Diffusible crosslinkers generate directed forces in microtubule networks
Lansky, Z. ; Braun, M. ; Diez, S. ; Janson, M.E. - \ 2015
Cell 160 (2015)6. - ISSN 0092-8674 - p. 1159 - 1168.
fission yeast - overlapping microtubules - kinesin motors - cell-division - protein - ase1p - friction - complex - cytokinesis - transport
Cytoskeletal remodeling is essential to eukaryotic cell division and morphogenesis. The mechanical forces driving the restructuring are attributed to the action of molecular motors and the dynamics of cytoskeletal filaments, which both consume chemical energy. By contrast, non-enzymatic filament crosslinkers are regarded as mere friction-generating entities. Here, we experimentally demonstrate that diffusible microtubule crosslinkers of the Ase1/PRC1/Map65 family generate directed microtubule sliding when confined between partially overlapping microtubules. The Ase1-generated forces, directly measured by optical tweezers to be in the piconewton-range, were sufficient to antagonize motor-protein driven microtubule sliding. Force generation is quantitatively explained by the entropic expansion of confined Ase1 molecules diffusing within the microtubule overlaps. The thermal motion of crosslinkers is thus harnessed to generate mechanical work analogous to compressed gas propelling a piston in a cylinder. As confinement of diffusible proteins is ubiquitous in cells, the associated entropic forces are likely of importance for cellular mechanics beyond cytoskeletal networks.
Nitrogen-depleted Chlorella zofingiensis produces astaxanthin, ketolutein and their fatty acid esters: a carotenoid metabolism study
Mulders, K.J.M. ; Weesepoel, Y.J.A. ; Bodenes, C. ; Lamers, P.P. ; Vincken, J.P. ; Martens, D.E. ; Gruppen, H. ; Wijffels, R.H. - \ 2015
Journal of Applied Phycology 27 (2015)1. - ISSN 0921-8971 - p. 125 - 140.
alga haematococcus-pluvialis - green-alga - triacylglycerol accumulation - biosynthetic-pathway - dunaliella-salina - light - chlorophyceae - inhibition - microalgae - complex
Natural carotenoids such as astaxanthin, ß,ß-carotene and lutein are pigments with a high market value. We studied the effects of nitrogen depletion on the carotenoid metabolism of Chlorella zofingiensis (Chlorophyta) and the subsequent treatment with diphenylamine (DPA), an inhibitor of the biosynthesis of secondary ketocarotenoids. Pigments were identified and quantified based on reversed phase ultrahigh performance liquid chromatography photodiode array tandem mass spectrometry (RP-UHPLC-PDA-MSn). Nitrogen depletion (without DPA) resulted in a degradation of chlorophylls and primary carotenoids and an accumulation of astaxanthin, ketolutein, canthaxanthin, adonixanthin and ß,ß-carotene. The DPA treatment decreased the overall production of ß,ß-carotene derivatives (sum of astaxanthin, canthaxanthin, echinenone and adonixanthin); however, the production of ketolutein and degradation of primary carotenoids were not modified. This suggests that the regulatory mechanisms controlling the flux towards ketolutein and primary carotenoids were not affected by the decreased levels of ß,ß-carotene derivatives. In addition, DPA increased production of the individual carotenoids, adonixanthin and echinenone. Insight into the regulation of microalgal carotenoid biosynthesis as demonstrated in this paper is essential when a large-scale carotenoid production process is to be optimised or a recombinant C. ofingiensis strain is to be designed with the intention of excessively producing primary or secondary carotenoids.
WOX5 Suppresses CYCLIN D Activity to Establish Quiescence at the Center of the Root Stem Cell Niche
Forzani, C. ; Aichinger, E. ; Willemsen, V. ; Murray, J.A. - \ 2014
Current Biology 24 (2014)16. - ISSN 0960-9822 - p. 1939 - 1944.
arabidopsis-thaliana root - division - meristem - differentiation - organization - transition - expression - complex - protein
In Arabidopsis, stem cells maintain the provision of new cells for root growth. They surround a group of slowly dividing cells named the quiescent center (QC), and, together, they form the stem cell niche (SCN). The QC acts as the signaling center of the SCN, repressing differentiation of the surrounding stem cells  and providing a pool of cells able to replace damaged stem cells [2, 3]. Maintenance of the stem cells depends on the transcription factor WUSCHEL-RELATED HOMEOBOX 5 (WOX5), which is specifically expressed in the QC . However, the molecular mechanisms by which WOX5 promotes stem cell fate and whether WOX5 regulates proliferation of the QC are unknown. Here, we reveal a new role for WOX5 in restraining cell division in the cells of the QC, thereby establishing quiescence. In contrast, WOX5 and CYCD3;3/CYCD1;1 both promote cell proliferation in the nascent columella. The additional QC divisions occurring in wox5 mutants are suppressed in mutant combinations with the D type cyclins CYCD3;3 and CYCD1;1. Moreover, ectopic expression of CYCD3;3 in the QC is sufficient to induce cell division in the QC. WOX5 thus suppresses QC divisions that are otherwise promoted by CYCD3;3 and CYCD1;1, in part by interacting with the CYCD3;3 promoter to repress CYCD3;3 expression in the QC. Therefore, we propose a specific role for WOX5 in initiating and maintaining quiescence of the QC by excluding CYCD activity from the QC.
Anti-inflammatory properties of the medicinal mushroom Cordyceps militaris might be related to its linear (1¿3)-ß-D-glucan.
Smiderle, F.R. ; Baggio, C.H. ; Borato, D.G. ; Santana-Filho, A.P. ; Sassaki, G.L. ; Iacomini, M. ; Griensven, L.J.L.D. van - \ 2014
PLoS ONE 9 (2014)10. - ISSN 1932-6203
formalin test - in-vivo - fruiting bodies - beta-glucans - congo-red - polysaccharides - mice - macrophages - extract - complex
The Ascomycete Cordyceps militaris, an entomopathogenic fungus, is one of the most important traditional Chinese medicines. Studies related to its pharmacological properties suggest that