reveals specific motor subnetworks.

    PubMed

    Sohn, William Seunghyun; Yoo, Kwangsun; Jeong, Yong

    2012-01-01

    Recent studies have shown that blood oxygen level-dependent low-frequency (<0.1 Hz) fluctuations (LFFs) during a resting-state exhibit a high degree of correlation with other regions that share cognitive function. Initial studies of resting-state network mapping have focused primarily on major networks such as the default mode network, primary motor, somatosensory, visual, and auditory networks. However, more specific or subnetworks, including those associated with specific motor functions, have yet to be properly addressed. We performed independent component analysis (ICA) in a specific target region of the brain, a process we name, "localized ICA." We demonstrated that when ICA is applied to localized fMRI data, it can be used to distinguish resting-state LFFs associated with specific motor functions (e.g., finger tapping, foot movement, or bilateral lip pulsing) in the primary motor cortex. These ICA components generated from localized data can then be used as functional regions of interest to map whole-brain connectivity. In addition, this method can be used to visualize inter-regional connectivity by expanding the localized region and identifying components that show connectivity between the two regions.

  16. Cells transplanted onto the surface of the glial scar reveal hidden potential for functional neural regeneration

    PubMed Central

    Sekiya, Tetsuji; Holley, Matthew C.; Hashido, Kento; Ono, Kazuya; Shimomura, Koichiro; Horie, Rie T.; Hamaguchi, Kiyomi; Yoshida, Atsuhiro; Sakamoto, Tatsunori; Ito, Juichi

    2015-01-01

    Cell transplantation therapy has long been investigated as a therapeutic intervention for neurodegenerative disorders, including spinal cord injury, Parkinson’s disease, and amyotrophic lateral sclerosis. Indeed, patients have high hopes for a cell-based therapy. However, there are numerous practical challenges for clinical translation. One major problem is that only very low numbers of donor cells survive and achieve functional integration into the host. Glial scar tissue in chronic neurodegenerative disorders strongly inhibits regeneration, and this inhibition must be overcome to accomplish successful cell transplantation. Intraneural cell transplantation is considered to be the best way to deliver cells to the host. We questioned this view with experiments in vivo on a rat glial scar model of the auditory system. Our results show that intraneural transplantation to the auditory nerve, preceded by chondroitinase ABC (ChABC)-treatment, is ineffective. There is no functional recovery, and almost all transplanted cells die within a few weeks. However, when donor cells are placed on the surface of a ChABC-treated gliotic auditory nerve, they autonomously migrate into it and recapitulate glia- and neuron-guided cell migration modes to repair the auditory pathway and recover auditory function. Surface transplantation may thus pave the way for improved functional integration of donor cells into host tissue, providing a less invasive approach to rescue clinically important neural tracts. PMID:26080415

  17. Muscle contraction phenotypic analysis enabled by optogenetics reveals functional relationships of sarcomere components in Caenorhabditis elegans.

    PubMed

    Hwang, Hyundoo; Barnes, Dawn E; Matsunaga, Yohei; Benian, Guy M; Ono, Shoichiro; Lu, Hang

    2016-01-29

    The sarcomere, the fundamental unit of muscle contraction, is a highly-ordered complex of hundreds of proteins. Despite decades of genetics work, the functional relationships and the roles of those sarcomeric proteins in animal behaviors remain unclear. In this paper, we demonstrate that optogenetic activation of the motor neurons that induce muscle contraction can facilitate quantitative studies of muscle kinetics in C. elegans. To increase the throughput of the study, we trapped multiple worms in parallel in a microfluidic device and illuminated for photoactivation of channelrhodopsin-2 to induce contractions in body wall muscles. Using image processing, the change in body size was quantified over time. A total of five parameters including rate constants for contraction and relaxation were extracted from the optogenetic assay as descriptors of sarcomere functions. To potentially relate the genes encoding the sarcomeric proteins functionally, a hierarchical clustering analysis was conducted on the basis of those parameters. Because it assesses physiological output different from conventional assays, this method provides a complement to the phenotypic analysis of C. elegans muscle mutants currently performed in many labs; the clusters may provide new insights and drive new hypotheses for functional relationships among the many sarcomere components.

  18. Resting state functional MRI reveals abnormal network connectivity in Neurofibromatosis 1

    PubMed Central

    Tomson, S.N.; Schreiner, M.; Narayan, M.; Rosser, Tena; Enrique, Nicole; Silva, Alcino J.; Allen, G.I.; Bookheimer, S.Y.; Bearden, C.E.

    2015-01-01

    Neurofibromatosis type I (NF1) is a genetic disorder caused by mutations in the neurofibromin 1 gene at locus 17q11.2. Individuals with NF1 have an increased incidence of learning disabilities, attention deficits and autism spectrum disorders. As a single gene disorder, NF1 represents a valuable model for understanding gene-brain-behavior relationships. While mouse models have elucidated molecular and cellular mechanisms underlying learning deficits associated with this mutation, little is known about functional brain architecture in human subjects with NF1. To address this question, we used resting state functional connectivity MRI (rs-fcMRI) to elucidate the intrinsic network structure of 30 NF1 participants compared with 30 healthy demographically matched controls during an eyes-open rs-fcMRI scan. Novel statistical methods were employed to quantify differences in local connectivity (edge strength) and modularity structure, in combination with traditional global graph theory applications. Our findings suggest that individuals with NF1 have reduced anterior-posterior connectivity, weaker bilateral edges, and altered modularity clustering relative to healthy controls. Further, edge strength and modular clustering indices were correlated with IQ and internalizing symptoms. These findings suggest that Ras signaling disruption may lead to abnormal functional brain connectivity; further investigation into the functional consequences of these alterations in both humans and in animal models is warranted. PMID:26304096

  19. Neutron Reflectometry reveals the interaction between functionalized SPIONs and the surface of lipid bilayers.

    PubMed

    Luchini, Alessandra; Gerelli, Yuri; Fragneto, Giovanna; Nylander, Tommy; Pálsson, Gunnar K; Appavou, Marie-Sousai; Paduano, Luigi

    2017-03-01

    The safe application of nanotechnology devices in biomedicine requires fundamental understanding on how they interact with and affect the different components of biological systems. In this respect, the cellular membrane, the cell envelope, certainly represents an important target or barrier for nanosystems. Here we report on the interaction between functionalized SuperParamagnetic Iron Oxide Nanoparticles (SPIONs), promising contrast agents for Magnetic Resonance Imaging (MRI), and lipid bilayers that mimic the plasma membrane. Neutron Reflectometry, supported by Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) experiments, was used to characterize this interaction by varying both SPION coating and lipid bilayer composition. In particular, the interaction of two different SPIONs, functionalized with a cationic surfactant and a zwitterionic phospholipid, and lipid bilayers, containing different amount of cholesterol, were compared. The obtained results were further validated by Dynamic Light Scattering (DLS) measurements and Cryogenic Transmission Electron Microscopy (Cryo-TEM) images. None of the investigated functionalized SPIONs were found to disrupt the lipid membrane. However, in all case we observed the attachment of the functionalized SPIONs onto the surface of the bilayers, which was affected by the bilayer rigidity, i.e. the cholesterol concentration.

  20. Diversity and functions of bacterial community in drinking water biofilms revealed by high-throughput sequencing

    NASA Astrophysics Data System (ADS)

    Chao, Yuanqing; Mao, Yanping; Wang, Zhiping; Zhang, Tong

    2015-06-01

    The development of biofilms in drinking water (DW) systems may cause various problems to water quality. To investigate the community structure of biofilms on different pipe materials and the global/specific metabolic functions of DW biofilms, PCR-based 454 pyrosequencing data for 16S rRNA genes and Illumina metagenomic data were generated and analysed. Considerable differences in bacterial diversity and taxonomic structure were identified between biofilms formed on stainless steel and biofilms formed on plastics, indicating that the metallic materials facilitate the formation of higher diversity biofilms. Moreover, variations in several dominant genera were observed during biofilm formation. Based on PCA analysis, the global functions in the DW biofilms were similar to other DW metagenomes. Beyond the global functions, the occurrences and abundances of specific protective genes involved in the glutathione metabolism, the SoxRS system, the OxyR system, RpoS regulated genes, and the production/degradation of extracellular polymeric substances were also evaluated. A near-complete and low-contamination draft genome was constructed from the metagenome of the DW biofilm, based on the coverage and tetranucleotide frequencies, and identified as a Bradyrhizobiaceae-like bacterium according to a phylogenetic analysis. Our findings provide new insight into DW biofilms, especially in terms of their metabolic functions.

  1. Metaproteomics Reveals Functional Shifts in Microbial and Human Proteins During Infant Gut Colonization Case

    DOE PAGES

    Young, Jacque C.; Pan, Chongle; Adams, Rachel M.; ...

    2015-01-01

    The microbial colonization of the human gastrointestinal tract plays an important role in establishing health and homeostasis. However, the time-dependent functional signatures of microbial and human proteins during early colonization of the gut have yet to be determined. Thus, we employed shotgun proteomics to simultaneously monitor microbial and human proteins in fecal samples from a preterm infant during the first month of life. Microbial community complexity and functions increased over time, with compositional changes that were consistent with previous metagenomic and rRNA gene data indicating three distinct colonization phases. Overall microbial community functions were established relatively early in development andmore » remained stable. Human proteins detected included those responsible for epithelial barrier function and antimicrobial activity. Some neutrophil-derived proteins increased in abundance early in the study period, suggesting activation of the innate immune system. Moreover, abundances of cytoskeletal and mucin proteins increased later in the time course, suggestive of subsequent adjustment to the increased microbial load. Our study provides the first snapshot of coordinated human and microbial protein expression in the infant gut during early development.« less

  2. Metaproteomics Reveals Functional Shifts in Microbial and Human Proteins During Infant Gut Colonization Case

    SciTech Connect

    Young, Jacque C.; Pan, Chongle; Adams, Rachel M.; Brooks, Brandon; Banfield, Jillian F.; Morowitz, Michael J.; Robert L. Hettich

    2015-01-01

    The microbial colonization of the human gastrointestinal tract plays an important role in establishing health and homeostasis. However, the time-dependent functional signatures of microbial and human proteins during early colonization of the gut have yet to be determined. Thus, we employed shotgun proteomics to simultaneously monitor microbial and human proteins in fecal samples from a preterm infant during the first month of life. Microbial community complexity and functions increased over time, with compositional changes that were consistent with previous metagenomic and rRNA gene data indicating three distinct colonization phases. Overall microbial community functions were established relatively early in development and remained stable. Human proteins detected included those responsible for epithelial barrier function and antimicrobial activity. Some neutrophil-derived proteins increased in abundance early in the study period, suggesting activation of the innate immune system. Moreover, abundances of cytoskeletal and mucin proteins increased later in the time course, suggestive of subsequent adjustment to the increased microbial load. Our study provides the first snapshot of coordinated human and microbial protein expression in the infant gut during early development.

  3. Resting-State Time-Varying Analysis Reveals Aberrant Variations of Functional Connectivity in Autism.

    PubMed

    Yao, Zhijun; Hu, Bin; Xie, Yuanwei; Zheng, Fang; Liu, Guangyao; Chen, Xuejiao; Zheng, Weihao

    2016-01-01

    Recently, studies based on time-varying functional connectivity have unveiled brain states diversity in some neuropsychiatric disorders, such as schizophrenia and major depressive disorder. However, time-varying functional connectivity analysis of resting-state functional Magnetic Resonance Imaging (fMRI) have been rarely performed on the Autism Spectrum Disorder (ASD). Hence, we performed time-varying connectivity analysis on resting-state fMRI data to investigate brain states mutation in ASD children. ASD showed an imbalance of connectivity state and aberrant ratio of connectivity with different strengths in the whole brain network, and decreased connectivity associated precuneus/posterior cingulate gyrus with medial prefrontal gyrus in default mode network. As compared to typical development children, weak relevance condition (the strength of a large number of connectivities in the state was less than means minus standard deviation of all connection strength) was maintained for a longer time between brain areas of ASD children, and ratios of weak connectivity in brain states varied dramatically in the ASD. In the ASD, the abnormal brain state might be related to repetitive behaviors and stereotypical interests, and macroscopically reflect disruption of gamma-aminobutyric acid at the cellular level. The detection of brain states based on time-varying functional connectivity analysis of resting-state fMRI might be conducive for diagnosis and early intervention of ASD before obvious clinical symptoms.

  4. fMRI of Cocaine Self-Administration in Macaques Reveals Functional Inhibition of Basal Ganglia

    PubMed Central

    Mandeville, Joseph B; Choi, Ji-Kyung; Jarraya, Bechir; Rosen, Bruce R; Jenkins, Bruce G; Vanduffel, Wim

    2011-01-01

    Disparities in cocaine-induced neurochemical and metabolic responses between human beings and rodents motivate the use of non-human primates (NHP) to model consequences of repeated cocaine exposure in human subjects. To characterize the functional response to cocaine infusion in NHP brain, we employed contrast-enhanced fMRI during both non-contingent injection of drug and self-administration of cocaine in the magnet. Cocaine robustly decreased cerebral blood volume (CBV) throughout basal ganglia and motor/pre-motor cortex and produced subtle functional inhibition of prefrontal cortex. No brain regions exhibited significant elevation of CBV in response to cocaine challenge. Theses effects in NHP brain are opposite in sign to the cocaine-induced fMRI response in rats, but consistent with previous measurements in NHP based on glucose metabolism. Because the striatal ratio of D2 to D1 receptors is larger in human beings and NHP than rats, we hypothesize that the inhibitory effects of D2 receptor binding dominate the functional response in primates, whereas excitatory D1 receptor stimulation predominates in the rat. If the NHP accurately models the human response to cocaine, downregulation of D2 receptors in human cocaine-abusing populations can be expected to blunt cocaine-induced functional responses, contributing to the weak and variable fMRI responses reported in human basal ganglia following cocaine infusion. PMID:21307843

  5. Resting state functional MRI reveals abnormal network connectivity in neurofibromatosis 1.

    PubMed

    Tomson, Steffie N; Schreiner, Matthew J; Narayan, Manjari; Rosser, Tena; Enrique, Nicole; Silva, Alcino J; Allen, Genevera I; Bookheimer, Susan Y; Bearden, Carrie E

    2015-11-01

    Neurofibromatosis type I (NF1) is a genetic disorder caused by mutations in the neurofibromin 1 gene at locus 17q11.2. Individuals with NF1 have an increased incidence of learning disabilities, attention deficits, and autism spectrum disorders. As a single-gene disorder, NF1 represents a valuable model for understanding gene-brain-behavior relationships. While mouse models have elucidated molecular and cellular mechanisms underlying learning deficits associated with this mutation, little is known about functional brain architecture in human subjects with NF1. To address this question, we used resting state functional connectivity magnetic resonance imaging (rs-fcMRI) to elucidate the intrinsic network structure of 30 NF1 participants compared with 30 healthy demographically matched controls during an eyes-open rs-fcMRI scan. Novel statistical methods were employed to quantify differences in local connectivity (edge strength) and modularity structure, in combination with traditional global graph theory applications. Our findings suggest that individuals with NF1 have reduced anterior-posterior connectivity, weaker bilateral edges, and altered modularity clustering relative to healthy controls. Further, edge strength and modular clustering indices were correlated with IQ and internalizing symptoms. These findings suggest that Ras signaling disruption may lead to abnormal functional brain connectivity; further investigation into the functional consequences of these alterations in both humans and in animal models is warranted.

  6. Functional Ecological Gene Networks to Reveal the Changes Among Microbial Interactions Under Elevated Carbon Dioxide Conditions

    SciTech Connect

    Deng, Ye; Zhou, Jizhong; Luo, Feng; He, Zhili; Tu, Qichao; Zhi, Xiaoyang

    2010-05-17

    Biodiversity and its responses to environmental changes is a central issue in ecology, and for society. Almost all microbial biodiversity researches focus on species richness and abundance but ignore the interactions among different microbial species/populations. However, determining the interactions and their relationships to environmental changes in microbial communities is a grand challenge, primarily due to the lack of information on the network structure among different microbial species/populations. Here, a novel random matrix theory (RMT)-based conceptual framework for identifying functional ecological gene networks (fEGNs) is developed with the high throughput functional gene array hybridization data from the grassland microbial communities in a long-term FACE (Free Air CO2 Enrichment) experiment. Both fEGNs under elevated CO2 (eCO2) and ambient CO2 (aCO2) possessed general characteristics of many complex systems such as scale-free, small-world, modular and hierarchical. However, the topological structure of the fEGNs is distinctly different between eCO2 and aCO2, suggesting that eCO2 dramatically altered the interactions among different microbial functional groups/populations. In addition, the changes in network structure were significantly correlated with soil carbon and nitrogen dynamics, and plant productivity, indicating the potential importance of network interactions in ecosystem functioning. Elucidating network interactions in microbial communities and their responses to environmental changes are fundamentally important for research in microbial ecology, systems microbiology, and global change.

  7. Functional Screening of Hydrolytic Activities Reveals an Extremely Thermostable Cellulase from a Deep-Sea Archaeon

    PubMed Central

    Leis, Benedikt; Heinze, Simon; Angelov, Angel; Pham, Vu Thuy Trang; Thürmer, Andrea; Jebbar, Mohamed; Golyshin, Peter N.; Streit, Wolfgang R.; Daniel, Rolf; Liebl, Wolfgang

    2015-01-01

    Extreme habitats serve as a source of enzymes that are active under extreme conditions and are candidates for industrial applications. In this work, six large-insert mixed genomic libraries were screened for hydrolase activities in a broad temperature range (8–70°C). Among a variety of hydrolytic activities, one fosmid clone, derived from a library of pooled isolates of hyperthermophilic archaea from deep sea vents, displayed hydrolytic activity on carboxymethyl cellulose substrate plates at 70°C but not at lower temperatures. Sequence analysis of the fosmid insert revealed a gene encoding a novel glycoside hydrolase family 12 (GHF12) endo-1,4-β-glucanase, termed Cel12E. The enzyme shares 45% sequence identity with a protein from the archaeon Thermococcus sp. AM4 and displays a unique multidomain architecture. Biochemical characterization of Cel12E revealed a remarkably thermostable protein, which appears to be of archaeal origin. The enzyme displayed maximum activity at 92°C and was active on a variety of linear 1,4-β-glucans like carboxymethyl cellulose, β-glucan, lichenan, and phosphoric acid swollen cellulose. The protein is able to bind to various insoluble β-glucans. Product pattern analysis indicated that Cel12E is an endo-cleaving β-glucanase. Cel12E expands the toolbox of hyperthermostable archaeal cellulases with biotechnological potential. PMID:26191525

  8. Replicated landscape genetic and network analyses reveal wide variation in functional connectivity for American pikas.

    PubMed

    Castillo, Jessica A; Epps, Clinton W; Jeffress, Mackenzie R; Ray, Chris; Rodhouse, Thomas J; Schwalm, Donelle

    2016-09-01

    Landscape connectivity is essential for maintaining viable populations, particularly for species restricted to fragmented habitats or naturally arrayed in metapopulations and facing rapid climate change. The importance of assessing both structural connectivity (physical distribution of favorable habitat patches) and functional connectivity (how species move among habitat patches) for managing such species is well understood. However, the degree to which functional connectivity for a species varies among landscapes, and the resulting implications for conservation, have rarely been assessed. We used a landscape genetics approach to evaluate resistance to gene flow and, thus, to determine how landscape and climate-related variables influence gene flow for American pikas (Ochotona princeps) in eight federally managed sites in the western United States. We used empirically derived, individual-based landscape resistance models in conjunction with predictive occupancy models to generate patch-based network models describing functional landscape connectivity. Metareplication across landscapes enabled identification of limiting factors for dispersal that would not otherwise have been apparent. Despite the cool microclimates characteristic of pika habitat, south-facing aspects consistently represented higher resistance to movement, supporting the previous hypothesis that exposure to relatively high temperatures may limit dispersal in American pikas. We found that other barriers to dispersal included areas with a high degree of topographic relief, such as cliffs and ravines, as well as streams and distances greater than 1-4 km depending on the site. Using the empirically derived network models of habitat patch connectivity, we identified habitat patches that were likely disproportionately important for maintaining functional connectivity, areas in which habitat appeared fragmented, and locations that could be targeted for management actions to improve functional connectivity

  9. Mutagenesis of conserved amino acids of Helicobacter pylori fur reveals residues important for function.

    PubMed

    Carpenter, Beth M; Gancz, Hanan; Benoit, Stéphane L; Evans, Sarah; Olsen, Cara H; Michel, Sarah L J; Maier, Robert J; Merrell, D Scott

    2010-10-01

    The ferric uptake regulator (Fur) of the medically important pathogen Helicobacter pylori is unique in that it has been shown to function as a repressor both in the presence of an Fe2+ cofactor and in its apo (non-Fe2+-bound) form. However, virtually nothing is known concerning the amino acid residues that are important for Fur functioning. Therefore, mutations in six conserved amino acid residues of H. pylori Fur were constructed and analyzed for their impact on both iron-bound and apo repression. In addition, accumulation of the mutant proteins, protein secondary structure, DNA binding ability, iron binding capacity, and the ability to form higher-order structures were also examined for each mutant protein. While none of the mutated residues completely abrogated the function of Fur, we were able to identify residues that were critical for both iron-bound and apo-Fur repression. One mutation, V64A, did not alter regulation of any target genes. However, each of the five remaining mutations showed an effect on either iron-bound or apo regulation. Of these, H96A, E110A, and E117A mutations altered iron-bound Fur regulation and were all shown to influence iron binding to different extents. Additionally, the H96A mutation was shown to alter Fur oligomerization, and the E110A mutation was shown to impact oligomerization and DNA binding. Conversely, the H134A mutant exhibited changes in apo-Fur regulation that were the result of alterations in DNA binding. Although the E90A mutant exhibited alterations in apo-Fur regulation, this mutation did not affect any of the assessed protein functions. This study is the first for H. pylori to analyze the roles of specific amino acid residues of Fur in function and continues to highlight the complexity of Fur regulation in this organism.

  10. Functional genomic screen and network analysis reveal novel modifiers of tauopathy dissociated from tau phosphorylation

    PubMed Central

    Ambegaokar, Surendra S.; Jackson, George R.

    2011-01-01

    A functional genetic screen using loss-of-function and gain-of-function alleles was performed to identify modifiers of tau-induced neurotoxicity using the 2N/4R (full-length) isoform of wild-type human tau expressed in the fly retina. We previously reported eye pigment mutations, which create dysfunctional lysosomes, as potent modifiers; here, we report 37 additional genes identified from ∼1900 genes screened, including the kinases shaggy/GSK-3beta, par-1/MARK, CamKI and Mekk1. Tau acts synergistically with Mekk1 and p38 to down-regulate extracellular regulated kinase activity, with a corresponding decrease in AT8 immunoreactivity (pS202/T205), suggesting that tau can participate in signaling pathways to regulate its own kinases. Modifiers showed poor correlation with tau phosphorylation (using the AT8, 12E8 and AT270 epitopes); moreover, tested suppressors of wild-type tau were equally effective in suppressing toxicity of a phosphorylation-resistant S11A tau construct, demonstrating that changes in tau phosphorylation state are not required to suppress or enhance its toxicity. Genes related to autophagy, the cell cycle, RNA-associated proteins and chromatin-binding proteins constitute a large percentage of identified modifiers. Other functional categories identified include mitochondrial proteins, lipid trafficking, Golgi proteins, kinesins and dynein and the Hsp70/Hsp90-organizing protein (Hop). Network analysis uncovered several other genes highly associated with the functional modifiers, including genes related to the PI3K, Notch, BMP/TGF-β and Hedgehog pathways, and nuclear trafficking. Activity of GSK-3β is strongly upregulated due to TDP-43 expression, and reduced GSK-3β dosage is also a common suppressor of Aβ42 and TDP-43 toxicity. These findings suggest therapeutic targets other than mitigation of tau phosphorylation. PMID:21949350

  11. Can sliding-window correlations reveal dynamic functional connectivity in resting-state fMRI?

    PubMed

    Hindriks, R; Adhikari, M H; Murayama, Y; Ganzetti, M; Mantini, D; Logothetis, N K; Deco, G

    2016-02-15

    During the last several years, the focus of research on resting-state functional magnetic resonance imaging (fMRI) has shifted from the analysis of functional connectivity averaged over the duration of scanning sessions to the analysis of changes of functional connectivity within sessions. Although several studies have reported the presence of dynamic functional connectivity (dFC), statistical assessment of the results is not always carried out in a sound way and, in some studies, is even omitted. In this study, we explain why appropriate statistical tests are needed to detect dFC, we describe how they can be carried out and how to assess the performance of dFC measures, and we illustrate the methodology using spontaneous blood-oxygen level-dependent (BOLD) fMRI recordings of macaque monkeys under general anesthesia and in human subjects under resting-state conditions. We mainly focus on sliding-window correlations since these are most widely used in assessing dFC, but also consider a recently proposed non-linear measure. The simulations and methodology, however, are general and can be applied to any measure. The results are twofold. First, through simulations, we show that in typical resting-state sessions of 10 min, it is almost impossible to detect dFC using sliding-window correlations. This prediction is validated by both the macaque and the human data: in none of the individual recording sessions was evidence for dFC found. Second, detection power can be considerably increased by session- or subject-averaging of the measures. In doing so, we found that most of the functional connections are in fact dynamic. With this study, we hope to raise awareness of the statistical pitfalls in the assessment of dFC and how they can be avoided by using appropriate statistical methods.

  12. Distinct Functional Roles of Cardiac Mitochondrial Subpopulations Revealed by a 3D Simulation Model

    PubMed Central

    Hatano, Asuka; Okada, Jun-ichi; Washio, Takumi; Hisada, Toshiaki; Sugiura, Seiryo

    2015-01-01

    Experimental characterization of two cardiac mitochondrial subpopulations, namely, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM), has been hampered by technical difficulties, and an alternative approach is eagerly awaited. We previously developed a three-dimensional computational cardiomyocyte model that integrates electrophysiology, metabolism, and mechanics with subcellular structure. In this study, we further developed our model to include intracellular oxygen diffusion, and determined whether mitochondrial localization or intrinsic properties cause functional variations. For this purpose, we created two models: one with equal SSM and IFM properties and one with IFM having higher activity levels. Using these two models to compare the SSM and IFM responses of [Ca2+], tricarboxylic acid cycle activity, [NADH], and mitochondrial inner membrane potential to abrupt changes in pacing frequency (0.25–2 Hz), we found that the reported functional differences between these subpopulations appear to be mostly related to local [Ca2+] heterogeneity, and variations in intrinsic properties only serve to augment these differences. We also examined the effect of hypoxia on mitochondrial function. Under normoxic conditions, intracellular oxygen is much higher throughout the cell than the half-saturation concentration for oxidative phosphorylation. However, under limited oxygen supply, oxygen is mostly exhausted in SSM, leaving the core region in an anoxic condition. Reflecting this heterogeneous oxygen environment, the inner membrane potential continues to decrease in IFM, whereas it is maintained to nearly normal levels in SSM, thereby ensuring ATP supply to this region. Our simulation results provide clues to understanding the origin of functional variations in two cardiac mitochondrial subpopulations and their differential roles in maintaining cardiomyocyte function as a whole. PMID:26039174

  13. Characterization of 4-HNE Modified L-FABP Reveals Alterations in Structural and Functional Dynamics

    PubMed Central

    Smathers, Rebecca L.; Fritz, Kristofer S.; Galligan, James J.; Shearn, Colin T.; Reigan, Philip; Marks, Michael J.; Petersen, Dennis R.

    2012-01-01

    4-Hydroxynonenal (4-HNE) is a reactive α,β-unsaturated aldehyde produced during oxidative stress and subsequent lipid peroxidation of polyunsaturated fatty acids. The reactivity of 4-HNE towards DNA and nucleophilic amino acids has been well established. In this report, using proteomic approaches, liver fatty acid-binding protein (L-FABP) is identified as a target for modification by 4-HNE. This lipid binding protein mediates the uptake and trafficking of hydrophobic ligands throughout cellular compartments. Ethanol caused a significant decrease in L-FABP protein (P<0.001) and mRNA (P<0.05), as well as increased poly-ubiquitinated L-FABP (P<0.001). Sites of 4-HNE adduction on mouse recombinant L-FABP were mapped using MALDI-TOF/TOF mass spectrometry on apo (Lys57 and Cys69) and holo (Lys6, Lys31, His43, Lys46, Lys57 and Cys69) L-FABP. The impact of 4-HNE adduction was found to occur in a concentration-dependent manner; affinity for the fluorescent ligand, anilinonaphthalene-8-sulfonic acid, was reduced from 0.347 µM to Kd1 = 0.395 µM and Kd2 = 34.20 µM. Saturation analyses revealed that capacity for ligand is reduced by approximately 50% when adducted by 4-HNE. Thermal stability curves of apo L-FABP was also found to be significantly affected by 4-HNE adduction (ΔTm = 5.44°C, P<0.01). Computational-based molecular modeling simulations of adducted protein revealed minor conformational changes in global protein structure of apo and holo L-FABP while more apparent differences were observed within the internal binding pocket, revealing reduced area and structural integrity. New solvent accessible portals on the periphery of the protein were observed following 4-HNE modification in both the apo and holo state, suggesting an adaptive response to carbonylation. The results from this study detail the dynamic process associated with L-FABP modification by 4-HNE and provide insight as to how alterations in structural integrity and ligand binding may a

  14. Community Structure Reveals Biologically Functional Modules in MEF2C Transcriptional Regulatory Network

    PubMed Central

    Alcalá-Corona, Sergio A.; Velázquez-Caldelas, Tadeo E.; Espinal-Enríquez, Jesús; Hernández-Lemus, Enrique

    2016-01-01

    Gene regulatory networks are useful to understand the activity behind the complex mechanisms in transcriptional regulation. A main goal in contemporary biology is using such networks to understand the systemic regulation of gene expression. In this work, we carried out a systematic study of a transcriptional regulatory network derived from a comprehensive selection of all potential transcription factor interactions downstream from MEF2C, a human transcription factor master regulator. By analyzing the connectivity structure of such network, we were able to find different biologically functional processes and specific biochemical pathways statistically enriched in communities of genes into the network, such processes are related to cell signaling, cell cycle and metabolism. In this way we further support the hypothesis that structural properties of biological networks encode an important part of their functional behavior in eukaryotic cells. PMID:27252657

  15. Single-cell RNA sequencing reveals molecular and functional platelet bias of aged haematopoietic stem cells

    PubMed Central

    Grover, Amit; Sanjuan-Pla, Alejandra; Thongjuea, Supat; Carrelha, Joana; Giustacchini, Alice; Gambardella, Adriana; Macaulay, Iain; Mancini, Elena; Luis, Tiago C.; Mead, Adam; Jacobsen, Sten Eirik W.; Nerlov, Claus

    2016-01-01

    Aged haematopoietic stem cells (HSCs) generate more myeloid cells and fewer lymphoid cells compared with young HSCs, contributing to decreased adaptive immunity in aged individuals. However, it is not known how intrinsic changes to HSCs and shifts in the balance between biased HSC subsets each contribute to the altered lineage output. Here, by analysing HSC transcriptomes and HSC function at the single-cell level, we identify increased molecular platelet priming and functional platelet bias as the predominant age-dependent change to HSCs, including a significant increase in a previously unrecognized class of HSCs that exclusively produce platelets. Depletion of HSC platelet programming through loss of the FOG-1 transcription factor is accompanied by increased lymphoid output. Therefore, increased platelet bias may contribute to the age-associated decrease in lymphopoiesis. PMID:27009448

  16. Conserved intergenic sequences revealed by CTAG-profiling in Salmonella: thermodynamic modeling for function prediction

    PubMed Central

    Tang, Le; Zhu, Songling; Mastriani, Emilio; Fang, Xin; Zhou, Yu-Jie; Li, Yong-Guo; Johnston, Randal N.; Guo, Zheng; Liu, Gui-Rong; Liu, Shu-Lin

    2017-01-01

    Highly conserved short sequences help identify functional genomic regions and facilitate genomic annotation. We used Salmonella as the model to search the genome for evolutionarily conserved regions and focused on the tetranucleotide sequence CTAG for its potentially important functions. In Salmonella, CTAG is highly conserved across the lineages and large numbers of CTAG-containing short sequences fall in intergenic regions, strongly indicating their biological importance. Computer modeling demonstrated stable stem-loop structures in some of the CTAG-containing intergenic regions, and substitution of a nucleotide of the CTAG sequence would radically rearrange the free energy and disrupt the structure. The postulated degeneration of CTAG takes distinct patterns among Salmonella lineages and provides novel information about genomic divergence and evolution of these bacterial pathogens. Comparison of the vertically and horizontally transmitted genomic segments showed different CTAG distribution landscapes, with the genome amelioration process to remove CTAG taking place inward from both terminals of the horizontally acquired segment. PMID:28262684

  17. Functional chromatography reveals three natural products that target the same protein with distinct mechanisms of action

    PubMed Central

    Kang, MinJin; Wu, Tongde; Wijeratne, E. M. Kithsiri; Lau, Eric C.; Mason, Damian J.; Mesa, Celestina; Tillotson, Joseph; Zhang, Donna D.; Gunatilaka, A. A. Leslie; La Clair, James J.

    2014-01-01

    Access to lead compounds with defined molecular targets continues to be a barrier to the translation of natural product resources. As a solution, we have developed a system that uses discreet, recombinant proteins as the vehicles for natural product isolation. Here, we describe the use of this functional chromatographic method to identify natural products that bind to the AAA+ chaperone, p97, a promising cancer target. Application of this method to a panel of fungal and plant extracts identified rheoemodin, 1-hydroxydehydroherbarin and phomapyrrolidone A as distinct p97 modulators. Excitingly, each of these molecules displayed a unique mechanism of p97 modulation. This discovery provides strong support for the application of functional chromatography to the discovery of protein modulators that would likely escape traditional high-throughput or phenotypic screening platforms. PMID:25125376

  18. High throughput estimation of functional cell activities reveals disease mechanisms and predicts relevant clinical outcomes.

    PubMed

    Hidalgo, Marta R; Cubuk, Cankut; Amadoz, Alicia; Salavert, Francisco; Carbonell-Caballero, José; Dopazo, Joaquin

    2017-01-17

    Understanding the aspects of the cell functionality that account for disease or drug action mechanisms is a main challenge for precision medicine. Here we propose a new method that models cell signaling using biological knowledge on signal transduction. The method recodes individual gene expression values (and/or gene mutations) into accurate measurements of changes in the activity of signaling circuits, which ultimately constitute high-throughput estimations of cell functionalities caused by gene activity within the pathway. Moreover, such estimations can be obtained either at cohort-level, in case/control comparisons, or personalized for individual patients. The accuracy of the method is demonstrated in an extensive analysis involving 5640 patients from 12 different cancer types. Circuit activity measurements not only have a high diagnostic value but also can be related to relevant disease outcomes such as survival, and can be used to assess therapeutic interventions.

  19. Conserved intergenic sequences revealed by CTAG-profiling in Salmonella: thermodynamic modeling for function prediction

    NASA Astrophysics Data System (ADS)

    Tang, Le; Zhu, Songling; Mastriani, Emilio; Fang, Xin; Zhou, Yu-Jie; Li, Yong-Guo; Johnston, Randal N.; Guo, Zheng; Liu, Gui-Rong; Liu, Shu-Lin

    2017-03-01

    Highly conserved short sequences help identify functional genomic regions and facilitate genomic annotation. We used Salmonella as the model to search the genome for evolutionarily conserved regions and focused on the tetranucleotide sequence CTAG for its potentially important functions. In Salmonella, CTAG is highly conserved across the lineages and large numbers of CTAG-containing short sequences fall in intergenic regions, strongly indicating their biological importance. Computer modeling demonstrated stable stem-loop structures in some of the CTAG-containing intergenic regions, and substitution of a nucleotide of the CTAG sequence would radically rearrange the free energy and disrupt the structure. The postulated degeneration of CTAG takes distinct patterns among Salmonella lineages and provides novel information about genomic divergence and evolution of these bacterial pathogens. Comparison of the vertically and horizontally transmitted genomic segments showed different CTAG distribution landscapes, with the genome amelioration process to remove CTAG taking place inward from both terminals of the horizontally acquired segment.

  20. Conserved intergenic sequences revealed by CTAG-profiling in Salmonella: thermodynamic modeling for function prediction.

    PubMed

    Tang, Le; Zhu, Songling; Mastriani, Emilio; Fang, Xin; Zhou, Yu-Jie; Li, Yong-Guo; Johnston, Randal N; Guo, Zheng; Liu, Gui-Rong; Liu, Shu-Lin

    2017-03-06

    Highly conserved short sequences help identify functional genomic regions and facilitate genomic annotation. We used Salmonella as the model to search the genome for evolutionarily conserved regions and focused on the tetranucleotide sequence CTAG for its potentially important functions. In Salmonella, CTAG is highly conserved across the lineages and large numbers of CTAG-containing short sequences fall in intergenic regions, strongly indicating their biological importance. Computer modeling demonstrated stable stem-loop structures in some of the CTAG-containing intergenic regions, and substitution of a nucleotide of the CTAG sequence would radically rearrange the free energy and disrupt the structure. The postulated degeneration of CTAG takes distinct patterns among Salmonella lineages and provides novel information about genomic divergence and evolution of these bacterial pathogens. Comparison of the vertically and horizontally transmitted genomic segments showed different CTAG distribution landscapes, with the genome amelioration process to remove CTAG taking place inward from both terminals of the horizontally acquired segment.

  1. Amino acid coevolution reveals three-dimensional structure and functional domains of insect odorant receptors.

    PubMed

    Hopf, Thomas A; Morinaga, Satoshi; Ihara, Sayoko; Touhara, Kazushige; Marks, Debora S; Benton, Richard

    2015-01-13

    Insect odorant receptors (ORs) comprise an enormous protein family that translates environmental chemical signals into neuronal electrical activity. These heptahelical receptors are proposed to function as ligand-gated ion channels and/or to act metabotropically as G protein-coupled receptors (GPCRs). Resolving their signalling mechanism has been hampered by the lack of tertiary structural information and primary sequence similarity to other proteins. We use amino acid evolutionary covariation across these ORs to define restraints on structural proximity of residue pairs, which permit de novo generation of three-dimensional models. The validity of our analysis is supported by the location of functionally important residues in highly constrained regions of the protein. Importantly, insect OR models exhibit a distinct transmembrane domain packing arrangement to that of canonical GPCRs, establishing the structural unrelatedness of these receptor families. The evolutionary couplings and models predict odour binding and ion conduction domains, and provide a template for rationale structure-activity dissection.

  2. Quantitative analysis of proteome and lipidome dynamics reveals functional regulation of global lipid metabolism.

    PubMed

    Casanovas, Albert; Sprenger, Richard R; Tarasov, Kirill; Ruckerbauer, David E; Hannibal-Bach, Hans Kristian; Zanghellini, Jürgen; Jensen, Ole N; Ejsing, Christer S

    2015-03-19

    Elucidating how and to what extent lipid metabolism is remodeled under changing conditions is essential for understanding cellular physiology. Here, we analyzed proteome and lipidome dynamics to investigate how regulation of lipid metabolism at the global scale supports remodeling of cellular architecture and processes during physiological adaptations in yeast. Our results reveal that activation of cardiolipin synthesis and remodeling supports mitochondrial biogenesis in the transition from fermentative to respiratory metabolism, that down-regulation of de novo sterol synthesis machinery prompts differential turnover of lipid droplet-associated triacylglycerols and sterol esters during respiratory growth, that sphingolipid metabolism is regulated in a previously unrecognized growth stage-specific manner, and that endogenous synthesis of unsaturated fatty acids constitutes an in vivo upstream activator of peroxisomal biogenesis, via the heterodimeric Oaf1/Pip2 transcription factor. Our work demonstrates the pivotal role of lipid metabolism in adaptive processes and provides a resource to investigate its regulation at the cellular level.

  3. The organization of thinking: what functional brain imaging reveals about the neuroarchitecture of complex cognition.

    PubMed

    Just, Marcel Adam; Varma, Sashank

    2007-09-01

    Recent findings in brain imaging, particularly in fMRI, are beginning to reveal some of the fundamental properties of the organization of the cortical systems that underpin complex cognition. We propose an emerging set of operating principles that govern this organization, characterizing the system as a set of collaborating cortical centers that operate as a large-scale cortical network. Two of the network's critical features are that it is resource constrained and dynamically configured, with resource constraints and demands dynamically shaping the network topology. The operating principles are embodied in a cognitive neuroarchitecture, 4CAPS, consisting of a number of interacting computational centers that correspond to activating cortical areas. Each 4CAPS center is a hybrid production system, possessing both symbolic and connectionist attributes. We describe 4CAPS models of sentence comprehension, spatial problem solving, and complex multitasking and compare the accounts of these models with brain activation and behavioral results. Finally, we compare 4CAPS with other proposed neuroarchitectures.

  4. Systematic perturbation of cytoskeletal function reveals a linear scaling relationship between cell geometry and fitness.

    PubMed

    Monds, Russell D; Lee, Timothy K; Colavin, Alexandre; Ursell, Tristan; Quan, Selwyn; Cooper, Tim F; Huang, Kerwyn Casey

    2014-11-20

    Diversification of cell size is hypothesized to have occurred through a process of evolutionary optimization, but direct demonstrations of causal relationships between cell geometry and fitness are lacking. Here, we identify a mutation from a laboratory-evolved bacterium that dramatically increases cell size through cytoskeletal perturbation and confers a large fitness advantage. We engineer a library of cytoskeletal mutants of different sizes and show that fitness scales linearly with respect to cell size over a wide physiological range. Quantification of the growth rates of single cells during the exit from stationary phase reveals that transitions between "feast-or-famine" growth regimes are a key determinant of cell-size-dependent fitness effects. We also uncover environments that suppress the fitness advantage of larger cells, indicating that cell-size-dependent fitness effects are subject to both biophysical and metabolic constraints. Together, our results highlight laboratory-based evolution as a powerful framework for studying the quantitative relationships between morphology and fitness.

  5. Functional photoreceptor loss revealed with adaptive optics: an alternate cause of color blindness.

    PubMed

    Carroll, Joseph; Neitz, Maureen; Hofer, Heidi; Neitz, Jay; Williams, David R

    2004-06-01

    There is enormous variation in the X-linked L/M (long/middle wavelength sensitive) gene array underlying "normal" color vision in humans. This variability has been shown to underlie individual variation in color matching behavior. Recently, red-green color blindness has also been shown to be associated with distinctly different genotypes. This has opened the possibility that there may be important phenotypic differences within classically defined groups of color blind individuals. Here, adaptive optics retinal imaging has revealed a mechanism for producing dichromatic color vision in which the expression of a mutant cone photopigment gene leads to the loss of the entire corresponding class of cone photoreceptor cells. Previously, the theory that common forms of inherited color blindness could be caused by the loss of photoreceptor cells had been discounted. We confirm that remarkably, this loss of one-third of the cones does not impair any aspect of vision other than color.

  6. Structure–function insights reveal the human ribosome as a cancer target for antibiotics

    PubMed Central

    Myasnikov, Alexander G.; Kundhavai Natchiar, S.; Nebout, Marielle; Hazemann, Isabelle; Imbert, Véronique; Khatter, Heena; Peyron, Jean-François; Klaholz, Bruno P.

    2016-01-01

    Many antibiotics in clinical use target the bacterial ribosome by interfering with the protein synthesis machinery. However, targeting the human ribosome in the case of protein synthesis deregulations such as in highly proliferating cancer cells has not been investigated at the molecular level up to now. Here we report the structure of the human 80S ribosome with a eukaryote-specific antibiotic and show its anti-proliferative effect on several cancer cell lines. The structure provides insights into the detailed interactions in a ligand-binding pocket of the human ribosome that are required for structure-assisted drug design. Furthermore, anti-proliferative dose response in leukaemic cells and interference with synthesis of c-myc and mcl-1 short-lived protein markers reveals specificity of a series of eukaryote-specific antibiotics towards cytosolic rather than mitochondrial ribosomes, uncovering the human ribosome as a promising cancer target. PMID:27665925

  7. Exome sequencing reveals germline gain-of-function EGFR mutation in an adult with Lhermitte–Duclos disease

    PubMed Central

    Colby, Samantha; Yehia, Lamis; Niazi, Farshad; Chen, JinLian; Ni, Ying

    2016-01-01

    Lhermitte–Duclos disease (LDD) is a rare cerebellar disorder believed to be pathognomonic for Cowden syndrome. Presently, the only known etiology is germline PTEN mutation. We report a 41-yr-old white female diagnosed with LDD and wild-type for PTEN. Exome sequencing revealed a germline heterozygous EGFR mutation that breaks a disulfide bond in the receptor's extracellular domain, resulting in constitutive activation. Functional studies demonstrate activation of ERK/AKT signaling pathways, mimicking PTEN loss-of-function downstream effects. The identification of EGFR as a candidate LDD susceptibility gene contributes to advancement of molecular diagnosis and targeted therapy for this rare condition with limited treatment options. PMID:27900366

  8. Functional metagenomics reveals novel β-galactosidases not predictable from gene sequences

    PubMed Central

    Cheng, Jiujun; Romantsov, Tatyana; Engel, Katja; Doxey, Andrew C.; Rose, David R.; Neufeld, Josh D.

    2017-01-01

    The techniques of metagenomics have allowed researchers to access the genomic potential of uncultivated microbes, but there remain significant barriers to determination of gene function based on DNA sequence alone. Functional metagenomics, in which DNA is cloned and expressed in surrogate hosts, can overcome these barriers, and make important contributions to the discovery of novel enzymes. In this study, a soil metagenomic library carried in an IncP cosmid was used for functional complementation for β-galactosidase activity in both Sinorhizobium meliloti (α-Proteobacteria) and Escherichia coli (γ-Proteobacteria) backgrounds. One β-galactosidase, encoded by six overlapping clones that were selected in both hosts, was identified as a member of glycoside hydrolase family 2. We could not identify ORFs obviously encoding possible β-galactosidases in 19 other sequenced clones that were only able to complement S. meliloti. Based on low sequence identity to other known glycoside hydrolases, yet not β-galactosidases, three of these ORFs were examined further. Biochemical analysis confirmed that all three encoded β-galactosidase activity. Lac36W_ORF11 and Lac161_ORF7 had conserved domains, but lacked similarities to known glycoside hydrolases. Lac161_ORF10 had neither conserved domains nor similarity to known glycoside hydrolases. Bioinformatic and structural modeling implied that Lac161_ORF10 protein represented a novel enzyme family with a five-bladed propeller glycoside hydrolase domain. By discovering founding members of three novel β-galactosidase families, we have reinforced the value of functional metagenomics for isolating novel genes that could not have been predicted from DNA sequence analysis alone. PMID:28273103

  9. Reconstitution of hepatic uricase in planar lipid bilayer reveals a functional organic anion channel.

    PubMed

    Leal-Pinto, E; London, R D; Knorr, B A; Abramson, R G

    1995-07-01

    Rat renal proximal tubule cell membranes have been reported to contain uricase-like proteins that function as electrogenic urate transporters. Although uricase, per se, has only been detected within peroxisomes in rat liver (where it functions as an oxidative enzyme) this protein has been shown to function as a urate transport protein when inserted into liposomes. Since both the uricase-like renal protein and hepatic uricase can transport urate, reconstitution studies were performed to further characterize the mechanism by which uricase may function as a transport protein. Ion channel activity was evaluated in planar lipid bilayers before and after fusion of uricase-containing proteoliposomes. In the presence of symmetrical solutions of urate and KCl, but absence of uricase, no current was generated when the voltage was ramped between +/- 100 mV. Following fusion of uricase with the bilayer, single channel activity was evident: the reconstituted channel rectified with a mean slope conductance of 8 pS, displayed voltage sensitivity, and demonstrated a marked selectivity for urate relative to K+ and Cl-. The channel was more selective to oxonate, an inhibitor of both enzymatic uricase activity and urate transport, than urate and it was equally selective to urate and pyrazinoate, an inhibitor of urate transport. With time, pyrazinoate blocked both its own movement and the movement of urate through the channel. Channel activity was also blocked by the IgG fraction of a polyclonal antibody to affinity purified pig liver uricase. These studies demonstrate that a highly selective, voltage dependent organic anion channel is formed when a purified preparation of uricase is reconstituted in lipid bilayers.

  10. The analysis of heterotaxy patients reveals new loss-of-function variants of GRK5

    PubMed Central

    Lessel, Davor; Muhammad, Tariq; Casar Tena, Teresa; Moepps, Barbara; Burkhalter, Martin D.; Hitz, Marc-Phillip; Toka, Okan; Rentzsch, Axel; Schubert, Stephan; Schalinski, Adelheid; Bauer, Ulrike M. M.; Kubisch, Christian; Ware, Stephanie M.; Philipp, Melanie

    2016-01-01

    G protein-coupled receptor kinase 5 (GRK5) is a regulator of cardiac performance and a potential therapeutic target in heart failure in the adult. Additionally, we have previously classified GRK5 as a determinant of left-right asymmetry and proper heart development using zebrafish. We thus aimed to identify GRK5 variants of functional significance by analysing 187 individuals with laterality defects (heterotaxy) that were associated with a congenital heart defect (CHD). Using Sanger sequencing we identified two moderately frequent variants in GRK5 with minor allele frequencies <10%, and seven very rare polymorphisms with minor allele frequencies <1%, two of which are novel variants. Given their evolutionarily conserved position in zebrafish, in-depth functional characterisation of four variants (p.Q41L, p.G298S, p.R304C and p.T425M) was performed. We tested the effects of these variants on normal subcellular localisation and the ability to desensitise receptor signalling as well as their ability to correct the left-right asymmetry defect upon Grk5l knockdown in zebrafish. While p.Q41L, p.R304C and p.T425M responded normally in the first two aspects, neither p.Q41L nor p.R304C were capable of rescuing the lateralisation phenotype. The fourth variant, p.G298S was identified as a complete loss-of-function variant in all assays and provides insight into the functions of GRK5. PMID:27618959

  11. The Structure of a Gene Co-Expression Network Reveals Biological Functions Underlying eQTLs

    PubMed Central

    Villa-Vialaneix, Nathalie; Liaubet, Laurence; Laurent, Thibault; Cherel, Pierre; Gamot, Adrien; SanCristobal, Magali

    2013-01-01

    What are the commonalities between genes, whose expression level is partially controlled by eQTL, especially with regard to biological functions? Moreover, how are these genes related to a phenotype of interest? These issues are particularly difficult to address when the genome annotation is incomplete, as is the case for mammalian species. Moreover, the direct link between gene expression and a phenotype of interest may be weak, and thus difficult to handle. In this framework, the use of a co-expression network has proven useful: it is a robust approach for modeling a complex system of genetic regulations, and to infer knowledge for yet unknown genes. In this article, a case study was conducted with a mammalian species. It showed that the use of a co-expression network based on partial correlation, combined with a relevant clustering of nodes, leads to an enrichment of biological functions of around 83%. Moreover, the use of a spatial statistics approach allowed us to superimpose additional information related to a phenotype; this lead to highlighting specific genes or gene clusters that are related to the network structure and the phenotype. Three main results are worth noting: first, key genes were highlighted as a potential focus for forthcoming biological experiments; second, a set of biological functions, which support a list of genes under partial eQTL control, was set up by an overview of the global structure of the gene expression network; third, pH was found correlated with gene clusters, and then with related biological functions, as a result of a spatial analysis of the network topology. PMID:23577081

  12. The structure of a gene co-expression network reveals biological functions underlying eQTLs.

    PubMed

    Villa-Vialaneix, Nathalie; Liaubet, Laurence; Laurent, Thibault; Cherel, Pierre; Gamot, Adrien; SanCristobal, Magali

    2013-01-01

    What are the commonalities between genes, whose expression level is partially controlled by eQTL, especially with regard to biological functions? Moreover, how are these genes related to a phenotype of interest? These issues are particularly difficult to address when the genome annotation is incomplete, as is the case for mammalian species. Moreover, the direct link between gene expression and a phenotype of interest may be weak, and thus difficult to handle. In this framework, the use of a co-expression network has proven useful: it is a robust approach for modeling a complex system of genetic regulations, and to infer knowledge for yet unknown genes. In this article, a case study was conducted with a mammalian species. It showed that the use of a co-expression network based on partial correlation, combined with a relevant clustering of nodes, leads to an enrichment of biological functions of around 83%. Moreover, the use of a spatial statistics approach allowed us to superimpose additional information related to a phenotype; this lead to highlighting specific genes or gene clusters that are related to the network structure and the phenotype. Three main results are worth noting: first, key genes were highlighted as a potential focus for forthcoming biological experiments; second, a set of biological functions, which support a list of genes under partial eQTL control, was set up by an overview of the global structure of the gene expression network; third, pH was found correlated with gene clusters, and then with related biological functions, as a result of a spatial analysis of the network topology.

  13. Feeding characteristics reveal functional distinctions among browsing herbivorous fishes on coral reefs

    NASA Astrophysics Data System (ADS)

    Streit, Robert P.; Hoey, Andrew S.; Bellwood, David R.

    2015-12-01

    The removal of macroalgal biomass by fishes is a key process on coral reefs. Numerous studies have identified the fish species responsible for removing mature macroalgae, and have identified how this varies spatially, temporally, and among different algal types. None, however, have considered the behavioural and morphological traits of the browsing fishes and how this may influence the removal of macroalgal material. Using video observations of fish feeding on the brown macroalga Sargassum polycystum, we quantified the feeding behaviour and morphology of the four dominant browsing species on the Great Barrier Reef ( Kyphosus vaigiensis, Naso unicornis, Siganus canaliculatus, and Siganus doliatus). The greatest distinction between species was the algal material they targeted. K. vaigiensis and N. unicornis bit on the entire macroalgal thallus in approximately 90 % of bites. In contrast, Si. canaliculatus and Si. doliatus avoided biting the stalks, with 80-98 % of bites being on the macroalgal leaves only. This distinctive grouping into `entire thallus-biters' versus `leaf-biters' was not supported by size-standardized measures of biting morphology. Rather, species-specific adult body sizes, tooth shape, and feeding behaviour appear to underpin this functional distinction, with adults of the two larger fish species ( N. unicornis and K. vaigiensis) eating the entire macroalgal thallus, while the two smaller species ( Si. canaliculatus and Si. doliatus) bite only leaves. These findings caution against assumed homogeneity within this, and potentially other, functional groups on coral reefs. As functional redundancy within the macroalgal browsers is limited, the smaller `leaf-biting' species are unlikely to be able to compensate functionally for the loss of larger `entire thallus-biting' species.

  14. Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site

    PubMed Central

    Sunden, Fanny; Peck, Ariana; Salzman, Julia; Ressl, Susanne; Herschlag, Daniel

    2015-01-01

    Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called ‘catalytic residues’ are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes. DOI: http://dx.doi.org/10.7554/eLife.06181.001 PMID:25902402

  15. Role of Survivin in cytokinesis revealed by a separation-of-function allele

    PubMed Central

    Szafer-Glusman, Edith; Fuller, Margaret T.; Giansanti, Maria Grazia

    2011-01-01

    The chromosomal passenger complex (CPC), containing Aurora B kinase, Inner Centromere Protein, Survivin, and Borealin, regulates chromosome condensation and interaction between kinetochores and microtubules at metaphase, then relocalizes to midzone microtubules at anaphase and regulates central spindle organization and cytokinesis. However, the precise role(s) played by the CPC in anaphase have been obscured by its prior functions in metaphase. Here we identify a missense allele of Drosophila Survivin that allows CPC localization and function during metaphase but not cytokinesis. Analysis of mutant cells showed that Survivin is essential to target the CPC and the mitotic kinesin-like protein 1 orthologue Pavarotti (Pav) to the central spindle and equatorial cell cortex during anaphase in both larval neuroblasts and spermatocytes. Survivin also enabled localization of Polo kinase and Rho at the equatorial cortex in spermatocytes, critical for contractile ring assembly. In neuroblasts, in contrast, Survivin function was not required for localization of Rho, Polo, or Myosin II to a broad equatorial cortical band but was required for Myosin II to transition to a compact, fully constricted ring. Analysis of this “separation-of-function” allele demonstrates the direct role of Survivin and the CPC in cytokinesis and highlights striking differences in regulation of cytokinesis in different cell systems. PMID:21865602

  16. Quantitative interaction mapping reveals an extended UBX domain in ASPL that disrupts functional p97 hexamers

    PubMed Central

    Arumughan, Anup; Roske, Yvette; Barth, Carolin; Forero, Laura Lleras; Bravo-Rodriguez, Kenny; Redel, Alexandra; Kostova, Simona; McShane, Erik; Opitz, Robert; Faelber, Katja; Rau, Kirstin; Mielke, Thorsten; Daumke, Oliver; Selbach, Matthias; Sanchez-Garcia, Elsa; Rocks, Oliver; Panáková, Daniela; Heinemann, Udo; Wanker, Erich E.

    2016-01-01

    Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity. PMID:27762274

  17. Analysis of regulatory network topology reveals functionally distinct classes of microRNAs

    PubMed Central

    Yu, Xueping; Lin, Jimmy; Zack, Donald J.; Mendell, Joshua T.; Qian, Jiang

    2008-01-01

    MicroRNAs (miRNAs) negatively regulate the expression of target genes at the post-transcriptional level. Little is known about the crosstalk between miRNAs and transcription factors (TFs). Here we provide data suggesting that the interaction patterns between TFs and miRNAs can influence the biological functions of miRNAs. From this global survey, we find that a regulated feedback loop, in which two TFs regulate each other and one miRNA regulates both of the factors, is the most significantly overrepresented network motif. Mathematical modeling shows that the miRNA in this motif stabilizes the feedback loop to resist environmental perturbation, providing one mechanism to explain the robustness of developmental programs that is contributed by miRNAs. Furthermore, on the basis of a network motif profile analysis, we demonstrate the existence of two classes of miRNAs with distinct network topological properties. The first class of miRNAs is regulated by a large number of TFs, whereas the second is regulated by only a few TFs. The differential expression level of the two classes of miRNAs in embryonic developmental stages versus adult tissues suggests that the two classes may have fundamentally different biological functions. Our results demonstrate that the TFs and miRNAs extensively interact with each other and the biological functions of miRNAs may be wired in the regulatory network topology. PMID:18927108

  18. Endozoicomonas genomes reveal functional adaptation and plasticity in bacterial strains symbiotically associated with diverse marine hosts

    PubMed Central

    Neave, Matthew J.; Michell, Craig T.; Apprill, Amy; Voolstra, Christian R.

    2017-01-01

    Endozoicomonas bacteria are globally distributed and often abundantly associated with diverse marine hosts including reef-building corals, yet their function remains unknown. In this study we generated novel Endozoicomonas genomes from single cells and metagenomes obtained directly from the corals Stylophora pistillata, Pocillopora verrucosa, and Acropora humilis. We then compared these culture-independent genomes to existing genomes of bacterial isolates acquired from a sponge, sea slug, and coral to examine the functional landscape of this enigmatic genus. Sequencing and analysis of single cells and metagenomes resulted in four novel genomes with 60–76% and 81–90% genome completeness, respectively. These data also confirmed that Endozoicomonas genomes are large and are not streamlined for an obligate endosymbiotic lifestyle, implying that they have free-living stages. All genomes show an enrichment of genes associated with carbon sugar transport and utilization and protein secretion, potentially indicating that Endozoicomonas contribute to the cycling of carbohydrates and the provision of proteins to their respective hosts. Importantly, besides these commonalities, the genomes showed evidence for differential functional specificity and diversification, including genes for the production of amino acids. Given this metabolic diversity of Endozoicomonas we propose that different genotypes play disparate roles and have diversified in concert with their hosts. PMID:28094347

  19. Functional near-infrared spectroscopy reveals reduced interhemispheric cortical communication after pediatric concussion.

    PubMed

    Urban, Karolina J; Barlow, Karen M; Jimenez, Jon J; Goodyear, Bradley G; Dunn, Jeff F

    2015-06-01

    Concussion, or mild traumatic brain injury (mTBI), is a growing concern, especially among the pediatric population. By age 25, as many as 30% of the population are likely to have had a concussion. Many result in long-term disability, with some evolving to postconcussion syndrome. Treatments are being developed, but are difficult to assess given the lack of measures to quantitatively monitor concussion. There is no accepted quantitative imaging metric for monitoring concussion. We hypothesized that because cognitive function and fiber tracks are often impacted in concussion, interhemispheric brain communication may be impaired. We used functional near-infrared spectroscopy (fNIRS) to quantify functional coherence between the left and right motor cortex as a marker of interhemispheric communication. Studies were undertaken during the resting state and with a finger-tapping task to activate the motor cortex. Pediatric patients (ages 12-18) had symptoms for 31-473 days, compared to controls, who have not had reported a previous concussion. We detected differences between patients and controls in coherence between the contralateral motor cortices using measurements of total hemoglobin and oxy-hemoglobin with a p<0.01 (n=8, control; n=12 mTBI). Given the critical need for a quantitative biomarker for recovery after a concussion, we present these data to highlight the potential of fNIRS coupled with interhemispheric coherence analysis as a biomarker of concussion injury.

  20. Comparative metagenomics reveals taxonomically idiosyncratic yet functionally congruent communities in periodontitis

    PubMed Central

    Dabdoub, Shareef M.; Ganesan, Sukirth M.; Kumar, Purnima S.

    2016-01-01

    The phylogenetic characteristics of microbial communities associated with periodontitis have been well studied, however, little is known about the functional endowments of this ecosystem. The present study examined 73 microbial assemblages from 25 individuals with generalized chronic periodontitis and 25 periodontally healthy individuals using whole genome shotgun sequencing. Core metabolic networks were computed from taxa and genes identified in at least 80% of individuals in each group. 50% of genes and species identified in health formed part of the core microbiome, while the disease-associated core microbiome contained 33% of genes and only 1% of taxa. Clinically healthy sites in individuals with periodontitis were more aligned with sites with disease than with health. 68% of the health-associated metagenome was dedicated to energy utilization through oxidative pathways, while in disease; fermentation and methanogenesis were predominant energy transfer mechanisms. Expanded functionality was observed in periodontitis, with unique- or over-representation of genes encoding for fermentation, antibiotic resistance, detoxification stress, adhesion, invasion and intracellular resistance, proteolysis, quorum sensing, Type III/IV secretion systems, phages and toxins in the disease-associated core microbiome. However, different species or consortia contributed to these functions in each individual. Several genes, but not species, demonstrated robust discriminating power between health and disease. PMID:27991530

  1. The functional micro-organization of grid cells revealed by cellular-resolution imaging

    PubMed Central

    Heys, James G.; Rangarajan, Krsna V.; Dombeck, Daniel A.

    2015-01-01

    Summary Establishing how grid cells are anatomically arranged, on a microscopic scale, in relation to their firing patterns in the environment would facilitate a greater micro-circuit level understanding of the brain’s representation of space. However, all previous grid cell recordings used electrode techniques that provide limited descriptions of fine-scale organization. We therefore developed a technique for cellular-resolution functional imaging of medial entorhinal cortex (MEC) neurons in mice navigating a virtual linear track, enabling a new experimental approach to study MEC. Using these methods, we show that grid cells are physically clustered in MEC compared to non-grid cells. Additionally, we demonstrate that grid cells are functionally micro-organized: The similarity between the environment firing locations of grid cell pairs varies as a function of the distance between them according to a “Mexican Hat” shaped profile. This suggests that, on average, nearby grid cells have more similar spatial firing phases than those further apart. PMID:25467986

  2. Fluorescence fluctuation microscopy to reveal 3D architecture and function in the cell nucleus.

    PubMed

    Lenser, Thorsten; Weisshart, Klaus; Ulbricht, Tobias; Klement, Karolin; Hemmerich, Peter

    2010-01-01

    The three-dimensional (3D) architecture of the cell nucleus is determined not only by the presence of subnuclear domains, such as the nuclear envelope, chromosome territories, and nuclear bodies, but also by smaller domains which form in response to specific functions, such as RNA transcription, DNA replication, and DNA repair. Since both stable and dynamic structures contribute to nuclear morphology, it is important to study the biophysical principles of the formation of macromolecular assemblies within the nucleus. For this purpose, a variety of fluorescence fluctuation microscopy techniques can be applied. Here, we summarize our current knowledge on the 3D architecture of the mammalian cell nucleus and describe in detail how the assembly of functional nuclear protein complexes can be analyzed in living cells using fluorescence bleaching techniques, fluorescence correlation spectroscopy, raster image correlation spectroscopy, and mathematical modeling. In conclusion, the application of all these techniques in combination is a powerful tool to assess the full spectrum of nuclear protein dynamics and to understand the biophysical principles underlying nuclear structure and function.

  3. Functional metagenomics reveals novel pathways of prebiotic breakdown by human gut bacteria.

    PubMed

    Cecchini, Davide A; Laville, Elisabeth; Laguerre, Sandrine; Robe, Patrick; Leclerc, Marion; Doré, Joël; Henrissat, Bernard; Remaud-Siméon, Magali; Monsan, Pierre; Potocki-Véronèse, Gabrielle

    2013-01-01

    The human intestine hosts a complex bacterial community that plays a major role in nutrition and in maintaining human health. A functional metagenomic approach was used to explore the prebiotic breakdown potential of human gut bacteria, including non-cultivated ones. Two metagenomic libraries, constructed from ileum mucosa and fecal microbiota, were screened for hydrolytic activities on the prebiotic carbohydrates inulin, fructo-oligosaccharides, xylo-oligosaccharides, galacto-oligosaccharides and lactulose. The DNA inserts of 17 clones, selected from the 167 hits that were identified, were pyrosequenced in-depth, yielding in total 407, 420 bp of metagenomic DNA. From these sequences, we discovered novel prebiotic degradation pathways containing carbohydrate transporters and hydrolysing enzymes, for which we provided the first experimental proof of function. Twenty of these proteins are encoded by genes that are also present in the gut metagenome of at least 100 subjects, whatever are their ages or their geographical origin. The sequence taxonomic assignment indicated that still unknown bacteria, for which neither culture conditions nor genome sequence are available, possess the enzymatic machinery to hydrolyse the prebiotic carbohydrates tested. The results expand the vision on how prebiotics are metabolized along the intestine, and open new perspectives for the design of functional foods.

  4. Continuous Descending Modulation of the Spinal Cord Revealed by Functional MRI

    PubMed Central

    Bosma, Rachael L.; Cotoi, Andreea I.; Leung, Roxanne H.; Kornelsen, Jennifer; Lawrence-Dewar, Jane M.; Pukall, Caroline F.; Staud, Roland

    2016-01-01

    Spontaneous variations in spinal cord activity may arise from regulation of any of a number of functions including sensory, motor, and autonomic control. Here, we use functional MRI (fMRI) of healthy participants to identify properties of blood oxygenation-level dependent (BOLD) variations in the spinal cord in response to knowledge that either a noxious stimulus is impending, or that no stimulus is to be expected. Expectation of a noxious stimulus, or no stimulus, is shown to have a significant effect on wide-spread BOLD signal variations in the spinal cord over the entire time period of the fMRI acquisition. Coordination of BOLD responses between/within spinal cord and brainstem regions are also influenced by this knowledge. We provide evidence that such signal variations are the result of continuous descending modulation of spinal cord function. BOLD signal variations in response to noxious stimulation of the hand are also shown, as in previous studies. The observation of both continuous and reactive BOLD responses to emotional/cognitive factors and noxious peripheral stimulation may have important implications, not only for our understanding of endogenous pain modulation, but also in showing that spinal cord activity is under continuous regulatory control. PMID:27907094

  5. Functional mutagenesis screens reveal the 'cap structure' formation in disulfide-bridge free TASK channels.

    PubMed

    Goldstein, Matthias; Rinné, Susanne; Kiper, Aytug K; Ramírez, David; Netter, Michael F; Bustos, Daniel; Ortiz-Bonnin, Beatriz; González, Wendy; Decher, Niels

    2016-01-22

    Two-pore-domain potassium (K2P) channels have a large extracellular cap structure formed by two M1-P1 linkers, containing a cysteine for dimerization. However, this cysteine is not present in the TASK-1/3/5 subfamily. The functional role of the cap is poorly understood and it remained unclear whether K2P channels assemble in the domain-swapped orientation or not. Functional alanine-mutagenesis screens of TASK-1 and TRAAK were used to build an in silico model of the TASK-1 cap. According to our data the cap structure of disulfide-bridge free TASK channels is similar to that of other K2P channels and is most likely assembled in the domain-swapped orientation. As the conserved cysteine is not essential for functional expression of all K2P channels tested, we propose that hydrophobic residues at the inner leaflets of the cap domains can interact with each other and that this way of stabilizing the cap is most likely conserved among K2P channels.

  6. Cornelia de Lange syndrome and molecular implications of the cohesin complex: Abstracts from the 7th biennial scientific and educational symposium 2016.

    PubMed

    Kline, Antonie D; Krantz, Ian D; Deardorff, Matthew A; Shirahige, Katsuhiko; Dorsett, Dale; Gerton, Jennifer L; Wu, Meng; Mehta, Devanshi; Mills, Jason A; Carrico, Cheri S; Noon, Sarah; Herrera, Pamela S; Horsfield, Julia A; Bettale, Chiara; Morgan, Jeremy; Huisman, Sylvia A; Moss, Jo; McCleery, Joseph; Grados, Marco; Hansen, Blake D; Srivastava, Siddharth; Taylor-Snell, Emily; Kerr, Lynne M; Katz, Olivia; Calof, Anne L; Musio, Antonio; Egense, Alena; Haaland, Richard E

    2017-02-12

    Cornelia de Lange Syndrome (CdLS) is due to mutations in the genes for the structural and regulatory proteins that make up the cohesin complex, and is considered a cohesinopathy disorder or, more recently, a transcriptomopathy. New phenotypes have been recognized in this expanding field. There are multiple clinical issues facing individuals with all forms of CdLS, particularly in the neurodevelopmental system, but also gastrointestinal, cardiac, and musculoskeletal. Aspects of developmental and cell biology have found common endpoints in the biology of the cohesin complex, with improved understanding of the mechanisms, easier diagnostic tests, and the possibility of potential therapeutics, all major clinical implications for the individual with CdLS. The following abstracts are the presentations from the 7th Cornelia de Lange Syndrome Scientific and Educational Symposium, June 22-23, 2016, in Orlando, FL, in conjunction with the Cornelia de Lange Syndrome Foundation National Meeting. In addition to the scientific and clinical discussions, there were talks related to practical aspects of behavior including autism, transitions, communication, access to medical care, and databases. At the end of the symposium, a panel was held, which included several parents, affected individuals and genetic counselors, and discussed the greatest challenges in life and how this information can assist in guiding future research. The Research Committee of the CdLS Foundation organizes this meeting, reviews, and accepts abstracts, and subsequently disseminates the information to the families through members of the Clinical Advisory Board and publications. AMA CME credits were provided by Greater Baltimore Medical Center, Baltimore, MD.

  7. In situ Expression of Functional Genes Reveals Nitrogen Cycling at High Temperatures in Terrestrial Hydrothermal Systems

    NASA Astrophysics Data System (ADS)

    Loiacono, S. T.; Meyer-Dombard, D. R.

    2011-12-01

    An essential element for life, nitrogen occurs in all living organisms and is critical for the synthesis of amino acids, proteins, nucleic acids, and other forms of biomass. Thus, nitrogen cycling likely plays a vital role in microbial metabolic processes as well as nutrient availability. For microorganisms in "extreme" environments, this means developing adaptations that allow them to survive in harsh conditions and still perform the metabolisms essential to sustain life. Recent studies have screened biofilms and thermal sediments of Yellowstone National Park (YNP) thermal features for the presence of nifH genes, which code for a key enzyme in the nitrogen fixation process [1-4]. Furthermore, analysis of nitrogen isotopes in biofilms across a temperature and chemical gradient revealed that nitrogen fixation likely varies across the chemosynthetic/photosynthetic ecotone [5]. Although research has evaluated and confirmed the presence of nifH genes in various thermophilic microbial communities, the existence of a gene in the DNA of an organism does not verify its use. Instead, other methods, such as culturing, isotope tracer assays, and gene expression studies are required to provide direct evidence of biological nitrogen fixation. Culturing and isotope tracer approaches have successfully revealed high-temperature biological nitrogen fixation in both marine hydrothermal vent microbial communities [6] and in acidic, terrestrial hydrothermal sediment [3]. Transcriptomics-based techniques (using mRNA extracted from samples to confirm in situ expression of targeted genes) have been much more limited in number, and only a few studies have, to date, investigated in situ expression of the nifH gene in thermophilic microbial communities [2, 7]. This study explores the presence and expression of nifH genes in several features of the Lower Geyser Basin (LGB) of YNP. Nucleic acids from chemosynthetic and photosynthetic microbial communities were extracted and then amplified

  8. Thermodynamic Characterization of a Triheme Cytochrome Family from Geobacter sulfurreducens Reveals Mechanistic and Functional Diversity

    PubMed Central

    Morgado, Leonor; Bruix, Marta; Pessanha, Miguel; Londer, Yuri Y.; Salgueiro, Carlos A.

    2010-01-01

    Abstract A family of five periplasmic triheme cytochromes (PpcA-E) was identified in Geobacter sulfurreducens, where they play a crucial role by driving electron transfer from the cytoplasm to the cell exterior and assisting the reduction of extracellular acceptors. The thermodynamic characterization of PpcA using NMR and visible spectroscopies was previously achieved under experimental conditions identical to those used for the triheme cytochrome c7 from Desulfuromonas acetoxidans. Under such conditions, attempts to obtain NMR data were complicated by the relatively fast intermolecular electron exchange. This work reports the detailed thermodynamic characterization of PpcB, PpcD, and PpcE under optimal experimental conditions. The thermodynamic characterization of PpcA was redone under these new conditions to allow a proper comparison of the redox properties with those of other members of this family. The heme reduction potentials of the four proteins are negative, differ from each other, and cover different functional ranges. These reduction potentials are strongly modulated by heme-heme interactions and by interactions with protonated groups (the redox-Bohr effect) establishing different cooperative networks for each protein, which indicates that they are designed to perform different functions in the cell. PpcA and PpcD appear to be optimized to interact with specific redox partners involving e−/H+ transfer via different mechanisms. Although no evidence of preferential electron transfer pathway or e−/H+ coupling was found for PpcB and PpcE, the difference in their working potential ranges suggests that they may also have different physiological redox partners. This is the first study, to our knowledge, to characterize homologous cytochromes from the same microorganism and provide evidence of their different mechanistic and functional properties. These findings provide an explanation for the coexistence of five periplasmic triheme cytochromes in G

  9. Evolution of the snake body form reveals homoplasy in amniote Hox gene function.

    PubMed

    Head, Jason J; Polly, P David

    2015-04-02

    Hox genes regulate regionalization of the axial skeleton in vertebrates, and changes in their expression have been proposed to be a fundamental mechanism driving the evolution of new body forms. The origin of the snake-like body form, with its deregionalized pre-cloacal axial skeleton, has been explained as either homogenization of Hox gene expression domains, or retention of standard vertebrate Hox domains with alteration of downstream expression that suppresses development of distinct regions. Both models assume a highly regionalized ancestor, but the extent of deregionalization of the primaxial domain (vertebrae, dorsal ribs) of the skeleton in snake-like body forms has never been analysed. Here we combine geometric morphometrics and maximum-likelihood analysis to show that the pre-cloacal primaxial domain of elongate, limb-reduced lizards and snakes is not deregionalized compared with limbed taxa, and that the phylogenetic structure of primaxial morphology in reptiles does not support a loss of regionalization in the evolution of snakes. We demonstrate that morphometric regional boundaries correspond to mapped gene expression domains in snakes, suggesting that their primaxial domain is patterned by a normally functional Hox code. Comparison of primaxial osteology in fossil and modern amniotes with Hox gene distributions within Amniota indicates that a functional, sequentially expressed Hox code patterned a subtle morphological gradient along the anterior-posterior axis in stem members of amniote clades and extant lizards, including snakes. The highly regionalized skeletons of extant archosaurs and mammals result from independent evolution in the Hox code and do not represent ancestral conditions for clades with snake-like body forms. The developmental origin of snakes is best explained by decoupling of the primaxial and abaxial domains and by increases in somite number, not by changes in the function of primaxial Hox genes.

  10. Functional characterization of chitinase-3 reveals involvement of chitinases in early embryo immunity in zebrafish.

    PubMed

    Teng, Zinan; Sun, Chen; Liu, Shousheng; Wang, Hongmiao; Zhang, Shicui

    2014-10-01

    The function and mechanism of chitinases in early embryonic development remain largely unknown. We show here that recombinant chitinase-3 (rChi3) is able to hydrolyze the artificial chitin substrate, 4-methylumbelliferyl-β-D-N,N',N″-triacetylchitotrioside, and to bind to and inhibit the growth of the fungus Candida albicans, implicating that Chi3 plays a dual function in innate immunity and chitin-bearing food digestion in zebrafish. This is further corroborated by the expression profile of Chi3 in the liver and gut, which are both immune- and digestion-relevant organs. Compared with rChi3, rChi3-CD lacking CBD still retains partial capacity to bind to C. albicans, but its enzymatic and antifungal activities are significantly reduced. By contrast, rChi3-E140N with the putative catalytic residue E140 mutated shows little affinity to chitin, and its enzymatic and antifungal activities are nearly completely lost. These suggest that both enzymatic and antifungal activities of Chi3 are dependent on the presence of CBD and E140. We also clearly demonstrate that in zebrafish, both the embryo extract and the developing embryo display antifungal activity against C. albicans, and all the findings point to chitinase-3 (Chi3) being a newly-identified factor involved in the antifungal activity. Taken together, a dual function in both innate immunity and food digestion in embryo is proposed for zebrafish Chi3. It also provides a new angle to understand the immune role of chitinases in early embryonic development of animals.

  11. A new therapeutic effect of simvastatin revealed by functional improvement in muscular dystrophy

    PubMed Central

    Whitehead, Nicholas P.; Kim, Min Jeong; Bible, Kenneth L.; Adams, Marvin E.; Froehner, Stanley C.

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a lethal, degenerative muscle disease with no effective treatment. DMD muscle pathogenesis is characterized by chronic inflammation, oxidative stress, and fibrosis. Statins, cholesterol-lowering drugs, inhibit these deleterious processes in ischemic diseases affecting skeletal muscle, and therefore have potential to improve DMD. However, statins have not been considered for DMD, or other muscular dystrophies, principally because skeletal-muscle-related symptoms are rare, but widely publicized, side effects of these drugs. Here we show positive effects of statins in dystrophic skeletal muscle. Simvastatin dramatically reduced damage and enhanced muscle function in dystrophic (mdx) mice. Long-term simvastatin treatment vastly improved overall muscle health in mdx mice, reducing plasma creatine kinase activity, an established measure of muscle damage, to near-normal levels. This reduction was accompanied by reduced inflammation, more oxidative muscle fibers, and improved strength of the weak diaphragm muscle. Shorter-term treatment protected against muscle fatigue and increased mdx hindlimb muscle force by 40%, a value comparable to current dystrophin gene-based therapies. Increased force correlated with reduced NADPH Oxidase 2 protein expression, the major source of oxidative stress in dystrophic muscle. Finally, in old mdx mice with severe muscle degeneration, simvastatin enhanced diaphragm force and halved fibrosis, a major cause of functional decline in DMD. These improvements were accompanied by autophagy activation, a recent therapeutic target for DMD, and less oxidative stress. Together, our findings highlight that simvastatin substantially improves the overall health and function of dystrophic skeletal muscles and may provide an unexpected, novel therapy for DMD and related neuromuscular diseases. PMID:26417069

  12. Computer Simulations Reveal Multiple Functions for Aromatic Residues in Cellulase Enzymes (Fact Sheet)

    SciTech Connect

    Not Available

    2012-07-01

    NREL researchers use high-performance computing to demonstrate fundamental roles of aromatic residues in cellulase enzyme tunnels. National Renewable Energy Laboratory (NREL) computer simulations of a key industrial enzyme, the Trichoderma reesei Family 6 cellulase (Cel6A), predict that aromatic residues near the enzyme's active site and at the entrance and exit tunnel perform different functions in substrate binding and catalysis, depending on their location in the enzyme. These results suggest that nature employs aromatic-carbohydrate interactions with a wide variety of binding affinities for diverse functions. Outcomes also suggest that protein engineering strategies in which mutations are made around the binding sites may require tailoring specific to the enzyme family. Cellulase enzymes ubiquitously exhibit tunnels or clefts lined with aromatic residues for processing carbohydrate polymers to monomers, but the molecular-level role of these aromatic residues remains unknown. In silico mutation of the aromatic residues near the catalytic site of Cel6A has little impact on the binding affinity, but simulation suggests that these residues play a major role in the glucopyranose ring distortion necessary for cleaving glycosidic bonds to produce fermentable sugars. Removal of aromatic residues at the entrance and exit of the cellulase tunnel, however, dramatically impacts the binding affinity. This suggests that these residues play a role in acquiring cellulose chains from the cellulose crystal and stabilizing the reaction product, respectively. These results illustrate that the role of aromatic-carbohydrate interactions varies dramatically depending on the position in the enzyme tunnel. As aromatic-carbohydrate interactions are present in all carbohydrate-active enzymes, the results have implications for understanding protein structure-function relationships in carbohydrate metabolism and recognition, carbon turnover in nature, and protein engineering strategies for

  13. Alanine scan of core positions in ubiquitin reveals links between dynamics, stability, and function.

    PubMed

    Lee, Shirley Y; Pullen, Lester; Virgil, Daniel J; Castañeda, Carlos A; Abeykoon, Dulith; Bolon, Daniel N A; Fushman, David

    2014-04-03

    Mutations at solvent-inaccessible core positions in proteins can impact function through many biophysical mechanisms including alterations to thermodynamic stability and protein dynamics. As these properties of proteins are difficult to investigate, the impacts of core mutations on protein function are poorly understood for most systems. Here, we determined the effects of alanine mutations at all 15 core positions in ubiquitin on function in yeast. The majority (13 of 15) of alanine substitutions supported yeast growth as the sole ubiquitin. Both the two null mutants (I30A and L43A) were less stable to temperature-induced unfolding in vitro than wild type (WT) but were well folded at physiological temperatures. Heteronuclear NMR studies indicated that the L43A mutation reduces temperature stability while retaining a ground-state structure similar to WT. This structure enables L43A to bind to common ubiquitin receptors in vitro. Many of the core alanine ubiquitin mutants, including one of the null variants (I30A), exhibited an increased accumulation of high-molecular-weight species, suggesting that these mutants caused a defect in the processing of ubiquitin-substrate conjugates. In contrast, L43A exhibited a unique accumulation pattern with reduced levels of high-molecular-weight species and undetectable levels of free ubiquitin. When conjugation to other proteins was blocked, L43A ubiquitin accumulated as free ubiquitin in yeast. Based on these findings, we speculate that ubiquitin's stability to unfolding may be required for efficient recycling during proteasome-mediated substrate degradation.

  14. Massive stars reveal variations of the stellar initial mass function in the Milky Way stellar clusters

    NASA Astrophysics Data System (ADS)

    Dib, Sami; Schmeja, Stefan; Hony, Sacha

    2017-01-01

    We investigate whether the stellar initial mass function (IMF) is universal, or whether it varies significantly among young stellar clusters in the Milky Way. We propose a method to uncover the range of variation of the parameters that describe the shape of the IMF for the population of young Galactic clusters.These parameters are the slopes in the low and high stellar mass regimes, γ and Γ, respectively, and the characteristic mass, Mch. The method relies exclusively on the high-mass content of the clusters, but is able to yield information on the distributions of parameters that describe the IMF over the entire stellar mass range. This is achieved by comparing the fractions of single and lonely massive O stars in a recent catalogue of the Milky Way clusters with a library of simulated clusters built with various distribution functions of the IMF parameters. The synthetic clusters are corrected for the effects of the binary population, stellar evolution, sample incompleteness, and ejected O stars. Our findings indicate that broad distributions of the IMF parameters are required in order to reproduce the fractions of single and lonely O stars in Galactic clusters. They also do not lend support to the existence of a cluster mass-maximum stellar mass relation. We propose a probabilistic formulation of the IMF whereby the parameters of the IMF are described by Gaussian distribution functions centred around γ = 0.91, Γ = 1.37, and Mch = 0.41 M⊙, and with dispersions of σγ = 0.25, σΓ = 0.60, and σ _{M_{ch}}=0.27 M⊙ around these values.

  15. A new therapeutic effect of simvastatin revealed by functional improvement in muscular dystrophy.

    PubMed

    Whitehead, Nicholas P; Kim, Min Jeong; Bible, Kenneth L; Adams, Marvin E; Froehner, Stanley C

    2015-10-13

    Duchenne muscular dystrophy (DMD) is a lethal, degenerative muscle disease with no effective treatment. DMD muscle pathogenesis is characterized by chronic inflammation, oxidative stress, and fibrosis. Statins, cholesterol-lowering drugs, inhibit these deleterious processes in ischemic diseases affecting skeletal muscle, and therefore have potential to improve DMD. However, statins have not been considered for DMD, or other muscular dystrophies, principally because skeletal-muscle-related symptoms are rare, but widely publicized, side effects of these drugs. Here we show positive effects of statins in dystrophic skeletal muscle. Simvastatin dramatically reduced damage and enhanced muscle function in dystrophic (mdx) mice. Long-term simvastatin treatment vastly improved overall muscle health in mdx mice, reducing plasma creatine kinase activity, an established measure of muscle damage, to near-normal levels. This reduction was accompanied by reduced inflammation, more oxidative muscle fibers, and improved strength of the weak diaphragm muscle. Shorter-term treatment protected against muscle fatigue and increased mdx hindlimb muscle force by 40%, a value comparable to current dystrophin gene-based therapies. Increased force correlated with reduced NADPH Oxidase 2 protein expression, the major source of oxidative stress in dystrophic muscle. Finally, in old mdx mice with severe muscle degeneration, simvastatin enhanced diaphragm force and halved fibrosis, a major cause of functional decline in DMD. These improvements were accompanied by autophagy activation, a recent therapeutic target for DMD, and less oxidative stress. Together, our findings highlight that simvastatin substantially improves the overall health and function of dystrophic skeletal muscles and may provide an unexpected, novel therapy for DMD and related neuromuscular diseases.

  16. Structural-Functional Analysis Reveals a Specific Domain Organization in Family GH20 Hexosaminidases.

    PubMed

    Val-Cid, Cristina; Biarnés, Xevi; Faijes, Magda; Planas, Antoni

    2015-01-01

    Hexosaminidases are involved in important biological processes catalyzing the hydrolysis of N-acetyl-hexosaminyl residues in glycosaminoglycans and glycoconjugates. The GH20 enzymes present diverse domain organizations for which we propose two minimal model architectures: Model A containing at least a non-catalytic GH20b domain and the catalytic one (GH20) always accompanied with an extra α-helix (GH20b-GH20-α), and Model B with only the catalytic GH20 domain. The large Bifidobacterium bifidum lacto-N-biosidase was used as a model protein to evaluate the minimal functional unit due to its interest and structural complexity. By expressing different truncated forms of this enzyme, we show that Model A architectures cannot be reduced to Model B. In particular, there are two structural requirements general to GH20 enzymes with Model A architecture. First, the non-catalytic domain GH20b at the N-terminus of the catalytic GH20 domain is required for expression and seems to stabilize it. Second, the substrate-binding cavity at the GH20 domain always involves a remote element provided by a long loop from the catalytic domain itself or, when this loop is short, by an element from another domain of the multidomain structure or from the dimeric partner. Particularly, the lacto-N-biosidase requires GH20b and the lectin-like domain at the N- and C-termini of the catalytic GH20 domain to be fully soluble and functional. The lectin domain provides this remote element to the active site. We demonstrate restoration of activity of the inactive GH20b-GH20-α construct (model A architecture) by a complementation assay with the lectin-like domain. The engineering of minimal functional units of multidomain GH20 enzymes must consider these structural requirements.

  17. Structural-Functional Analysis Reveals a Specific Domain Organization in Family GH20 Hexosaminidases

    PubMed Central

    Val-Cid, Cristina; Biarnés, Xevi; Faijes, Magda; Planas, Antoni

    2015-01-01

    Hexosaminidases are involved in important biological processes catalyzing the hydrolysis of N-acetyl-hexosaminyl residues in glycosaminoglycans and glycoconjugates. The GH20 enzymes present diverse domain organizations for which we propose two minimal model architectures: Model A containing at least a non-catalytic GH20b domain and the catalytic one (GH20) always accompanied with an extra α-helix (GH20b-GH20-α), and Model B with only the catalytic GH20 domain. The large Bifidobacterium bifidum lacto-N-biosidase was used as a model protein to evaluate the minimal functional unit due to its interest and structural complexity. By expressing different truncated forms of this enzyme, we show that Model A architectures cannot be reduced to Model B. In particular, there are two structural requirements general to GH20 enzymes with Model A architecture. First, the non-catalytic domain GH20b at the N-terminus of the catalytic GH20 domain is required for expression and seems to stabilize it. Second, the substrate-binding cavity at the GH20 domain always involves a remote element provided by a long loop from the catalytic domain itself or, when this loop is short, by an element from another domain of the multidomain structure or from the dimeric partner. Particularly, the lacto-N-biosidase requires GH20b and the lectin-like domain at the N- and C-termini of the catalytic GH20 domain to be fully soluble and functional. The lectin domain provides this remote element to the active site. We demonstrate restoration of activity of the inactive GH20b-GH20-α construct (model A architecture) by a complementation assay with the lectin-like domain. The engineering of minimal functional units of multidomain GH20 enzymes must consider these structural requirements. PMID:26024355

  18. Nitrogen cycling in Yellowstone National Park thermal features: using gene expression to reveal ecological function

    NASA Astrophysics Data System (ADS)

    Lafree, S. T.; Burton, M. S.; Meyer-Dombard, D. R.

    2010-12-01

    Studies of biodiversity, metabolic strategies, and functional ecology in modern hydrothermal systems have the potential to provide insight into the metabolism and evolution of life. The geochemical and microbial diversity present at Yellowstone National Park (YNP), Wyoming, USA, makes it an ideal place for studying the functional ecology and metabolic processes of prokaryotic organisms. While much work in terrestrial hydrothermal features is focused on phylogenetic and geochemical analyses, a few recent investigations in YNP and other hydrothermal areas have focused on “gene hunting”: screening thermal sediment and biofilm samples for the presence of genes utilized in specific metabolic processes [2, 3, 6, 7, 8]. Although research has evaluated and confirmed the presence of many of these genes in various thermophilic microbial communities, the existence of a gene in the DNA of an organism does not verify its use, and few researchers have done work to confirm the utilization (expression) of the genes discovered in thermal samples [1, 6, 7, 8]. Disequilibrium between reduced hydrothermal fluid of YNP thermal features and the atmosphere provides a copious source of potential energy to be harnessed through microbial metabolic processes, with NO3- and NO2- serving as the preferred electron acceptors and top energy sources after O2 [4, 5]. Consequentially, nitrogen cycling likely plays a vital role in microbial metabolic processes, as well as nutrient availability. This study explores the presence and utilization of functional genes that are key in steps of the nitrogen cycle, such as nitrogen fixation (NifH), denitrification (nirKS), and ammonia oxidation (amoA). Both DNA and RNA were extracted from thermal sediment and streamer biofilm communities collected in the chemosynthetic zone of various thermal features of the Sentinel Meadows Group in Lower Geyser Basin, YNP. Extracted DNA and reverse transcribed RNA (cDNA) were amplified using the polymerase chain

  19. Functional Magnetic Resonance Imaging of Rats with Experimental Autoimmune Encephalomyelitis Reveals Brain Cortex Remodeling

    PubMed Central

    Tambalo, Stefano; Peruzzotti-Jametti, Luca; Rigolio, Roberta; Fiorini, Silvia; Bontempi, Pietro; Mallucci, Giulia; Balzarotti, Beatrice; Marmiroli, Paola; Sbarbati, Andrea; Cavaletti, Guido

    2015-01-01

    Cortical reorganization occurring in multiple sclerosis (MS) patients is thought to play a key role in limiting the effect of structural tissue damage. Conversely, its exhaustion may contribute to the irreversible disability that accumulates with disease progression. Several aspects of MS-related cortical reorganization, including the overall functional effect and likely modulation by therapies, still remain to be elucidated. The aim of this work was to assess the extent of functional cortical reorganization and its brain structural/pathological correlates in Dark Agouti rats with experimental autoimmune encephalomyelitis (EAE), a widely accepted preclinical model of chronic MS. Morphological and functional MRI (fMRI) were performed before disease induction and during the relapsing and chronic phases of EAE. During somatosensory stimulation of the right forepaw, fMRI demonstrated that cortical reorganization occurs in both relapsing and chronic phases of EAE with increased activated volume and decreased laterality index versus baseline values. Voxel-based morphometry demonstrated gray matter (GM) atrophy in the cerebral cortex, and both GM and white matter atrophy were assessed by ex vivo pathology of the sensorimotor cortex and corpus callosum. Neuroinflammation persisted in the relapsing and chronic phases, with dendritic spine density in the layer IV sensory neurons inversely correlating with the number of cluster of differentiation 45-positive inflammatory lesions. Our work provides an innovative experimental platform that may be pivotal for the comprehension of key mechanisms responsible for the accumulation of irreversible brain damage and for the development of innovative therapies to reduce disability in EAE/MS. SIGNIFICANCE STATEMENT Since the early 2000s, functional MRI (fMRI) has demonstrated profound modifications in the recruitment of cortical areas during motor, cognitive, and sensory tasks in multiple sclerosis (MS) patients. Experimental autoimmune

  20. Lipid Profiling and Transcriptomic Analysis Reveals a Functional Interplay between Estradiol and Growth Hormone in Liver

    PubMed Central

    Fernández-Pérez, Leandro; Santana-Farré, Ruymán; de Mirecki-Garrido, Mercedes; García, Irma; Guerra, Borja; Mateo-Díaz, Carlos; Iglesias-Gato, Diego; Díaz-Chico, Juan Carlos; Flores-Morales, Amilcar; Díaz, Mario

    2014-01-01

    17β-estradiol (E2) may interfere with endocrine, metabolic, and gender-differentiated functions in liver in both females and males. Indirect mechanisms play a crucial role because of the E2 influence on the pituitary GH secretion and the GHR-JAK2-STAT5 signaling pathway in the target tissues. E2, through its interaction with the estrogen receptor, exerts direct effects on liver. Hypothyroidism also affects endocrine and metabolic functions of the liver, rendering a metabolic phenotype with features that mimic deficiencies in E2 or GH. In this work, we combined the lipid and transcriptomic analysis to obtain comprehensive information on the molecular mechanisms of E2 effects, alone and in combination with GH, to regulate liver functions in males. We used the adult hypothyroid-orchidectomized rat model to minimize the influence of internal hormones on E2 treatment and to explore its role in male-differentiated functions. E2 influenced genes involved in metabolism of lipids and endo-xenobiotics, and the GH-regulated endocrine, metabolic, immune, and male-specific responses. E2 induced a female-pattern of gene expression and inhibited GH-regulated STAT5b targeted genes. E2 did not prevent the inhibitory effects of GH on urea and amino acid metabolism-related genes. The combination of E2 and GH decreased transcriptional immune responses. E2 decreased the hepatic content of saturated fatty acids and induced a transcriptional program that seems to be mediated by the activation of PPARα. In contrast, GH inhibited fatty acid oxidation. Both E2 and GH replacements reduced hepatic CHO levels and increased the formation of cholesterol esters and triacylglycerols. Notably, the hepatic lipid profiles were endowed with singular fingerprints that may be used to segregate the effects of different hormonal replacements. In summary, we provide in vivo evidence that E2 has a significant impact on lipid content and transcriptome in male liver and that E2 exerts a marked influence on

  1. Functional screening of antibiotic resistance genes from human gut microbiota reveals a novel gene fusion.

    PubMed

    Cheng, Gong; Hu, Yongfei; Yin, Yeshi; Yang, Xi; Xiang, Chunsheng; Wang, Baohong; Chen, Yanfei; Yang, Fengling; Lei, Fang; Wu, Na; Lu, Na; Li, Jing; Chen, Quanze; Li, Lanjuan; Zhu, Baoli

    2012-11-01

    The human gut microbiota has a high density of bacteria that are considered a reservoir for antibiotic resistance genes (ARGs). In this study, one fosmid metagenomic library generated from the gut microbiota of four healthy humans was used to screen for ARGs against seven antibiotics. Eight new ARGs were obtained: one against amoxicillin, six against d-cycloserine, and one against kanamycin. The new amoxicillin resistance gene encodes a protein with 53% identity to a class D β-lactamase from Riemerella anatipestifer RA-GD. The six new d-cycloserine resistance genes encode proteins with 73-81% identity to known d-alanine-d-alanine ligases. The new kanamycin resistance gene encodes a protein of 274 amino acids with an N-terminus (amino acids 1-189) that has 42% identity to the 6'-aminoglycoside acetyltransferase [AAC(6')] from Enterococcus hirae and a C-terminus (amino acids 190-274) with 35% identity to a hypothetical protein from Clostridiales sp. SSC/2. A functional study on the novel kanamycin resistance gene showed that only the N-terminus conferred kanamycin resistance. Our results showed that functional metagenomics is a useful tool for the identification of new ARGs.

  2. Instantaneous inactivation of cofilin reveals its function of F-actin disassembly in lamellipodia.

    PubMed

    Vitriol, Eric A; Wise, Ariel L; Berginski, Mathew E; Bamburg, James R; Zheng, James Q

    2013-07-01

    Cofilin is a key regulator of the actin cytoskeleton. It can sever actin filaments, accelerate filament disassembly, act as a nucleation factor, recruit or antagonize other actin regulators, and control the pool of polymerization-competent actin monomers. In cells these actions have complex functional outputs. The timing and localization of cofilin activity are carefully regulated, and thus global, long-term perturbations may not be sufficient to probe its precise function. To better understand cofilin's spatiotemporal action in cells, we implemented chromophore-assisted laser inactivation (CALI) to instantly and specifically inactivate it. In addition to globally inhibiting actin turnover, CALI of cofilin generated several profound effects on the lamellipodia, including an increase of F-actin, a rearward expansion of the actin network, and a reduction in retrograde flow speed. These results support the hypothesis that the principal role of cofilin in lamellipodia at steady state is to break down F-actin, control filament turnover, and regulate the rate of retrograde flow.

  3. A symbiotic mutant of Sinorhizobium meliloti reveals a novel genetic pathway involving succinoglycan biosynthetic functions.

    PubMed

    Griffitts, Joel S; Long, Sharon R

    2008-03-01

    A large-scale screen for symbiotic mutants was carried out using the model root nodulating bacterium Sinorhizobium meliloti. Several mutations in the previously uncharacterized gene msbA2 were isolated. msbA2 encodes a member of the ATP-binding cassette exporter family. This protein family is known to export a wide variety of compounds from bacterial cells. S. meliloti MsbA2 is required for the invasion of nodule tissue, with msbA2 mutant cells stimulating nodule primordium morphogenesis, but failing to invade plant tissue beyond the epidermal cell layer. msbA2 mutants do not exhibit any of the free-living traits often found to correlate with symbiotic defects, suggesting that MsbA2 may take part in a specifically symbiotic function. In strains that overproduce the symbiotic signalling polysaccharide succinoglycan, loss of MsbA2 function is extremely deleterious. This synthetic lethal phenotype can be suppressed by disrupting the succinoglycan biosynthetic genes exoY or exoA. It can also be suppressed by disrupting putative glycosyltransferase-encoding genes found upstream of msbA2. Finally, the symbiotic phenotype of a msbA2 null mutant is suppressed by secondary mutations in these upstream transferase genes, indicating that the msbA2 mutant phenotype may be caused by an inhibitory accumulation of a novel polysaccharide that is synthesized from succinoglycan precursors.

  4. Axonal and dendritic synaptotagmin isoforms revealed by a pHluorin-syt functional screen.

    PubMed

    Dean, Camin; Dunning, F Mark; Liu, Huisheng; Bomba-Warczak, Ewa; Martens, Henrik; Bharat, Vinita; Ahmed, Saheeb; Chapman, Edwin R

    2012-05-01

    The synaptotagmins (syts) are a family of molecules that regulate membrane fusion. There are 17 mammalian syt isoforms, most of which are expressed in the brain. However, little is known regarding the subcellular location and function of the majority of these syts in neurons, largely due to a lack of isoform-specific antibodies. Here we generated pHluorin-syt constructs harboring a luminal domain pH sensor, which reports localization, pH of organelles to which syts are targeted, and the kinetics and sites of exocytosis and endocytosis. Of interest, only syt-1 and 2 are targeted to synaptic vesicles, whereas other isoforms selectively recycle in dendrites (syt-3 and 11), axons (syt-5, 7, 10, and 17), or both axons and dendrites (syt-4, 6, 9, and 12), where they undergo exocytosis and endocytosis with distinctive kinetics. Hence most syt isoforms localize to distinct secretory organelles in both axons and dendrites and may regulate neuropeptide/neurotrophin release to modulate neuronal function.

  5. Single-molecule imaging reveals a switch between spurious and functional ncRNA transcription

    PubMed Central

    Lenstra, Tineke L.; Coulon, Antoine; Chow, Carson C.; Larson, Daniel R.

    2015-01-01

    Summary Eukaryotic transcription is pervasive, and many of the resulting RNAs are non-coding. It is unknown if ubiquitous transcription is functional or simply reflects stochastic transcriptional noise. By single-molecule visualization of the dynamic interplay between coding and non-coding transcription at the GAL locus in living yeast cells, we show that antisense GAL10 ncRNA transcription can switch between functional and spurious under different conditions. During galactose induction, GAL10 sense transcription occurs in short stochastic bursts which are unaffected by transcription of antisense GAL10 ncRNA, even when both are present simultaneously at the same locus. In contrast, when GAL10 is not induced, ncRNA transcription is critical to prevent transcriptional leakage of GAL1 and GAL10. Suppression of ncRNA transcription by strand-specific CRISPR/dCas9 results in transcriptional leakage of the inducer GAL1, leading to a more sensitive transcription activation threshold, an alteration of metabolic switching, and a fitness defect in competition experiments. PMID:26549684

  6. Structural and functional insights into the conductive pili of Geobacter sulfurreducens revealed in molecular dynamics simulations.

    PubMed

    Feliciano, G T; Steidl, R J; Reguera, G

    2015-09-14

    Geobacter sulfurreducens (GS) electronically connects with extracellular electron acceptors using conductive protein filaments or pili. To gain insights into their role as biological nanowires, we investigated the structural dynamics of the GS pilus in solution via molecular dynamics simulations. In the model, all of the pilin's aromatics clustered as a right-handed helical band along the pilus, maintaining inter-aromatic distances and dimer configurations optimal for multistep hopping. The aromatics were interspersed within the regions of highest negative potential, which influenced the type and configuration of the aromatic contacts and the rates of electron transfer. Small foci of positive potential were also present but were neutralized within uncharged regions, thus minimizing charge trapping. Consistent with the model predictions, mutant strains with reduced aromatic contacts or negative potentials had defects in pili functions such as the reduction of Fe(III) oxides and electrodes. The results therefore support the notion of a pilus fiber evolved to function as an electronic conduit between the cell and extracellular electron acceptors.

  7. Purification and characterization of progenitor and mature human astrocytes reveals transcriptional and functional differences with mouse

    PubMed Central

    Zhang, Ye; Sloan, Steven A.; Clarke, Laura E.; Caneda, Christine; Plaza, Colton A.; Blumenthal, Paul D.; Vogel, Hannes; Steinberg, Gary K.; Edwards, Michael S. B.; Li, Gordon; Duncan, John A.; Cheshier, Samuel H.; Shuer, Lawrence M.; Chang, Edward F.; Grant, Gerald A.; Hayden Gephart, Melanie G.; Barres, Ben A.

    2015-01-01

    Summary The functional and molecular similarities and distinctions between human and murine astrocytes are poorly understood. Here we report the development of an immunopanning method to acutely purify astrocytes from fetal, juvenile, and adult human brains, and to maintain these cells in serum-free cultures. We found that human astrocytes have similar abilities to murine astrocytes in promoting neuronal survival, inducing functional synapse formation, and engulfing synaptosomes. In contrast to existing observations in mice, we found that mature human astrocytes respond robustly to glutamate. We next performed RNA-sequencing of healthy human astrocytes along with astrocytes from epileptic and tumor foci, and compared these to human neurons, oligodendrocytes, microglia, and endothelial cells. With these profiles, we identified novel human-specific astrocyte genes, and discovered a transcriptome-wide transformation between astrocyte precursor cells and mature post-mitotic astrocytes. These data represent some of the first cell type-specific molecular profiles of the healthy and diseased human brain. PMID:26687838

  8. Multivariate sequence analysis reveals additional function impacting residues in the SDR superfamily.

    PubMed

    Tiwari, Pratibha; Singh, Noopur; Dixit, Aparna; Choudhury, Devapriya

    2014-10-01

    The "extended" type of short chain dehydrogenases/reductases (SDR), share a remarkable similarity in their tertiary structures inspite of being highly divergent in their functions and sequences. We have carried out principal component analysis (PCA) on structurally equivalent residue positions of 10 SDR families using information theoretic measures like Jensen-Shannon divergence and average shannon entropy as variables. The results classify residue positions in the SDR fold into six groups, one of which is characterized by low Shannon entropies but high Jensen-Shannon divergence against the reference family SDR1E, suggesting that these positions are responsible for the specific functional identities of individual SDR families, distinguishing them from the reference family SDR1E. Site directed mutagenesis of three residues from this group in the enzyme UDP-Galactose 4-epimerase belonging to SDR1E shows that the mutants promote the formation of NADH containing abortive complexes. Finally, molecular dynamics simulations have been used to suggest a mechanism by which the mutants interfere with the re-oxidation of NADH leading to the formation of abortive complexes.

  9. Fractionation of the Gulf Toadfish Intestinal Precipitate Organic Matrix Reveals Potential Functions of Individual Proteins.

    PubMed

    Schauer, Kevin L; Grosell, Martin

    2017-03-15

    The regulatory mechanisms behind the production of CaCO3 in the marine teleost intestine are poorly studied despite being essential for osmoregulation and responsible for a conservatively estimated 3-15% of annual oceanic CaCO3 production. It has recently been reported that the intestinally derived precipitates produced by fish as a byproduct of their osmoregulatory strategy form in conjunction with a proteinaceous matrix containing nearly 150 unique proteins. The individual functions of these proteins have not been the subject of investigation until now. Here, organic matrix was extracted from precipitates produced by Gulf toadfish (Opsanus beta) and the matrix proteins were fractionated by their charge using strong anion exchange chromatography. The precipitation regulatory abilities of the individual fractions were then analyzed using a recently developed in vitro calcification assay, and the protein constituents of each fraction were determined by mass spectrometry. The different fractions were found to have differing effects on both the rate of carbonate mineral production, as well as the morphology of the crystals that form. Using data collected from the calcification assay as well as the mass spectrometry experiments, individual calcification promotional indices were calculated for each protein, giving the first insight into the functions each of these matrix proteins may play in regulating precipitation.

  10. Genetic analysis of yeast RPA1 reveals its multiple functions in DNA metabolism.

    PubMed Central

    Umezu, K; Sugawara, N; Chen, C; Haber, J E; Kolodner, R D

    1998-01-01

    Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 10(4) to 10(5) times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages. PMID:9539419

  11. Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function.

    PubMed

    Del Campo, Cristian; Bartholomäus, Alexander; Fedyunin, Ivan; Ignatova, Zoya

    2015-10-01

    Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation.

  12. Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function

    PubMed Central

    Fedyunin, Ivan; Ignatova, Zoya

    2015-01-01

    Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation. PMID:26495981

  13. Who is who in litter decomposition? Metaproteomics reveals major microbial players and their biogeochemical functions.

    PubMed

    Schneider, Thomas; Keiblinger, Katharina M; Schmid, Emanuel; Sterflinger-Gleixner, Katja; Ellersdorfer, Günther; Roschitzki, Bernd; Richter, Andreas; Eberl, Leo; Zechmeister-Boltenstern, Sophie; Riedel, Kathrin

    2012-09-01

    Leaf-litter decomposition is a central process in carbon cycling; however, our knowledge about the microbial regulation of this process is still scarce. Metaproteomics allows us to link the abundance and activity of enzymes during nutrient cycling to their phylogenetic origin based on proteins, the 'active building blocks' in the system. Moreover, we employed metaproteomics to investigate the influence of environmental factors and nutrients on the decomposer structure and function during beech litter decomposition. Litter was collected at forest sites in Austria with different litter nutrient content. Proteins were analyzed by 1-D-SDS-PAGE followed by liquid-chromatography and tandem mass-spectrometry. Mass spectra were assigned to phylogenetic and functional groups by a newly developed bioinformatics workflow, assignments being validated by complementary approaches. We provide evidence that the litter nutrient content and the stoichiometry of C:N:P affect the decomposer community structure and activity. Fungi were found to be the main producers of extracellular hydrolytic enzymes, with no bacterial hydrolases being detected by our metaproteomics approach. Detailed investigation of microbial succession suggests that it is influenced by litter nutrient content. Microbial activity was stimulated at higher litter nutrient contents via a higher abundance and activity of extracellular enzymes.

  14. Microbial structures, functions, and metabolic pathways in wastewater treatment bioreactors revealed using high-throughput sequencing.

    PubMed

    Ye, Lin; Zhang, Tong; Wang, Taitao; Fang, Zhiwei

    2012-12-18

    The objective of this study was to explore microbial community structures, functional profiles, and metabolic pathways in a lab-scale and a full-scale wastewater treatment bioreactors. In order to do this, over 12 gigabases of metagenomic sequence data and 600,000 paired-end sequences of bacterial 16S rRNA gene were generated with the Illumina HiSeq 2000 platform, using DNA extracted from activated sludge in the two bioreactors. Three kinds of sequences (16S rRNA gene amplicons, 16S rRNA gene sequences obtained from metagenomic sequencing, and predicted proteins) were used to conduct taxonomic assignments. Specially, relative abundances of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) were analyzed. Compared with quantitative real-time PCR (qPCR), metagenomic sequencing was demonstrated to be a better approach to quantify AOA and AOB in activated sludge samples. It was found that AOB were more abundant than AOA in both reactors. Furthermore, the analysis of the metabolic profiles indicated that the overall patterns of metabolic pathways in the two reactors were quite similar (73.3% of functions shared). However, for some pathways (such as carbohydrate metabolism and membrane transport), the two reactors differed in the number of pathway-specific genes.

  15. RNA interference in the appendicularian Oikopleura dioica reveals the function of the Brachyury gene.

    PubMed

    Omotezako, Tatsuya; Nishino, Atsuo; Onuma, Takeshi A; Nishida, Hiroki

    2013-07-01

    The appendicularian Oikopleura dioica is a chordate that has a remarkably simple adult body with small cell number. Its transparency, stereotyped cell lineages, short life cycle, and small genome make it a promising new experimental model of chordate developmental biology. However, the functions of its various genes are still poorly understood due to lack of a tool for suppression of gene expression. Here, we applied a double-stranded RNA (dsRNA)-based-RNA interference (RNAi) method in O. dioica. For introducing dsRNA into eggs and embryos, we injected dsRNAs into the ovary. dsRNA, which is specific to EGFP or mCherry mRNA, decreased the exogenous mRNA-derived fluorescence in both eggs and embryos. dsRNA specific to the Brachyury gene of O. dioica, which is a homologous gene of a key notochord transcriptional factor in ascidians, triggered degradation of endogenous Brachyury mRNA and induced malformation or loss of the notochord in the tail. This effect was Brachyury sequence specific, as three dsRNAs covering different sequences produced the same phenotype. The result is in accordance with its expression site and also with the key regulatory function of Brachyury in notochord formation in other chordates. RNAi in O. dioica would be a useful tool for gaining insight into the oogenesis and early developmental processes in chordates.

  16. Global analysis of SUMO chain function reveals multiple roles in chromatin regulation

    PubMed Central

    Srikumar, Tharan; Lewicki, Megan C.; Costanzo, Michael; Tkach, Johnny M.; van Bakel, Harm; Tsui, Kyle; Johnson, Erica S.; Brown, Grant W.; Andrews, Brenda J.; Boone, Charles; Giaever, Guri; Nislow, Corey

    2013-01-01

    Like ubiquitin, the small ubiquitin-related modifier (SUMO) proteins can form oligomeric “chains,” but the biological functions of these superstructures are not well understood. Here, we created mutant yeast strains unable to synthesize SUMO chains (smt3allR) and subjected them to high-content microscopic screening, synthetic genetic array (SGA) analysis, and high-density transcript profiling to perform the first global analysis of SUMO chain function. This comprehensive assessment identified 144 proteins with altered localization or intensity in smt3allR cells, 149 synthetic genetic interactions, and 225 mRNA transcripts (primarily consisting of stress- and nutrient-response genes) that displayed a >1.5-fold increase in expression levels. This information-rich resource strongly implicates SUMO chains in the regulation of chromatin. Indeed, using several different approaches, we demonstrate that SUMO chains are required for the maintenance of normal higher-order chromatin structure and transcriptional repression of environmental stress response genes in budding yeast. PMID:23547032

  17. Revealing the spiral arms through radial migration and the shape of the metallicity distribution function

    NASA Astrophysics Data System (ADS)

    Martinez-Medina, L. A.; Pichardo, B.; Moreno, E.; Peimbert, A.

    2016-11-01

    Recent observations show that the Milky Way's metallicity distribution function (MDF) changes its shape as a function of radius. This new evidence of radial migration within the stellar disc sets additional constraints on Galactic models. By performing controlled test particle simulations in a very detailed, observationally motivated model of the Milky Way, we demonstrate that, in the inner region of the disc, the MDF is shaped by the joint action of the bar and spiral arms, while at outer radii the MDF is mainly shaped by the spiral arms. We show that the spiral arms are able to imprint their signature in the radial migration, shaping the MDF in the outskirts of the Galactic disc with a minimal participation of the bar. Conversely, this work has the potential to characterize some structural and dynamical parameters of the spiral arms based on radial migration and the shape of the MDF. Finally, the resemblance obtained with this approximation to the MDF curves of the Galaxy as seen by APOGEE, show that a fundamental factor influencing their shape is the Galactic potential.

  18. Metagenomic analysis reveals microbial diversity and function in the rhizosphere soil of a constructed wetland.

    PubMed

    Bai, Yaohui; Liang, Jinsong; Liu, Ruiping; Hu, Chengzhi; Qu, Jiuhui

    2014-01-01

    Microbial communities play a critical role in the degradation of effluent contaminants in constructed wetlands. Many questions remain, however, regarding the role ofmicrobial communities in rhizospheric soil. In this study, we used metagenomic analysis to assess microbial community composition and function in a constructed wetland receiving surface water. The diversity of the microbial community of rhizosphere soil was found to be significantly greater than that of the wetland influent water. This enhancement is likely due to the availability of diverse habitats and nutrients provided by the wetland plants. From function annotation of metagenomic data, a number of biodegradation pathways associated with 14 xenobiotic compounds were identified in soil. Nitrogen fixation, nitrification and denitrification genes were semi-quantitatively analysed. By screening of manganese transformation genes, we found that the biological oxidation of Mn2+ (mainly catalysed by multicopper oxidase) in the influent water yielded insoluble Mn4+, which subsequently precipitated and were incorporated into the wetland soil. These data show that the use of metagenomic analysis can provide important new insights for the study of wetland ecosystems and, in particular, how biologically mediated transformation or degradation can be used to reduce contamination of point and non-point source wastewater.

  19. Global analysis of SUMO chain function reveals multiple roles in chromatin regulation.

    PubMed

    Srikumar, Tharan; Lewicki, Megan C; Costanzo, Michael; Tkach, Johnny M; van Bakel, Harm; Tsui, Kyle; Johnson, Erica S; Brown, Grant W; Andrews, Brenda J; Boone, Charles; Giaever, Guri; Nislow, Corey; Raught, Brian

    2013-04-01

    Like ubiquitin, the small ubiquitin-related modifier (SUMO) proteins can form oligomeric "chains," but the biological functions of these superstructures are not well understood. Here, we created mutant yeast strains unable to synthesize SUMO chains (smt3(allR)) and subjected them to high-content microscopic screening, synthetic genetic array (SGA) analysis, and high-density transcript profiling to perform the first global analysis of SUMO chain function. This comprehensive assessment identified 144 proteins with altered localization or intensity in smt3(allR) cells, 149 synthetic genetic interactions, and 225 mRNA transcripts (primarily consisting of stress- and nutrient-response genes) that displayed a >1.5-fold increase in expression levels. This information-rich resource strongly implicates SUMO chains in the regulation of chromatin. Indeed, using several different approaches, we demonstrate that SUMO chains are required for the maintenance of normal higher-order chromatin structure and transcriptional repression of environmental stress response genes in budding yeast.

  20. Microarray Technology Reveals Potentially Novel Genes and Pathways Involved in Non-Functioning Pituitary Adenomas

    PubMed Central

    Qiao, X; Wang, H; Wang, X; Zhao, B; Liu, J

    2016-01-01

    Abstract Microarray data of non-functioning pituitary adenomas (NFPAs) were analyzed to disclose novel genes and pathways involved in NFPA tumorigenesis. Raw microarray data were downloaded from Gene Expression Omnibus. Data pre-treatment and differential analysis were conducted using packages in R. Functional and pathway enrichment analyses were performed using package GOs-tats. A protein-protein interaction (PPI) network was constructed using server STRING and Cytoscape. Known genes involved in pituitary adenomas (PAs), were obtained from the Comparative Toxicogenomics Database. A total of 604 differentially expressed genes (DEGs) were identifed between NFPAs and controls, including 177 up- and 427 down-regulated genes. Jak-STAT and p53 signaling pathways were significantly enriched by DEGs. The PPI network of DEGs was constructed, containing 99 up- and 288 down-regulated known disease genes (e.g. EGFR and ESR1) as well as 16 up- and 17 down-regulated potential novel NFPAs-related genes (e.g. COL4A5, LHX3, MSN, and GHSR). Genes like COL4A5, LHX3, MSN, and GHSR and pathways such as p53 signaling and Jak-STAT signaling, might participate in NFPA development. Although further validations are required, these findings might provide guidance for future basic and therapy researches. PMID:28289583

  1. Optical nanoscopy to reveal structural and functional properties of liver cells (Presentation Recording)

    NASA Astrophysics Data System (ADS)

    McCourt, Peter; Huser, Thomas R.; Sørensen, Karen K.; Øie, Cristina I.; Mönkemöller, Viola; Ahluwalia, Balpreet S.

    2015-08-01

    The advent of optical nanoscopy has provided an opportunity to study fundamental properties of nanoscale biological functions, such as liver sinusoidal endothelial cells (LSEC) and their fenestrations. The fenestrations are nano-pores (50-200 nm) on the LSEC plasma membrane that allow free passage of molecules through cells. The fenestrated LSEC also hase a voracious appetite for waste molecules, viruses and nanoparticles. LSEC daily remove huge amounts of waste, nanoparticles and virus from the blood. Pharmaceuticals also need to pass through these fenestrations to be activated (e.g. cholesterol reducing statins) or detoxified by hepatocytes. And, when we age, our LSEC fenestrations become smaller and fewer. Today, we study these cells and structures using either conventional light microscopy on living cells, or high-resolution (but static) methods such as transmission and scanning electron microscopy on fixed (i.e. dead) tissue. Such methods, while very powerful, yield no real time information about the uptake of virus or nanoparticles, nor any information about fenestration dynamics. Therefore, to study LS-SEC, we are now using optical nanoscopy methods, and developing our own, to map their functions in 4 dimensions. Attaining this goal will shed new light on the cell biology of the liver and how it keeps us alive. This paper describes the challenges of studying LS-SEC with light microscopy, as well as current and potential solutions to this challenge using optical nanoscopy.

  2. The functional readthrough extension of malate dehydrogenase reveals a modification of the genetic code

    PubMed Central

    Hofhuis, Julia; Schueren, Fabian; Nötzel, Christopher; Lingner, Thomas; Gärtner, Jutta; Jahn, Olaf

    2016-01-01

    Translational readthrough gives rise to C-terminally extended proteins, thereby providing the cell with new protein isoforms. These may have different properties from the parental proteins if the extensions contain functional domains. While for most genes amino acid incorporation at the stop codon is far lower than 0.1%, about 4% of malate dehydrogenase (MDH1) is physiologically extended by translational readthrough and the actual ratio of MDH1x (extended protein) to ‘normal' MDH1 is dependent on the cell type. In human cells, arginine and tryptophan are co-encoded by the MDH1x UGA stop codon. Readthrough is controlled by the 7-nucleotide high-readthrough stop codon context without contribution of the subsequent 50 nucleotides encoding the extension. All vertebrate MDH1x is directed to peroxisomes via a hidden peroxisomal targeting signal (PTS) in the readthrough extension, which is more highly conserved than the extension of lactate dehydrogenase B. The hidden PTS of non-mammalian MDH1x evolved to be more efficient than the PTS of mammalian MDH1x. These results provide insight into the genetic and functional co-evolution of these dually localized dehydrogenases. PMID:27881739

  3. Speech processing asymmetry revealed by dichotic listening and functional brain imaging.

    PubMed

    Hugdahl, Kenneth; Westerhausen, René

    2016-12-01

    In this article, we review research in our laboratory from the last 25 to 30 years on the neuronal basis for laterality of speech perception focusing on the upper, posterior parts of the temporal lobes, and its functional and structural connections to other brain regions. We review both behavioral and brain imaging data, with a focus on dichotic listening experiments, and using a variety of imaging modalities. The data have come in most parts from healthy individuals and from studies on normally functioning brain, although we also review a few selected clinical examples. We first review and discuss the structural model for the explanation of the right-ear advantage (REA) and left hemisphere asymmetry for auditory language processing. A common theme across many studies have been our interest in the interaction between bottom-up, stimulus-driven, and top-down, instruction-driven, aspects of hemispheric asymmetry, and how perceptual factors interact with cognitive factors to shape asymmetry of auditory language information processing. In summary, our research have shown laterality for the initial processing of consonant-vowel syllables, first observed as a behavioral REA when subjects are required to report which syllable of a dichotic syllable-pair they perceive. In subsequent work we have corroborated the REA with brain imaging, and have shown that the REA is modulated through both bottom-up manipulations of stimulus properties, like sound intensity, and top-down manipulations of cognitive properties, like attention focus.

  4. Molecular dissection of zyxin function reveals its involvement in cell motility.

    PubMed

    Drees, B E; Andrews, K M; Beckerle, M C

    1999-12-27

    Spatially controlled actin filament assembly is critical for numerous processes, including the vectorial cell migration required for wound healing, cell- mediated immunity, and embryogenesis. One protein implicated in the regulation of actin assembly is zyxin, a protein concentrated at sites where the fast growing ends of actin filaments are enriched. To evaluate the role of zyxin in vivo, we developed a specific peptide inhibitor of zyxin function that blocks its interaction with alpha-actinin and displaces it from its normal subcellular location. Mislocalization of zyxin perturbs cell migration and spreading, and affects the behavior of the cell edge, a structure maintained by assembly of actin at sites proximal to the plasma membrane. These results support a role for zyxin in cell motility, and demonstrate that the correct positioning of zyxin within the cell is critical for its physiological function. Interestingly, the mislocalization of zyxin in the peptide-injected cells is accompanied by disturbances in the distribution of Ena/VASP family members, proteins that have a well-established role in promoting actin assembly. In concert with previous work, our findings suggest that zyxin promotes the spatially restricted assembly of protein complexes necessary for cell motility.

  5. Structural and functional insights into caseinolytic proteases reveal an unprecedented regulation principle of their catalytic triad.

    PubMed

    Zeiler, Evelyn; List, Anja; Alte, Ferdinand; Gersch, Malte; Wachtel, Rudolf; Poreba, Marcin; Drag, Marcin; Groll, Michael; Sieber, Stephan A

    2013-07-09

    Caseinolytic proteases (ClpPs) are large oligomeric protein complexes that contribute to cell homeostasis as well as virulence regulation in bacteria. Although most organisms possess a single ClpP protein, some organisms encode two or more ClpP isoforms. Here, we elucidated the crystal structures of ClpP1 and ClpP2 from pathogenic Listeria monocytogenes and observe an unprecedented regulation principle by the catalytic triad. Whereas L. monocytogenes (Lm)ClpP2 is both structurally and functionally similar to previously studied tetradecameric ClpP proteins from Escherichia coli and Staphylococcus aureus, heptameric LmClpP1 features an asparagine in its catalytic triad. Mutation of this asparagine to aspartate increased the reactivity of the active site and led to the assembly of a tetradecameric complex. We analyzed the heterooligomeric complex of LmClpP1 and LmClpP2 via coexpression and subsequent labeling studies with natural product-derived probes. Notably, the LmClpP1 peptidase activity is stimulated 75-fold in the complex providing insights into heterooligomerization as a regulatory mechanism. Collectively, our data point toward different preferences for substrates and inhibitors of the two ClpP enzymes and highlight their structural and functional characteristics.

  6. Comparative Genomic Analysis Reveals Organization, Function and Evolution of ars Genes in Pantoea spp.

    PubMed Central

    Wang, Liying; Wang, Jin; Jing, Chuanyong

    2017-01-01

    Numerous genes are involved in various strategies to resist toxic arsenic (As). However, the As resistance strategy in genus Pantoea is poorly understood. In this study, a comparative genome analysis of 23 Pantoea genomes was conducted. Two vertical genetic arsC-like genes without any contribution to As resistance were found to exist in the 23 Pantoea strains. Besides the two arsC-like genes, As resistance gene clusters arsRBC or arsRBCH were found in 15 Pantoea genomes. These ars clusters were found to be acquired by horizontal gene transfer (HGT) from sources related to Franconibacter helveticus, Serratia marcescens, and Citrobacter freundii. During the history of evolution, the ars clusters were acquired more than once in some species, and were lost in some strains, producing strains without As resistance capability. This study revealed the organization, distribution and the complex evolutionary history of As resistance genes in Pantoea spp.. The insights gained in this study improved our understanding on the As resistance strategy of Pantoea spp. and its roles in the biogeochemical cycling of As. PMID:28377759

  7. ‘Candidatus Competibacter'-lineage genomes retrieved from metagenomes reveal functional metabolic diversity

    PubMed Central

    McIlroy, Simon J; Albertsen, Mads; Andresen, Eva K; Saunders, Aaron M; Kristiansen, Rikke; Stokholm-Bjerregaard, Mikkel; Nielsen, Kåre L; Nielsen, Per H

    2014-01-01

    The glycogen-accumulating organism (GAO) ‘Candidatus Competibacter' (Competibacter) uses aerobically stored glycogen to enable anaerobic carbon uptake, which is subsequently stored as polyhydroxyalkanoates (PHAs). This biphasic metabolism is key for the Competibacter to survive under the cyclic anaerobic-‘feast': aerobic-‘famine' regime of enhanced biological phosphorus removal (EBPR) wastewater treatment systems. As they do not contribute to phosphorus (P) removal, but compete for resources with the polyphosphate-accumulating organisms (PAO), thought responsible for P removal, their proliferation theoretically reduces the EBPR capacity. In this study, two complete genomes from Competibacter were obtained from laboratory-scale enrichment reactors through metagenomics. Phylogenetic analysis identified the two genomes, ‘Candidatus Competibacter denitrificans' and ‘Candidatus Contendobacter odensis', as being affiliated with Competibacter-lineage subgroups 1 and 5, respectively. Both have genes for glycogen and PHA cycling and for the metabolism of volatile fatty acids. Marked differences were found in their potential for the Embden–Meyerhof–Parnas and Entner–Doudoroff glycolytic pathways, as well as for denitrification, nitrogen fixation, fermentation, trehalose synthesis and utilisation of glucose and lactate. Genetic comparison of P metabolism pathways with sequenced PAOs revealed the absence of the Pit phosphate transporter in the Competibacter-lineage genomes—identifying a key metabolic difference with the PAO physiology. These genomes are the first from any GAO organism and provide new insights into the complex interaction and niche competition between PAOs and GAOs in EBPR systems. PMID:24173461

  8. Novel gene function revealed by mouse mutagenesis screens for models of age-related disease

    PubMed Central

    Potter, Paul K.; Bowl, Michael R.; Jeyarajan, Prashanthini; Wisby, Laura; Blease, Andrew; Goldsworthy, Michelle E.; Simon, Michelle M.; Greenaway, Simon; Michel, Vincent; Barnard, Alun; Aguilar, Carlos; Agnew, Thomas; Banks, Gareth; Blake, Andrew; Chessum, Lauren; Dorning, Joanne; Falcone, Sara; Goosey, Laurence; Harris, Shelley; Haynes, Andy; Heise, Ines; Hillier, Rosie; Hough, Tertius; Hoslin, Angela; Hutchison, Marie; King, Ruairidh; Kumar, Saumya; Lad, Heena V.; Law, Gemma; MacLaren, Robert E.; Morse, Susan; Nicol, Thomas; Parker, Andrew; Pickford, Karen; Sethi, Siddharth; Starbuck, Becky; Stelma, Femke; Cheeseman, Michael; Cross, Sally H.; Foster, Russell G.; Jackson, Ian J.; Peirson, Stuart N.; Thakker, Rajesh V.; Vincent, Tonia; Scudamore, Cheryl; Wells, Sara; El-Amraoui, Aziz; Petit, Christine; Acevedo-Arozena, Abraham; Nolan, Patrick M.; Cox, Roger; Mallon, Anne-Marie; Brown, Steve D. M.

    2016-01-01

    Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss. PMID:27534441

  9. Function of the hydration layer around an antifreeze protein revealed by atomistic molecular dynamics simulations

    SciTech Connect

    Nutt, David; Smith, Jeremy C

    2008-10-01

    Atomistic molecular dynamics simulations are used to investigate the mechanism by which the antifreeze protein from the spruce budworm, Choristoneura fumiferana, binds to ice. Comparison of structural and dynamic properties of the water around the three faces of the triangular prism-shaped protein in aqueous solution reveals that at low temperature the water structure is ordered and the dynamics slowed down around the ice-binding face of the protein, with a disordering effect observed around the other two faces. These results suggest a dual role for the solvation water around the protein. The preconfigured solvation shell around the ice-binding face is involved in the initial recognition and binding of the antifreeze protein to ice by lowering the barrier for binding and consolidation of the protein:ice interaction surface. Thus, the antifreeze protein can bind to the molecularly rough ice surface by becoming actively involved in the formation of its own binding site. Also, the disruption of water structure around the rest of the protein helps prevent the adsorbed protein becoming covered by further ice growth.

  10. Sequencing of transcriptomes from two Miscanthus species reveals functional specificity in rhizomes, and clarifies evolutionary relationships

    PubMed Central

    2014-01-01

    Background Miscanthus is a promising biomass crop for temperate regions. Despite the increasing interest in this plant, limited sequence information has constrained research into its biology, physiology, and breeding. The whole genome transcriptomes of M. sinensis and M. sacchariflorus presented in this study may provide good resources to understand functional compositions of two important Miscanthus genomes and their evolutionary relationships. Results For M. sinensis, a total of 457,891 and 512,950 expressed sequence tags (ESTs) were produced from leaf and rhizome tissues, respectively, which were assembled into 12,166 contigs and 89,648 singletons for leaf, and 13,170 contigs and 112,138 singletons for rhizome. For M. sacchariflorus, a total of 288,806 and 267,952 ESTs from leaf and rhizome tissues, respectively, were assembled into 8,732 contigs and 66,881 singletons for leaf, and 8,104 contigs and 63,212 singletons for rhizome. Based on the distributions of synonymous nucleotide substitution (Ks), sorghum and Miscanthus diverged about 6.2 million years ago (MYA), Saccharum and Miscanthus diverged 4.6 MYA, and M. sinensis and M. sacchariflorus diverged 1.5 MYA. The pairwise alignment of predicted protein sequences from sorghum-Miscanthus and two Miscanthus species found a total of 43,770 and 35,818 nsSNPs, respectively. The impacts of striking mutations found by nsSNPs were much lower between sorghum and Miscanthus than those between the two Miscanthus species, perhaps as a consequence of the much higher level of gene duplication in Miscanthus and resulting ability to buffer essential functions against disturbance. Conclusions The ESTs generated in the present study represent a significant addition to Miscanthus functional genomics resources, permitting us to discover some candidate genes associated with enhanced biomass production. Ks distributions based on orthologous ESTs may serve as a guideline for future research into the evolution of Miscanthus species

  11. A FRET biosensor reveals spatiotemporal activation and functions of aurora kinase A in living cells.

    PubMed

    Bertolin, Giulia; Sizaire, Florian; Herbomel, Gaëtan; Reboutier, David; Prigent, Claude; Tramier, Marc

    2016-09-14

    Overexpression of AURKA is a major hallmark of epithelial cancers. It encodes the multifunctional serine/threonine kinase aurora A, which is activated at metaphase and is required for cell cycle progression; assessing its activation in living cells is mandatory for next-generation drug design. We describe here a Förster's resonance energy transfer (FRET) biosensor detecting the conformational changes of aurora kinase A induced by its autophosphorylation on Thr288. The biosensor functionally replaces the endogenous kinase in cells and allows the activation of the kinase to be followed throughout the cell cycle. Inhibiting the catalytic activity of the kinase prevents the conformational changes of the biosensor. Using this approach, we discover that aurora kinase A activates during G1 to regulate the stability of microtubules in cooperation with TPX2 and CEP192. These results demonstrate that the aurora kinase A biosensor is a powerful tool to identify new regulatory pathways controlling aurora kinase A activation.

  12. The crystal structures of the eukaryotic chaperonin CCT reveal its functional partitioning.

    PubMed

    Kalisman, Nir; Schröder, Gunnar F; Levitt, Michael

    2013-04-02

    In eukaryotes, CCT is essential for the correct and efficient folding of many cytosolic proteins, most notably actin and tubulin. Structural studies of CCT have been hindered by the failure of standard crystallographic analysis to resolve its eight different subunit types at low resolutions. Here, we exhaustively assess the R value fit of all possible CCT models to available crystallographic data of the closed and open forms with resolutions of 3.8 Å and 5.5 Å, respectively. This unbiased analysis finds the native subunit arrangements with overwhelming significance. The resulting structures provide independent crystallographic proof of the subunit arrangement of CCT and map major asymmetrical features of the particle onto specific subunits. The actin and tubulin substrates both bind around subunit CCT6, which shows other structural anomalies. CCT is thus clearly partitioned, both functionally and evolutionary, into a substrate-binding side that is opposite to the ATP-hydrolyzing side.

  13. Comparative analysis of human and bovine protein kinases reveals unique relationship and functional diversity.

    PubMed

    Kabir, Nuzhat N; Kazi, Julhash U

    2011-10-01

    Reversible protein phosphorylation by protein kinases and phosphatases is a common event in various cellular processes. The eukaryotic protein kinase superfamily, which is one of the largest superfamilies of eukaryotic proteins, plays several roles in cell signaling and diseases. We identified 482 eukaryotic protein kinases and 39 atypical protein kinases in the bovine genome, by searching publicly accessible genetic-sequence databases. Bovines have 512 putative protein kinases, each orthologous to a human kinase. Whereas orthologous kinase pairs are, on an average, 90.6% identical, orthologous kinase catalytic domain pairs are, on an average, 95.9% identical at the amino acid level. This bioinformatic study of bovine protein kinases provides a suitable framework for further characterization of their functional and structural properties.

  14. Single-molecule measurements of replisome composition and function reveal the mechanism of polymerase exchange

    NASA Astrophysics Data System (ADS)

    Loparo, Joseph

    2011-03-01

    A complete understanding of the molecular mechanisms underlying the functioning of large, multiprotein complexes requires experimental tools capable of simultaneously visualizing molecular architecture and enzymatic activity in real time. I will describe a novel single-molecule assay that combines the flow-stretching of individual DNA molecules to measure the activity of the DNA-replication machinery with the visualization of fluorescently labeled DNA polymerases at the replication fork. By correlating polymerase stoichiometry with DNA synthesis of T7 bacteriophage replisomes, we are able to quantitatively describe the mechanism of polymerase exchange. We find that even at relatively modest polymerase concentration (2 nM), soluble polymerases are recruited to an actively synthesizing replisome, dramatically increasing local polymerase concentration. These excess polymerases remain passively associated with the replisome through electrostatic interactions with the T7 helicase for 50 seconds until a stochastic and transient dissociation of the synthesizing polymerase from the primer-template allows for a polymerase exchange event to occur.

  15. Revealing the Supramolecular Nature of Side-Chain Terpyridine-Functionalized Polymer Networks

    PubMed Central

    Brassinne, Jérémy; Jochum, Florian D.; Fustin, Charles-André; Gohy, Jean-François

    2015-01-01

    Nowadays, finely controlling the mechanical properties of polymeric materials is possible by incorporating supramolecular motifs into their architecture. In this context, the synthesis of a side-chain terpyridine-functionalized poly(2-(dimethylamino)ethyl methacrylate) is reported via reversible addition-fragmentation chain transfer polymerization. By addition of transition metal ions, concentrated aqueous solutions of this polymer turn into metallo-supramolecular hydrogels whose dynamic mechanical properties are investigated by rotational rheometry. Hence, the possibility for the material to relax mechanical constrains via dissociation of transient cross-links is brought into light. In addition, the complex phenomena occurring under large oscillatory shear are interpreted in the context of transient networks. PMID:25569082

  16. Crystal structure of folliculin reveals a hidDENN function in genetically inherited renal cancer.

    PubMed

    Nookala, Ravi K; Langemeyer, Lars; Pacitto, Angela; Ochoa-Montaño, Bernardo; Donaldson, Jane C; Blaszczyk, Beata K; Chirgadze, Dimitri Y; Barr, Francis A; Bazan, J Fernando; Blundell, Tom L

    2012-08-01

    Mutations in the renal tumour suppressor protein, folliculin, lead to proliferative skin lesions, lung complications and renal cell carcinoma. Folliculin has been reported to interact with AMP-activated kinase, a key component of the mammalian target of rapamycin pathway. Most cancer-causing mutations lead to a carboxy-terminal truncation of folliculin, pointing to a functional importance of this domain in tumour suppression. We present here the crystal structure of folliculin carboxy-terminal domain and demonstrate that it is distantly related to differentially expressed in normal cells and neoplasia (DENN) domain proteins, a family of Rab guanine nucleotide exchange factors (GEFs). Using biochemical analysis, we show that folliculin has GEF activity, indicating that folliculin is probably a distantly related member of this class of Rab GEFs.

  17. High-resolution temporal analysis reveals a functional timeline for the molecular regulation of cytokinesis

    PubMed Central

    Davies, Tim; Jordan, Shawn N.; Chand, Vandana; Sees, Jennifer A.; Laband, Kimberley; Carvalho, Ana; Shirasu-Hiza, Mimi; Kovar, David R.; Dumont, Julien; Canman, Julie C.

    2014-01-01

    Summary To take full advantage of fast-acting temperature-sensitive mutations, thermal control must be extremely rapid. We developed the Therminator, a device capable of shifting sample temperature in ~17s while simultaneously imaging cell division in vivo. Applying this technology to six key regulators of cytokinesis, we found that each has a distinct temporal requirement in the C. elegans zygote. Specifically, myosin-II is required throughout cytokinesis until contractile ring closure. In contrast, formin-mediated actin nucleation is only required during assembly and early contractile ring constriction. Centralspindlin is required to maintain division after ring closure, though its GAP activity is only required until just prior to closure. Finally, the Chromosomal Passenger Complex is required for cytokinesis only early in mitosis, but not during metaphase or cytokinesis. Together, our results provide a precise functional timeline for molecular regulators of cytokinesis using the Therminator, a powerful tool for ultra-rapid protein inactivation. PMID:25073157

  18. High-resolution temporal analysis reveals a functional timeline for the molecular regulation of cytokinesis.

    PubMed

    Davies, Tim; Jordan, Shawn N; Chand, Vandana; Sees, Jennifer A; Laband, Kimberley; Carvalho, Ana X; Shirasu-Hiza, Mimi; Kovar, David R; Dumont, Julien; Canman, Julie C

    2014-07-28

    To take full advantage of fast-acting temperature-sensitive mutations, thermal control must be extremely rapid. We developed the Therminator, a device capable of shifting sample temperature in ~17 s while simultaneously imaging cell division in vivo. Applying this technology to six key regulators of cytokinesis, we found that each has a distinct temporal requirement in the Caenorhabditis elegans zygote. Specifically, myosin-II is required throughout cytokinesis until contractile ring closure. In contrast, formin-mediated actin nucleation is only required during assembly and early contractile ring constriction. Centralspindlin is required to maintain division after ring closure, although its GAP activity is only required until just prior to closure. Finally, the chromosomal passenger complex is required for cytokinesis only early in mitosis, but not during metaphase or cytokinesis. Together, our results provide a precise functional timeline for molecular regulators of cytokinesis using the Therminator, a powerful tool for ultra-rapid protein inactivation.

  19. Agonistic antibodies reveal the function of GPR56 in human glioma U87-MG cells.

    PubMed

    Ohta, Shigeyuki; Sakaguchi, Sayaka; Kobayashi, Yuki; Mizuno, Norikazu; Tago, Kenji; Itoh, Hiroshi

    2015-01-01

    GPR56 is a member of the adhesion G protein-coupled receptor (GPCR) and is highly expressed in parts of tumor cells. The involvement of GPR56 in tumorigenesis has been reported. We generated agonistic monoclonal antibodies against human GPR56 and analyzed the action and signaling pathway of GPR56. The antibodies inhibited cell migration through the Gq and Rho pathway in human glioma U87-MG cells. Co-immunoprecipitation analysis indicated that the interaction between the GPR56 extracellular domain and transmembrane domain was potentiated by agonistic antibodies. These results demonstrated that functional antibodies are invaluable tools for GPCR research and should open a new avenue for therapeutic treatment of tumors.

  20. Identical expression profiling of human and murine TIPE3 protein reveals links to its functions.

    PubMed

    Cui, Jian; Hao, Chunyan; Zhang, Wenqian; Shao, Jie; Zhang, Na; Zhang, Guizhong; Liu, Suxia

    2015-03-01

    Tumor necrosis factor-alpha-induced protein-8 like-3 (TNFAIP8L3, TIPE3) is a newly discovered member of TNFAIP8 family and regarded as a lipid second messenger transfer protein that promotes cancer. Yet the nature of the cells and tissues that express TIPE3 protein has not been determined. In this study, we examined TIPE3 expression in various murine and human tissues by immunohistochemistry and quantitative PCR. We found that TIPE3 expression was almost identical in most organs from human and mice. TIPE3 is a cytoplasmic protein expressed preferentially in epithelial-derived cells with secretory functions. Furthermore, TIPE3 protein is highly expressed in most human carcinoma cell lines. These results suggest that TIPE3 may play important roles in carcinogenesis and cell secretion.

  1. A FRET biosensor reveals spatiotemporal activation and functions of aurora kinase A in living cells

    PubMed Central

    Bertolin, Giulia; Sizaire, Florian; Herbomel, Gaëtan; Reboutier, David; Prigent, Claude; Tramier, Marc

    2016-01-01

    Overexpression of AURKA is a major hallmark of epithelial cancers. It encodes the multifunctional serine/threonine kinase aurora A, which is activated at metaphase and is required for cell cycle progression; assessing its activation in living cells is mandatory for next-generation drug design. We describe here a Förster's resonance energy transfer (FRET) biosensor detecting the conformational changes of aurora kinase A induced by its autophosphorylation on Thr288. The biosensor functionally replaces the endogenous kinase in cells and allows the activation of the kinase to be followed throughout the cell cycle. Inhibiting the catalytic activity of the kinase prevents the conformational changes of the biosensor. Using this approach, we discover that aurora kinase A activates during G1 to regulate the stability of microtubules in cooperation with TPX2 and CEP192. These results demonstrate that the aurora kinase A biosensor is a powerful tool to identify new regulatory pathways controlling aurora kinase A activation. PMID:27624869

  2. Metatranscriptomics reveals temperature-driven functional changes in microbiome impacting cheese maturation rate.

    PubMed

    De Filippis, Francesca; Genovese, Alessandro; Ferranti, Pasquale; Gilbert, Jack A; Ercolini, Danilo

    2016-02-25

    Traditional cheeses harbour complex microbial consortia that play an important role in shaping typical sensorial properties. However, the microbial metabolism is considered difficult to control. Microbial community succession and the related gene expression were analysed during ripening of a traditional Italian cheese, identifying parameters that could be modified to accelerate ripening. Afterwards, we modulated ripening conditions and observed consistent changes in microbial community structure and function. We provide concrete evidence of the essential contribution of non-starter lactic acid bacteria in ripening-related activities. An increase in the ripening temperature promoted the expression of genes related to proteolysis, lipolysis and amino acid/lipid catabolism and significantly increases the cheese maturation rate. Moreover, temperature-promoted microbial metabolisms were consistent with the metabolomic profiles of proteins and volatile organic compounds in the cheese. The results clearly indicate how processing-driven microbiome responses can be modulated in order to optimize production efficiency and product quality.

  3. Metatranscriptomics reveals temperature-driven functional changes in microbiome impacting cheese maturation rate

    SciTech Connect

    De Filippis, Francesca; Genovese, Alessandro; Ferranti, Pasquale; Gilbert, Jack A.; Ercolini, Danilo

    2016-02-25

    Traditional cheeses harbour complex microbial consortia that play an important role in shaping typical sensorial properties. However, the microbial metabolism is considered difficult to control. Microbial community succession and the related gene expression were analysed during ripening of a traditional Italian cheese, identifying parameters that could be modified to accelerate ripening. Afterwards, we modulated ripening conditions and observed consistent changes in microbial community structure and function. We provide concrete evidence of the essential contribution of non-starter lactic acid bacteria in ripening-related activities. An increase in the ripening temperature promoted the expression of genes related to proteolysis, lipolysis and amino acid/lipid catabolism and significantly increases the cheese maturation rate. Moreover, temperature-promoted microbial metabolisms were consistent with the metabolomic profiles of proteins and volatile organic compounds in the cheese. Finally, the results clearly indicate how processing-driven microbiome responses can be modulated in order to optimize production efficiency and product quality.

  4. Identical Expression Profiling of Human and Murine TIPE3 Protein Reveals Links to Its Functions

    PubMed Central

    Cui, Jian; Hao, Chunyan; Zhang, Wenqian; Shao, Jie; Zhang, Na; Zhang, Guizhong

    2015-01-01

    Tumor necrosis factor-alpha-induced protein-8 like-3 (TNFAIP8L3, TIPE3) is a newly discovered member of TNFAIP8 family and regarded as a lipid second messenger transfer protein that promotes cancer. Yet the nature of the cells and tissues that express TIPE3 protein has not been determined. In this study, we examined TIPE3 expression in various murine and human tissues by immunohistochemistry and quantitative PCR. We found that TIPE3 expression was almost identical in most organs from human and mice. TIPE3 is a cytoplasmic protein expressed preferentially in epithelial-derived cells with secretory functions. Furthermore, TIPE3 protein is highly expressed in most human carcinoma cell lines. These results suggest that TIPE3 may play important roles in carcinogenesis and cell secretion. PMID:25479791

  5. Metatranscriptomics reveals temperature-driven functional changes in microbiome impacting cheese maturation rate

    PubMed Central

    De Filippis, Francesca; Genovese, Alessandro; Ferranti, Pasquale; Gilbert, Jack A.; Ercolini, Danilo

    2016-01-01

    Traditional cheeses harbour complex microbial consortia that play an important role in shaping typical sensorial properties. However, the microbial metabolism is considered difficult to control. Microbial community succession and the related gene expression were analysed during ripening of a traditional Italian cheese, identifying parameters that could be modified to accelerate ripening. Afterwards, we modulated ripening conditions and observed consistent changes in microbial community structure and function. We provide concrete evidence of the essential contribution of non-starter lactic acid bacteria in ripening-related activities. An increase in the ripening temperature promoted the expression of genes related to proteolysis, lipolysis and amino acid/lipid catabolism and significantly increases the cheese maturation rate. Moreover, temperature-promoted microbial metabolisms were consistent with the metabolomic profiles of proteins and volatile organic compounds in the cheese. The results clearly indicate how processing-driven microbiome responses can be modulated in order to optimize production efficiency and product quality. PMID:26911915

  6. Principles of motivation revealed by the diverse functions of neuropharmacological and neuroanatomical substrates underlying feeding behavior

    PubMed Central

    Baldo, Brian A.; Pratt, Wayne E.; Will, Matthew J.; Hanlon, Erin C.; Bakshi, Vaishali P.; Cador, Martine

    2013-01-01

    Circuits that participate in specific subcomponents of feeding (e.g., gustatory perception, peripheral feedback relevant to satiety and energy balance, reward coding, etc.) are found at all levels of the neural axis. Further complexity is conferred by the wide variety of feeding-modulatory neurotransmitters and neuropeptides that act within these circuits. An ongoing challenge has been to refine the understanding of the functional specificity of these neurotransmitters and circuits, and there have been exciting advances in recent years. We focus here on foundational work of Dr. Ann Kelley that identified distinguishable actions of striatal opioid peptide modulation and dopamine transmission in subcomponents of reward processing. We also discuss her work in overlaying these neuropharmacological effects upon anatomical pathways that link the telencephalon (cortex and basal ganglia) with feeding-control circuits in the hypothalamus. Using these seminal contributions as a starting point, we will discuss new findings that expand our understanding of (1) the specific, differentiable motivational processes that are governed by central dopamine and opioid transmission, (2) the manner in which other striatal neuromodulators, specifically acetylcholine, endocannabinoids and adenosine, modulate these motivational processes (including via interactions with opioid systems), and (3) the organization of the cortical-subcortical network that subserves opioid-driven feeding. The findings discussed here strengthen the view that incentive-motivational properties of food are coded by substrates and neural circuits that are distinguishable from those that mediate the acute hedonic experience of food reward. Striatal opioid transmission modulates reward processing by engaging frontotemporal circuits, possibly via a hypothalamic-thalamic axis, that ultimately impinges upon hypothalamic modules dedicated to autonomic function and motor pattern control. We will conclude by discussing

  7. Otx2 ChIP-seq Reveals Unique and Redundant Functions in the Mature Mouse Retina

    PubMed Central

    Fant, Bruno; Lamonerie, Thomas

    2014-01-01

    During mouse retinal development and into adulthood, the transcription factor Otx2 is expressed in pigment epithelium, photoreceptors and bipolar cells. In the mature retina, Otx2 ablation causes photoreceptor degeneration through a non-cell-autonomous mechanism involving Otx2 function in the supporting RPE. Surprisingly, photoreceptor survival does not require Otx2 expression in the neural retina, where the related Crx homeobox gene, a major regulator of photoreceptor development, is also expressed. To get a deeper view of mouse Otx2 activities in the neural retina, we performed chromatin-immunoprecipitation followed by massively parallel sequencing (ChIP-seq) on Otx2. Using two independent ChIP-seq assays, we identified consistent sets of Otx2-bound cis-regulatory elements. Comparison with our previous RPE-specific Otx2 ChIP-seq data shows that Otx2 occupies different functional domains of the genome in RPE cells and in neural retina cells and regulates mostly different sets of genes. To assess the potential redundancy of Otx2 and Crx, we compared our data with Crx ChIP-seq data. While Crx genome occupancy markedly differs from Otx2 genome occupancy in the RPE, it largely overlaps that of Otx2 in the neural retina. Thus, in accordance with its essential role in the RPE and its non-essential role in the neural retina, Otx2 regulates different gene sets in the RPE and the neural retina, and shares an important part of its repertoire with Crx in the neural retina. Overall, this study provides a better understanding of gene-regulatory networks controlling photoreceptor homeostasis and disease. PMID:24558479

  8. Crustal structure and deformation under the Longmenshan and its surroundings revealed by receiver function data

    NASA Astrophysics Data System (ADS)

    Sun, Ya; Liu, Jianxin; Zhou, Keping; Chen, Bo; Guo, Rongwen

    2015-07-01

    The convergence of India and Eurasia and the obstruction from the rigid Sichuan Basin cause the Longmenshan (LMS) to have the steepest topographic gradient at the eastern margin of the Tibetan Plateau. However, the mechanisms of surface uplift are still controversial. In this paper, we estimate the crustal structure and deformation under the LMS and its surroundings by analyzing a large amount of receiver function data recorded by regional seismic networks of the China Earthquake Administration. We apply a comprehensive splitting measurement technique on Ps conversion phase at the Moho (Moho Ps splitting) to calculate crustal anisotropy from azimuthal variations of receiver functions. Our results show that most of the seismic stations beneath the LMS area exhibit significant seismic anisotropy with the splitting time of 0.22-0.94 s and a fast polarization direction of NW-SE, while less or even no crustal anisotropy has been observed under the Sichuan Basin. Comparing the fast polarization directions of Moho Ps splitting with the indicators of lithospheric deformation (such as shear wave splitting, absolute plate motion, and global positioning system) imply a consistent tendency of deformation between the lower crust and upper mantle, but decoupling deformation in the crust beneath the LMS area. We further compare Moho Ps splitting time to that estimated from previous SKS splitting, indicating that crustal anisotropy is an important source of the SKS splitting time in this study area. In addition, a thick crust (>50 km) with high Vp/Vs values (1.74-1.86) is also observed using the H-κ stacking method. These seismic observations are consistent with the scenario that the LMS area has been built by the lower crustal flow. Combined with the seismic reflection/refraction profile and geology studies, we further suggest that the lower crustal flow may extrude upward into the upper crust along the steeply dipping strike faults under the LMS area, resulting in the surface

  9. Functional somatotopy revealed across multiple cortical regions using a model of complex motor task

    PubMed Central

    Cunningham, David A.; Machado, Andre; Yue, Guang H.; Carey, Jim R.; Plow, Ela B.

    2014-01-01

    The primary motor cortex (M1) possesses a functional somatotopic structure -representations of adjacent within-limb joints overlap to facilitate coordination while maintaining discrete centers for individuated movement. We examined whether similar organization exists across other sensorimotor cortices. Twenty-four right-handed healthy subjects underwent functional Magnetic Resonance Imaging (fMRI) while tracking complex targets with flexion/extension at right finger, elbow and ankle separately. Activation related to each joint at false discovery rate of .005 served as its representation across multiple regions. Within each region, we identified the Center of Mass (COM) for each representation, and overlap between representations of within-limb (finger and elbow) and between-limb joints (finger and ankle). Somatosensory (S1) and premotor cortices (PMC) demonstrated greater distinction of COM and minimal overlap for within- and between-limb representations. Contrarily, M1 and supplementary motor area (SMA) showed more integrative somatotopy with higher sharing for within-limb representations. Superior and inferior parietal lobule (SPL and IPL) possessed both types of structure. Some clusters exhibited extensive overlap of within- and between-limb representations, while others showed discrete COMs for within-limb representations. Our results help infer hierarchy in motor control. Areas as S1 may be associated with individuated movements, while M1 may be more integrative for coordinated motion; parietal associative regions may allow switch between both modes of control. Such hierarchy creates redundant opportunities to exploit in stroke rehabilitation. Use of complex rather than traditionally used simple movements was integral to illustrating comprehensive somatotopic structure; complex tasks can potentially help understand cortical representation of skill and learning-related plasticity. PMID:23920009

  10. Metabolic Profiling of Right Ventricular-Pulmonary Vascular Function Reveals Circulating Biomarkers of Pulmonary Hypertension

    PubMed Central

    Lewis, Gregory D.; Ngo, Debby; Hemnes, Anna R.; Farrell, Laurie; Domos, Carly; Pappagianopoulos, Paul P.; Dhakal, Bishnu P.; Souza, Amanda; Shi, Xu; Pugh, Meredith E.; Beloiartsev, Arkadi; Sinha, Sumita; Clish, Clary B.; Gerszten, Robert E.

    2016-01-01

    BACKGROUND Pulmonary hypertension and associated right ventricular (RV) dysfunction are important determinants of morbidity and mortality, which are optimally characterized by invasive hemodynamic measurements. OBJECTIVES This study sought to determine whether metabolite profiling could identify plasma signatures of right ventricular-pulmonary vascular (RV-PV) dysfunction. METHODS We measured plasma concentrations of 105 metabolites using targeted mass spectrometry in 71 individuals (discovery cohort) who underwent comprehensive physiological assessment with right-sided heart catheterization and radionuclide ventriculography at rest and during exercise. Our findings were validated in a second cohort undergoing invasive hemodynamic evaluations (n = 71), as well as in an independent cohort with or without known pulmonary arterial (PA) hypertension (n = 30). RESULTS In the discovery cohort, 21 metabolites were associated with 2 or more hemodynamic indicators of RV-PV function (i.e., resting right atrial pressure, mean PA pressure, pulmonary vascular resistance [PVR], and PVR and PA pressure-flow response [ΔPQ] during exercise). We identified novel associations of RV-PV dysfunction with circulating indoleamine 2,3-dioxygenase (IDO)–dependent tryptophan metabolites (TMs), tricarboxylic acid intermediates, and purine metabolites and confirmed previously described associations with arginine–nitric oxide metabolic pathway constituents. IDO-TM levels were inversely related to RV ejection fraction and were particularly well correlated with exercise PVR and ΔPQ. Multisite sampling demonstrated transpulmonary release of IDO-TMs. IDO-TMs also identified RV-PV dysfunction in a validation cohort with known risk factors for pulmonary hypertension and in patients with established PA hypertension. CONCLUSIONS Metabolic profiling identified reproducible signatures of RV-PV dysfunction, highlighting both new biomarkers and pathways for further functional characterization. PMID

  11. Functional specialization among insect chitinase family genes revealed by RNA interference.

    PubMed

    Zhu, Qingsong; Arakane, Yasuyuki; Beeman, Richard W; Kramer, Karl J; Muthukrishnan, Subbaratnam

    2008-05-06

    The biological functions of individual members of the large family of chitinase-like proteins from the red flour beetle, Tribolium castaneum (Tc), were examined by using gene-specific RNAi. One chitinase, TcCHT5, was found to be required for pupal-adult molting only. A lethal phenotype was observed when the transcript level of TcCHT5 was down-regulated by injection of TcCHT5-specific dsRNA into larvae. The larvae had metamorphosed into pupae and then to pharate adults but did not complete adult eclosion. Specific knockdown of transcripts for another chitinase, TcCHT10, which has multiple catalytic domains, prevented embryo hatch, larval molting, pupation, and adult metamorphosis, indicating a vital role for TcCHT10 during each of these processes. A third chitinase-like protein, TcCHT7, was required for abdominal contraction and wing/elytra extension immediately after pupation but was dispensable for larval-larval molting, pupation, and adult eclosion. The wing/elytra abnormalities found in TcCHT7-silenced pupae were also manifest in the ensuing adults. A fourth chitinase-like protein, TcIDGF4, exhibited no chitinolytic activity but contributed to adult eclosion. No phenotypic effects were observed after knockdown of transcripts for several other chitinase-like proteins, including imaginal disk growth factor IDGF2. These data indicate functional specialization among insect chitinase family genes, primarily during the molting process, and provide a biological rationale for the presence of a large assortment of chitinase-like proteins.

  12. Comparative proteomics reveals a significant bias toward alternative protein isoforms with conserved structure and function.

    PubMed

    Ezkurdia, Iakes; del Pozo, Angela; Frankish, Adam; Rodriguez, Jose Manuel; Harrow, Jennifer; Ashman, Keith; Valencia, Alfonso; Tress, Michael L

    2012-09-01

    Advances in high-throughput mass spectrometry are making proteomics an increasingly important tool in genome annotation projects. Peptides detected in mass spectrometry experiments can be used to validate gene models and verify the translation of putative coding sequences (CDSs). Here, we have identified peptides that cover 35% of the genes annotated by the GENCODE consortium for the human genome as part of a comprehensive analysis of experimental spectra from two large publicly available mass spectrometry databases. We detected the translation to protein of "novel" and "putative" protein-coding transcripts as well as transcripts annotated as pseudogenes and nonsense-mediated decay targets. We provide a detailed overview of the population of alternatively spliced protein isoforms that are detectable by peptide identification methods. We found that 150 genes expressed multiple alternative protein isoforms. This constitutes the largest set of reliably confirmed alternatively spliced proteins yet discovered. Three groups of genes were highly overrepresented. We detected alternative isoforms for 10 of the 25 possible heterogeneous nuclear ribonucleoproteins, proteins with a key role in the splicing process. Alternative isoforms generated from interchangeable homologous exons and from short indels were also significantly enriched, both in human experiments and in parallel analyses of mouse and Drosophila proteomics experiments. Our results show that a surprisingly high proportion (almost 25%) of the detected alternative isoforms are only subtly different from their constitutive counterparts. Many of the alternative splicing events that give rise to these alternative isoforms are conserved in mouse. It was striking that very few of these conserved splicing events broke Pfam functional domains or would damage globular protein structures. This evidence of a strong bias toward subtle differences in CDS and likely conserved cellular function and structure is remarkable and

  13. Loft features reveal the functioning of the young pigeon's navigational system

    NASA Astrophysics Data System (ADS)

    Jorge, Paulo; Silva, Inês; Vicente, Luis

    2008-03-01

    It is thought that young homing pigeons are able to use information acquired en route for their initial homeward orientation. However, the cues involved and mechanisms utilised are under discussion. Blocking light-dependent route-specific information during the first leg of an outward journey detour, together with analysis of pigeons that were raised under different loft conditions, allowed us to correctly evaluate the functioning of this mechanism and, more generally, the navigational map of birds. Pigeons from the same stock were raised and kept in two different lofts. The birds in the experimental groups were transported to the release sites via detours, and light-dependent information was denied during the first half of the outward journey (no compass information was available). Control birds were transported by the most direct route and had access to all available information. In general, the results showed that the low-loft birds preferred to use magnetic compass cues, whereas the high-loft birds preferred to use navigational map cues to collect information of the first part of the outward journey. The impairments observed in the homing performances of the experimental groups highlight the reliability of information collected inside the map area. Relevant to an understanding of the route-reversal mechanism was the evidence that this mechanism is able to function in the absence of compass information (birds raised in a wind-exposed loft show a detour effect). In systems where directional information could be provided by multiple sources, processing and extracting accurate course trajectories through a common mechanism may prove more efficient and reliable.

  14. Combined structural and functional imaging reveals cortical deactivations in grapheme-color synaesthesia

    PubMed Central

    O'Hanlon, Erik; Newell, Fiona N.; Mitchell, Kevin J.

    2013-01-01

    Synaesthesia is a heritable condition in which particular stimuli generate specific and consistent sensory percepts or associations in another modality or processing stream. Functional neuroimaging studies have identified potential correlates of these experiences, including, in some but not all cases, the hyperactivation of visuotemporal areas and of parietal areas thought to be involved in perceptual binding. Structural studies have identified a similarly variable spectrum of differences between synaesthetes and controls. However, it remains unclear the extent to which these neural correlates reflect the synaesthetic experience itself or additional phenotypes associated with the condition. Here, we acquired both structural and functional neuroimaging data comparing thirteen grapheme-color synaesthetes with eleven non-synaesthetes. Using voxel-based morphometry and diffusion tensor imaging, we identify a number of clusters of increased volume of gray matter, of white matter or of increased fractional anisotropy in synaesthetes vs. controls. To assess the possible involvement of these areas in the synaesthetic experience, we used nine areas of increased gray matter volume as regions of interest in an fMRI experiment that characterized the contrast in response to stimuli which induced synaesthesia (i.e., letters) vs. those which did not (non-meaningful symbols). Four of these areas showed sensitivity to this contrast in synaesthetes but not controls. Unexpectedly, in two of them, in left lateral occipital cortex and in postcentral gyrus, the letter stimuli produced a strong negative BOLD signal in synaesthetes. An additional whole-brain fMRI analysis identified 14 areas, three of which were driven mainly by a negative BOLD response to letters in synaesthetes. Our findings suggest that cortical deactivations may be involved in the conscious experience of internally generated synaesthetic percepts. PMID:24198794

  15. Metatranscriptomes reveal functional variation in diatom communities from the Antarctic Peninsula.

    PubMed

    Pearson, Gareth A; Lago-Leston, Asuncion; Cánovas, Fernando; Cox, Cymon J; Verret, Frederic; Lasternas, Sebastian; Duarte, Carlos M; Agusti, Susana; Serrão, Ester A

    2015-10-01

    Functional genomics of diatom-dominated communities from the Antarctic Peninsula was studied using comparative metatranscriptomics. Samples obtained from diatom-rich communities in the Bransfield Strait, the western Weddell Sea and sea ice in the Bellingshausen Sea/Wilkins Ice Shelf yielded more than 500K pyrosequencing reads that were combined to produce a global metatranscriptome assembly. Multi-gene phylogenies recovered three distinct communities, and diatom-assigned contigs further indicated little read-sharing between communities, validating an assembly-based annotation and analysis approach. Although functional analysis recovered a core of abundant shared annotations that were expressed across the three diatom communities, over 40% of annotations (but accounting for <10% of sequences) were community-specific. The two pelagic communities differed in their expression of N-metabolism and acquisition genes, which was almost absent in post-bloom conditions in the Weddell Sea community, while enrichment of transporters for ammonia and urea in Bransfield Strait diatoms suggests a physiological stance towards acquisition of reduced N-sources. The depletion of carbohydrate and energy metabolism pathways in sea ice relative to pelagic communities, together with increased light energy dissipation (via LHCSR proteins), photorespiration, and NO3(-) uptake and utilization all pointed to irradiance stress and/or inorganic carbon limitation within sea ice. Ice-binding proteins and cold-shock transcription factors were also enriched in sea ice diatoms. Surprisingly, the abundance of gene transcripts for the translational machinery tracked decreasing environmental temperature across only a 4 °C range, possibly reflecting constraints on translational efficiency and protein production in cold environments.

  16. Functional Metagenomics of a Biostimulated Petroleum-Contaminated Soil Reveals an Extraordinary Diversity of Extradiol Dioxygenases.

    PubMed

    Terrón-González, Laura; Martín-Cabello, Guadalupe; Ferrer, Manuel; Santero, Eduardo

    2016-04-01

    A metagenomic library of a petroleum-contaminated soil was constructed in a fosmid vector that allowed heterologous expression of metagenomic DNA. The library, consisting of 6.5 Gb of metagenomic DNA, was screened for extradiol dioxygenase (Edo) activity using catechol and 2,3-dihydroxybiphenyl as the substrates. Fifty-eight independent clones encoding extradiol dioxygenase activity were identified. Forty-one different Edo-encoding genes were identified. The population of Edo genes was not dominated by a particular gene or by highly similar genes; rather, the genes had an even distribution and high diversity. Phylogenetic analyses revealed that most of the genes could not be ascribed to previously defined subfamilies of Edos. Rather, the Edo genes led to the definition of 10 new subfamilies of type I Edos. Phylogenetic analysis of type II enzymes defined 7 families, 2 of which harbored the type II Edos that were found in this work. Particularly striking was the diversity found in family I.3 Edos; 15 out of the 17 sequences assigned to this family belonged to 7 newly defined subfamilies. A strong bias was found that depended on the substrate used for the screening: catechol mainly led to the detection of Edos belonging to the I.2 family, while 2,3-dihydroxybiphenyl led to the detection of most other Edos. Members of the I.2 family showed a clear substrate preference for monocyclic substrates, while those from the I.3 family showed a broader substrate range and high activity toward 2,3-dihydroxybiphenyl. This metagenomic analysis has substantially increased our knowledge of the existing biodiversity of Edos.

  17. Functional electric field changes in photoactivated proteins revealed by ultrafast Stark spectroscopy of the Trp residues

    PubMed Central

    Léonard, Jérémie; Portuondo-Campa, Erwin; Cannizzo, Andrea; van Mourik, Frank; van der Zwan, Gert; Tittor, Jörg; Haacke, Stefan; Chergui, Majed

    2009-01-01

    Ultrafast transient absorption spectroscopy of wild-type bacteriorhodopsin (WT bR) and 2 tryptophan mutants (W86F and W182F) is performed with visible light excitation (pump) and UV probe. The aim is to investigate the photoinduced change in the charge distribution with 50-fs time resolution by probing the effects on the tryptophan absorption bands. A systematic, quantitative comparison of the transient absorption of the 3 samples is carried out. The main result is the absence in the W86F mutant of a transient induced absorption band observed at ≈300–310 nm in WT bR and W182F. A simple model describing the dipolar interaction of the retinal moiety with the 2 tryptophan residues of interest allows us to reproduce the dominant features of the transient signals observed in the 3 samples at ultrashort pump-probe delays. In particular, we show that Trp86 undergoes a significant Stark shift induced by the transient retinal dipole moment. The corresponding transient signal can be isolated by direct subtraction of experimental data obtained for WT bR and W86F. It shows an instantaneous rise, followed by a decay over ≈500 fs corresponding to the isomerization time. Interestingly, it does not decay back to zero, thus revealing a change in the local electrostatic environment that remains long after isomerization, in the K intermediate state of the protein cycle. The comparison of WT bR and W86F also leads to a revised interpretation of the overall transient UV absorption of bR. PMID:19416877

  18. Comprehensive Gene Expression Profiling Reveals Synergistic Functional Networks in Cerebral Vessels after Hypertension or Hypercholesterolemia

    PubMed Central

    Ong, Wei-Yi; Ng, Mary Pei-Ern; Loke, Sau-Yeen; Jin, Shalai; Wu, Ya-Jun; Tanaka, Kazuhiro; Wong, Peter Tsun-Hon

    2013-01-01

    Atherosclerotic stenosis of cerebral arteries or intracranial large artery disease (ICLAD) is a major cause of stroke especially in Asians, Hispanics and Africans, but relatively little is known about gene expression changes in vessels at risk. This study compares comprehensive gene expression profiles in the middle cerebral artery (MCA) of New Zealand White rabbits exposed to two stroke risk factors i.e. hypertension and/or hypercholesterolemia, by the 2-Kidney-1-Clip method, or dietary supplementation with cholesterol. Microarray and Ingenuity Pathway Analyses of the MCA of the hypertensive rabbits showed up-regulated genes in networks containing the node molecules: UBC (ubiquitin), P38 MAPK, ERK, NFkB, SERPINB2, MMP1 and APP (amyloid precursor protein); and down-regulated genes related to MAPK, ERK 1/2, Akt, 26 s proteasome, histone H3 and UBC. The MCA of hypercholesterolemic rabbits showed differentially expressed genes that are surprisingly, linked to almost the same node molecules as the hypertensive rabbits, despite a relatively low percentage of ‘common genes’ (21 and 7%) between the two conditions. Up-regulated common genes were related to: UBC, SERPINB2, TNF, HNF4A (hepatocyte nuclear factor 4A) and APP, and down-regulated genes, related to UBC. Increased HNF4A message and protein were verified in the aorta. Together, these findings reveal similar nodal molecules and gene pathways in cerebral vessels affected by hypertension or hypercholesterolemia, which could be a basis for synergistic action of risk factors in the pathogenesis of ICLAD. PMID:23874591

  19. Functional Metagenomics of a Biostimulated Petroleum-Contaminated Soil Reveals an Extraordinary Diversity of Extradiol Dioxygenases

    PubMed Central

    Terrón-González, Laura; Martín-Cabello, Guadalupe; Ferrer, Manuel

    2016-01-01

    A metagenomic library of a petroleum-contaminated soil was constructed in a fosmid vector that allowed heterologous expression of metagenomic DNA. The library, consisting of 6.5 Gb of metagenomic DNA, was screened for extradiol dioxygenase (Edo) activity using catechol and 2,3-dihydroxybiphenyl as the substrates. Fifty-eight independent clones encoding extradiol dioxygenase activity were identified. Forty-one different Edo-encoding genes were identified. The population of Edo genes was not dominated by a particular gene or by highly similar genes; rather, the genes had an even distribution and high diversity. Phylogenetic analyses revealed that most of the genes could not be ascribed to previously defined subfamilies of Edos. Rather, the Edo genes led to the definition of 10 new subfamilies of type I Edos. Phylogenetic analysis of type II enzymes defined 7 families, 2 of which harbored the type II Edos that were found in this work. Particularly striking was the diversity found in family I.3 Edos; 15 out of the 17 sequences assigned to this family belonged to 7 newly defined subfamilies. A strong bias was found that depended on the substrate used for the screening: catechol mainly led to the detection of Edos belonging to the I.2 family, while 2,3-dihydroxybiphenyl led to the detection of most other Edos. Members of the I.2 family showed a clear substrate preference for monocyclic substrates, while those from the I.3 family showed a broader substrate range and high activity toward 2,3-dihydroxybiphenyl. This metagenomic analysis has substantially increased our knowledge of the existing biodiversity of Edos. PMID:26896130

  20. The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology

    PubMed Central

    Schmitt-Engel, Christian; Schultheis, Dorothea; Schwirz, Jonas; Ströhlein, Nadi; Troelenberg, Nicole; Majumdar, Upalparna; Dao, Van Anh; Grossmann, Daniela; Richter, Tobias; Tech, Maike; Dönitz, Jürgen; Gerischer, Lizzy; Theis, Mirko; Schild, Inga; Trauner, Jochen; Koniszewski, Nikolaus D. B.; Küster, Elke; Kittelmann, Sebastian; Hu, Yonggang; Lehmann, Sabrina; Siemanowski, Janna; Ulrich, Julia; Panfilio, Kristen A.; Schröder, Reinhard; Morgenstern, Burkhard; Stanke, Mario; Buchhholz, Frank; Frasch, Manfred; Roth, Siegfried; Wimmer, Ernst A.; Schoppmeier, Michael; Klingler, Martin; Bucher, Gregor

    2015-01-01

    Genetic screens are powerful tools to identify the genes required for a given biological process. However, for technical reasons, comprehensive screens have been restricted to very few model organisms. Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum, which identifies gene functions in embryonic and postembryonic development, physiology and cell biology. The utility of Tribolium as a screening platform is demonstrated by the identification of genes involved in insect epithelial adhesion. This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila. PMID:26215380

  1. The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology.

    PubMed

    Schmitt-Engel, Christian; Schultheis, Dorothea; Schwirz, Jonas; Ströhlein, Nadi; Troelenberg, Nicole; Majumdar, Upalparna; Dao, Van Anh; Grossmann, Daniela; Richter, Tobias; Tech, Maike; Dönitz, Jürgen; Gerischer, Lizzy; Theis, Mirko; Schild, Inga; Trauner, Jochen; Koniszewski, Nikolaus D B; Küster, Elke; Kittelmann, Sebastian; Hu, Yonggang; Lehmann, Sabrina; Siemanowski, Janna; Ulrich, Julia; Panfilio, Kristen A; Schröder, Reinhard; Morgenstern, Burkhard; Stanke, Mario; Buchhholz, Frank; Frasch, Manfred; Roth, Siegfried; Wimmer, Ernst A; Schoppmeier, Michael; Klingler, Martin; Bucher, Gregor

    2015-07-28

    Genetic screens are powerful tools to identify the genes required for a given biological process. However, for technical reasons, comprehensive screens have been restricted to very few model organisms. Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum, which identifies gene functions in embryonic and postembryonic development, physiology and cell biology. The utility of Tribolium as a screening platform is demonstrated by the identification of genes involved in insect epithelial adhesion. This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila.

  2. Aristotle's illusion reveals interdigit functional somatosensory alterations in focal hand dystonia.

    PubMed

    Tinazzi, Michele; Marotta, Angela; Fasano, Alfonso; Bove, Francesco; Bentivoglio, Anna Rita; Squintani, Giovanna; Pozzer, Lara; Fiorio, Mirta

    2013-03-01

    In focal hand dystonia, the cortical somatosensory representation of the fingers is abnormal, with overlapping receptive fields and reduced interdigit separation. These abnormalities are associated with deficits in sensory perception, as previously demonstrated by applying tactile stimuli to one finger at a time. What is still unknown is whether the sensory deficits can be observed when tactile perception involves more than one finger. To address this issue, we applied 'Aristotle's illusion' to 15 patients with focal hand dystonia, 15 patients with dystonia not affecting the hand (blepharospasm and cervical dystonia) and 15 healthy control subjects. In this illusion, one object touching the contact point of two crossed fingertips is perceived as two objects by a blindfolded subject. The same object placed between two parallel fingertips is correctly perceived as one. The illusory doubling sensation is because of the fact that the contact point between the crossed fingers consists of non-adjacent and functionally unrelated skin regions, which usually send sensory signals to separate spots in the somatosensory cortex. In our study, participants were touched by one sphere between the second-third digits, the second-fourth digits and the fourth-fifth digits of both hands, either in crossed or in parallel position, and had to refer whether they felt one or two stimuli. The percentage of 'two stimuli' responses was an index of the illusory doubling. Both healthy control subjects and dystonic patients presented Aristotle's illusion when the fingers were crossed. However, patients with focal hand dystonia presented a significant reduction of the illusion when the sphere was placed between the crossed fourth and fifth digits of the affected hand. This reduction correlated with the severity of motor disease at the fingers. Similar findings were not observed in non-hand dystonia and control groups. The reduction of Aristotle's illusion in non-affected fingers and its

  3. Cyclin Y-mediated transcript profiling reveals several important functional pathways regulated by Cyclin Y in hippocampal neurons

    PubMed Central

    Kim, Hanna; Hong, Jung-Hwa; Kim, Mirang; Park, Mikyoung

    2017-01-01

    Cyclin Y (CCNY), which is a cyclin protein known to play a role in cell division, is unexpectedly and thus interestingly expressed in non-proliferating neuronal cells. There have been only a few studies reporting the neuronal functions of CCNY in synapse remodeling and hippocampal long-term potentiation. Therefore, we here provide global and comprehensive information on the putative functions of CCNY in biological and functional pathways in neuronal systems. We adopted high-throughput RNA-sequencing technology for analyzing transcriptomes regulated by CCNY and utilized bioinformatics for identifying putative molecules, biological processes, and functional pathways that are possibly connected to CCNY functions in hippocampal neuronal cells of rats. We revealed that several enriched annotation terms and pathways associated with CCNY expression within neurons, including apoptosis, learning or memory, synaptic plasticity, actin cytoskeleton, focal adhesion, extracellular matrix-receptor interaction and chemokine signaling pathway are targeted by CCNY. In addition, the mRNA levels of some genes enriched for those annotation terms and pathways or genes reported to be altered in Alzheimer’s disease mouse model were further validated by quantitative real-time PCR in hippocampal neuronal cells. The present study provides an excellent resource for future investigations of CCNY functions in neuronal systems. PMID:28241067

  4. Phylogeny and phylogeography of functional genes shared among seven terrestrial subsurface metagenomes reveal N-cycling and microbial evolutionary relationships

    PubMed Central

    Lau, Maggie C. Y.; Cameron, Connor; Magnabosco, Cara; Brown, C. Titus; Schilkey, Faye; Grim, Sharon; Hendrickson, Sarah; Pullin, Michael; Sherwood Lollar, Barbara; van Heerden, Esta; Kieft, Thomas L.; Onstott, Tullis C.

    2014-01-01

    Comparative studies on community phylogenetics and phylogeography of microorganisms living in extreme environments are rare. Terrestrial subsurface habitats are valuable for studying microbial biogeographical patterns due to their isolation and the restricted dispersal mechanisms. Since the taxonomic identity of a microorganism does not always correspond well with its functional role in a particular community, the use of taxonomic assignments or patterns may give limited inference on how microbial functions are affected by historical, geographical and environmental factors. With seven metagenomic libraries generated from fracture water samples collected from five South African mines, this study was carried out to (1) screen for ubiquitous functions or pathways of biogeochemical cycling of CH4, S, and N; (2) to characterize the biodiversity represented by the common functional genes; (3) to investigate the subsurface biogeography as revealed by this subset of genes; and (4) to explore the possibility of using metagenomic data for evolutionary study. The ubiquitous functional genes are NarV, NPD, PAPS reductase, NifH, NifD, NifK, NifE, and NifN genes. Although these eight common functional genes were taxonomically and phylogenetically diverse and distinct from each other, the dissimilarity between samples did not correlate strongly with geographical or environmental parameters or residence time of the water. Por genes homologous to those of Thermodesulfovibrio yellowstonii detected in all metagenomes were deep lineages of Nitrospirae, suggesting that subsurface habitats have preserved ancestral genetic signatures that inform the study of the origin and evolution of prokaryotes. PMID:25400621

  5. Phylogeny and phylogeography of functional genes shared among seven terrestrial subsurface metagenomes reveal N-cycling and microbial evolutionary relationships.

    PubMed

    Lau, Maggie C Y; Cameron, Connor; Magnabosco, Cara; Brown, C Titus; Schilkey, Faye; Grim, Sharon; Hendrickson, Sarah; Pullin, Michael; Sherwood Lollar, Barbara; van Heerden, Esta; Kieft, Thomas L; Onstott, Tullis C

    2014-01-01

    Comparative studies on community phylogenetics and phylogeography of microorganisms living in extreme environments are rare. Terrestrial subsurface habitats are valuable for studying microbial biogeographical patterns due to their isolation and the restricted dispersal mechanisms. Since the taxonomic identity of a microorganism does not always correspond well with its functional role in a particular community, the use of taxonomic assignments or patterns may give limited inference on how microbial functions are affected by historical, geographical and environmental factors. With seven metagenomic libraries generated from fracture water samples collected from five South African mines, this study was carried out to (1) screen for ubiquitous functions or pathways of biogeochemical cycling of CH4, S, and N; (2) to characterize the biodiversity represented by the common functional genes; (3) to investigate the subsurface biogeography as revealed by this subset of genes; and (4) to explore the possibility of using metagenomic data for evolutionary study. The ubiquitous functional genes are NarV, NPD, PAPS reductase, NifH, NifD, NifK, NifE, and NifN genes. Although these eight common functional genes were taxonomically and phylogenetically diverse and distinct from each other, the dissimilarity between samples did not correlate strongly with geographical or environmental parameters or residence time of the water. Por genes homologous to those of Thermodesulfovibrio yellowstonii detected in all metagenomes were deep lineages of Nitrospirae, suggesting that subsurface habitats have preserved ancestral genetic signatures that inform the study of the origin and evolution of prokaryotes.

  6. Tracking of plus-ends reveals microtubule functional diversity in different cell types

    NASA Astrophysics Data System (ADS)

    Shaebani, M. Reza; Pasula, Aravind; Ott, Albrecht; Santen, Ludger

    2016-07-01

    Many cellular processes are tightly connected to the dynamics of microtubules (MTs). While in neuronal axons MTs mainly regulate intracellular trafficking, they participate in cytoskeleton reorganization in many other eukaryotic cells, enabling the cell to efficiently adapt to changes in the environment. We show that the functional differences of MTs in different cell types and regions is reflected in the dynamic properties of MT tips. Using plus-end tracking proteins EB1 to monitor growing MT plus-ends, we show that MT dynamics and life cycle in axons of human neurons significantly differ from that of fibroblast cells. The density of plus-ends, as well as the rescue and catastrophe frequencies increase while the growth rate decreases toward the fibroblast cell margin. This results in a rather stable filamentous network structure and maintains the connection between nucleus and membrane. In contrast, plus-ends are uniformly distributed along the axons and exhibit diverse polymerization run times and spatially homogeneous rescue and catastrophe frequencies, leading to MT segments of various lengths. The probability distributions of the excursion length of polymerization and the MT length both follow nearly exponential tails, in agreement with the analytical predictions of a two-state model of MT dynamics.

  7. Functional studies of the Ciona intestinalis myogenic regulatory factor reveal conserved features of chordate myogenesis.

    PubMed

    Izzi, Stephanie A; Colantuono, Bonnie J; Sullivan, Kelly; Khare, Parul; Meedel, Thomas H

    2013-04-15

    Ci-MRF is the sole myogenic regulatory factor (MRF) of the ascidian Ciona intestinalis, an invertebrate chordate. In order to investigate its properties we developed a simple in vivo assay based on misexpressing Ci-MRF in the notochord of Ciona embryos. We used this assay to examine the roles of three structural motifs that are conserved among MRFs: an alanine-threonine (Ala-Thr) dipeptide of the basic domain that is known in vertebrates as the myogenic code, a cysteine/histidine-rich (C/H) domain found just N-terminal to the basic domain, and a carboxy-terminal amphipathic α-helix referred to as Helix III. We show that the Ala-Thr dipeptide is necessary for normal Ci-MRF function, and that while eliminating the C/H domain or Helix III individually has no demonstrable effect on Ci-MRF, simultaneous loss of both motifs significantly reduces its activity. Our studies also indicate that direct interaction between CiMRF and an essential E-box of Ciona Troponin I is required for the expression of this muscle-specific gene and that multiple classes of MRF-regulated genes exist in Ciona. These findings are consistent with substantial conservation of MRF-directed myogenesis in chordates and demonstrate for the first time that the Ala/Thr dipeptide of the basic domain of an invertebrate MRF behaves as a myogenic code.

  8. A compartmentalized microfluidic neuromuscular co-culture system reveals spatial aspects of GDNF functions

    PubMed Central

    Zahavi, Eitan Erez; Ionescu, Ariel; Gluska, Shani; Gradus, Tal; Ben-Yaakov, Keren; Perlson, Eran

    2015-01-01

    ABSTRACT Bidirectional molecular communication between the motoneuron and the muscle is vital for neuromuscular junction (NMJ) formation and maintenance. The molecular mechanisms underlying such communication are of keen interest and could provide new targets for intervention in motoneuron disease. Here, we developed a microfluidic platform with motoneuron cell bodies on one side and muscle cells on the other, connected by motor axons extending through microgrooves to form functional NMJs. Using this system, we were able to differentiate between the proximal and distal effects of oxidative stress and glial-derived neurotrophic factor (GDNF), demonstrating a dying-back degeneration and retrograde transmission of pro-survival signaling, respectively. Furthermore, we show that GDNF acts differently on motoneuron axons versus soma, promoting axonal growth and innervation only when applied locally to axons. Finally, we track for the first time the retrograde transport of secreted GDNF from muscle to neuron. Thus, our data suggests spatially distinct effects of GDNF – facilitating growth and muscle innervation at axon terminals and survival pathways in the soma. PMID:25632161

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

    PubMed Central

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

    2014-01-01

    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 exhibited 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 wildtype 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 wildtype synaptic plasticity and sufficient to rescue PAM+/− LTP. These experiments support roles for PAM in Cu homeostasis and for synaptic Cu in amygdalar function. PMID:24593825

  10. Situating and teaching 21st century zoology: revealing pattern in the form and function of animals.

    PubMed

    Russell, Anthony P

    2009-09-01

    The current challenges (increasing levels of integration in the biological sciences) facing the teaching of zoology and the structure of the zoology curriculum are explored herein. General context is provided and a more focused scrutiny of the situation in North America is presented. The changing emphases in more broadly-based biological sciences programs in North America are outlined, and their influence on the role of zoology as part of fundamental biological training is considered. The longer term impact of such changes in emphasis on the teaching of zoology is discussed, and the central role that zoology can play in dealing with both science content and science education is advanced. Based upon a focal workshop on the future of the zoology curriculum in Canada, a perspective on the challenges facing curriculum evolution is provided. Extensive curriculum redesign is called for to ensure that zoology provides a broad-scale integrative approach to the understanding of biodiversity in evolutionary, ecological and functional contexts. Barriers to, and drivers of change are identified and the need for collaborative approaches to curricular evolution is emphasized.

  11. Temporal lobe abnormalities in semantic processing by criminal psychopaths as revealed by functional magnetic resonance imaging.

    PubMed

    Kiehl, Kent A; Smith, Andra M; Mendrek, Adrianna; Forster, Bruce B; Hare, Robert D; Liddle, Peter F

    2004-04-30

    We tested the hypothesis that psychopathy is associated with abnormalities in semantic processing of linguistic information. Functional magnetic resonance imaging (fMRI) was used to elucidate and characterize the neural architecture underlying lexico-semantic processes in criminal psychopathic individuals and in a group of matched control participants. Participants performed a lexical decision task in which blocks of linguistic stimuli alternated with a resting baseline condition. In each lexical decision block, the stimuli were either concrete words and pseudowords or abstract words and pseudowords. Consistent with our hypothesis, psychopathic individuals, relative to controls, showed poorer behavioral performance for processing abstract words. Analysis of the fMRI data for both groups indicated that processing of word stimuli, compared with the resting baseline condition, was associated with neural activation in bilateral fusiform gyrus, anterior cingulate, left middle temporal gyrus, right posterior superior temporal gyrus, and left and right inferior frontal gyrus. Analyses confirmed our prediction that psychopathic individuals would fail to show the appropriate neural differentiation between abstract and concrete stimuli in the right anterior temporal gyrus and surrounding cortex. The results are consistent with other studies of semantic processing in psychopathy and support the theory that psychopathy is associated with right hemisphere abnormalities for processing conceptually abstract material.

  12. Temporal lobe abnormalities in semantic processing by criminal psychopaths as revealed by functional magnetic resonance imaging.

    PubMed

    Kiehl, Kent A; Smith, Andra M; Mendrek, Adrianna; Forster, Bruce B; Hare, Robert D; Liddle, Peter F

    2004-01-15

    We tested the hypothesis that psychopathy is associated with abnormalities in semantic processing of linguistic information. Functional magnetic resonance imaging (fMRI) was used to elucidate and characterize the neural architecture underlying lexico-semantic processes in criminal psychopathic individuals and in a group of matched control participants. Participants performed a lexical decision task in which blocks of linguistic stimuli alternated with a resting baseline condition. In each lexical decision block, the stimuli were either concrete words and pseudowords or abstract words and pseudowords. Consistent with our hypothesis, psychopathic individuals, relative to controls, showed poorer behavioral performance for processing abstract words. Analysis of the fMRI data for both groups indicated that processing of word stimuli, compared with the resting baseline condition, was associated with neural activation in bilateral fusiform gyrus, anterior cingulate, left middle temporal gyrus, right posterior superior temporal gyrus, and left and right inferior frontal gyrus. Analyses confirmed our prediction that psychopathic individuals would fail to show the appropriate neural differentiation between abstract and concrete stimuli in the right anterior temporal gyrus and surrounding cortex. The results are consistent with other studies of semantic processing in psychopathy and support the theory that psychopathy is associated with right hemisphere abnormalities for processing conceptually abstract material.

  13. Fluorescent false neurotransmitter reveals functionally silent dopamine vesicle clusters in the striatum

    PubMed Central

    Pereira, Daniela B.; Schmitz, Yvonne; Mészáros, József; Merchant, Paolomi; Hu, Gang; Li, Shu; Henke, Adam; Lizardi-Ortiz, José E.; Karpowicz, Richard J.; Morgenstern, Travis J.; Sonders, Mark S.; Kanter, Ellen; Rodriguez, Pamela C.; Mosharov, Eugene V.; Sames, Dalibor; Sulzer, David

    2016-01-01

    Neurotransmission at dopaminergic synapses has been studied with techniques that provide high temporal resolution but cannot resolve individual synapses. To elucidate the spatial dynamics and heterogeneity of individual dopamine boutons, we developed fluorescent false neurotransmitter 200 (FFN200), a vesicular monoamine transporter 2 (VMAT2) substrate that is the first probe to selectively trace monoamine exocytosis in both neuronal cell culture and brain tissue. By monitoring electrically-evoked Ca2+ transients with GCaMP3 and FFN200 release simultaneously, we find that only a small fraction of dopamine boutons that exhibit Ca2+ influx engage in exocytosis, a result confirmed with activity-dependent loading of the endocytic probe FM 1-43. Thus, only a low fraction of striatal dopamine axonal sites with uptake-competent VMAT2 vesicles are capable of transmitter release. This is consistent with the presence of functionally “silent” dopamine vesicle clusters and represents a first report suggestive of presynaptically silent neuromodulatory synapses. PMID:26900925

  14. The naris muscles in tiger salamander. I. Potential functions and innervation as revealed by biocytin tracing.

    PubMed

    Wirsig-Wiechmann, Celeste R; Holliday, Katherine R

    2002-06-01

    The naris constrictor muscle, along with naris dilator and naris accessory muscles, controls the opening and closing of the external naris in tiger salamanders. It has been hypothesized that contraction of the naris constrictor muscle also causes the external nasal gland to secrete its contents inside the lateral wall of the external naris opening. This location is just rostral to vomeronasal organ and thus secretion in this region may be important for access of odorous compounds to vomeronasal organ. Little is known about the innervation of the naris muscles. To elucidate the neural control of these muscles, their innervation was examined using retrograde tract tracing with biocytin. Following application of biocytin to the naris constrictor muscle, labeling was observed in a ventral axonal plexus of the palatine nerve and numerous neuronal cell bodies distributed along this peripheral nerve plexus and within the main portion of the palatine ganglion. If the naris accessory and/or dilator muscles were also exposed to the tracer, the lateral-most branch of the palatine nerve and its associated neural cell bodies were labeled. To confirm the functional innervation of the muscles by the palatine nerve, the nerve was cut and the contraction of the muscles was eliminated. These findings demonstrate that the muscles controlling the external naris are under the control of palatine ganglion neurons. We hypothesize that this innervation of the naris constrictor muscle controls both muscle contraction and glandular secretion that may facilitate access of chemosensory substances to the vomeronasal organ.

  15. Integrative proteomic profiling of ovarian cancer cell lines reveals precursor cell associated proteins and functional status

    PubMed Central

    Coscia, F.; Watters, K. M.; Curtis, M.; Eckert, M. A.; Chiang, C. Y.; Tyanova, S.; Montag, A.; Lastra, R. R.; Lengyel, E.; Mann, M.

    2016-01-01

    A cell line representative of human high-grade serous ovarian cancer (HGSOC) should not only resemble its tumour of origin at the molecular level, but also demonstrate functional utility in pre-clinical investigations. Here, we report the integrated proteomic analysis of 26 ovarian cancer cell lines, HGSOC tumours, immortalized ovarian surface epithelial cells and fallopian tube epithelial cells via a single-run mass spectrometric workflow. The in-depth quantification of >10,000 proteins results in three distinct cell line categories: epithelial (group I), clear cell (group II) and mesenchymal (group III). We identify a 67-protein cell line signature, which separates our entire proteomic data set, as well as a confirmatory publicly available CPTAC/TCGA tumour proteome data set, into a predominantly epithelial and mesenchymal HGSOC tumour cluster. This proteomics-based epithelial/mesenchymal stratification of cell lines and human tumours indicates a possible origin of HGSOC either from the fallopian tube or from the ovarian surface epithelium. PMID:27561551

  16. Gene knockouts reveal separate functions for two cytoplasmic dyneins in Tetrahymena thermophila.

    PubMed

    Lee, S; Wisniewski, J C; Dentler, W L; Asai, D J

    1999-03-01

    In many organisms, there are multiple isoforms of cytoplasmic dynein heavy chains, and division of labor among the isoforms would provide a mechanism to regulate dynein function. The targeted disruption of somatic genes in Tetrahymena thermophila presents the opportunity to determine the contributions of individual dynein isoforms in a single cell that expresses multiple dynein heavy chain genes. Substantial portions of two Tetrahymena cytoplasmic dynein heavy chain genes were cloned, and their motor domains were sequenced. Tetrahymena DYH1 encodes the ubiquitous cytoplasmic dynein Dyh1, and DYH2 encodes a second cytoplasmic dynein isoform, Dyh2. The disruption of DYH1, but not DYH2, resulted in cells with two detectable defects: 1) phagocytic activity was inhibited, and 2) the cells failed to distribute their chromosomes correctly during micronuclear mitosis. In contrast, the disruption of DYH2 resulted in a loss of regulation of cell size and cell shape and in the apparent inability of the cells to repair their cortical cytoskeletons. We conclude that the two dyneins perform separate tasks in Tetrahymena.

  17. Fine functional organization of auditory cortex revealed by Fourier optical imaging.

    PubMed

    Kalatsky, Valery A; Polley, Daniel B; Merzenich, Michael M; Schreiner, Christoph E; Stryker, Michael P

    2005-09-13

    We provide an overall view of the functional tonotopic organization of the auditory cortex in the rat. We apply a recently developed technique for acquiring intrinsic signal optical maps, Fourier imaging, in the rat auditory cortex. These highly detailed maps, derived in a several-minute-long recording procedure, delineate multiple auditory cortical areas and demonstrate their shapes, sizes, and tonotopic order. Beyond the primary auditory cortex, there are at least three distinct areas with fine-scale tonotopic organization, as well as at least one additional high-frequency field. The arrangement of all of these cortical areas is consistent across subjects. The accuracy of these optical maps was confirmed by microelectrode mapping in the same subjects. This imaging method allows fast mapping of the auditory cortex at high spatial resolution comparable to that provided by conventional microelectrode technique. Although spiking activity is largely responsible for the evoked intrinsic signals, certain features of the optical signal cannot be explained by spiking activity only, and should probably be attributed to other mechanisms inducing metabolic activity, such as subthreshold membrane phenomena.

  18. Temporal dynamics of hot desert microbial communities reveal structural and functional responses to water input

    PubMed Central

    Armstrong, Alacia; Valverde, Angel; Ramond, Jean-Baptiste; Makhalanyane, Thulani P.; Jansson, Janet K.; Hopkins, David W.; Aspray, Thomas J.; Seely, Mary; Trindade, Marla I.; Cowan, Don A.

    2016-01-01

    The temporal dynamics of desert soil microbial communities are poorly understood. Given the implications for ecosystem functioning under a global change scenario, a better understanding of desert microbial community stability is crucial. Here, we sampled soils in the central Namib Desert on sixteen different occasions over a one-year period. Using Illumina-based amplicon sequencing of the 16S rRNA gene, we found that α-diversity (richness) was more variable at a given sampling date (spatial variability) than over the course of one year (temporal variability). Community composition remained essentially unchanged across the first 10 months, indicating that spatial sampling might be more important than temporal sampling when assessing β-diversity patterns in desert soils. However, a major shift in microbial community composition was found following a single precipitation event. This shift in composition was associated with a rapid increase in CO2 respiration and productivity, supporting the view that desert soil microbial communities respond rapidly to re-wetting and that this response may be the result of both taxon-specific selection and changes in the availability or accessibility of organic substrates. Recovery to quasi pre-disturbance community composition was achieved within one month after rainfall. PMID:27680878

  19. Regulation of apoptosis by fau revealed by functional expression cloning and antisense expression.

    PubMed

    Mourtada-Maarabouni, Mirna; Kirkham, Lucy; Farzaneh, Farzin; Williams, Gwyn T

    2004-12-16

    Functional expression cloning is a powerful strategy for identifying critical steps in biological pathways independently of prior assumptions. It is particularly suitable for the identification of molecules crucial to the control of apoptosis. Our screen for sequences suppressing T-cell apoptosis isolated a sequence antisense to fau (Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV)-associated ubiquitously expressed gene). The fox gene in FBR murine osteosarcoma virus is also antisense to fau and several reports have indicated that fau displays tumour suppressor and oncogenic properties in different contexts. Our observations indicate that the fau antisense sequence suppresses expression of endogenous fau mRNA and produces resistance to apoptosis induced both by the glucocorticoid analogue dexamethasone' by ul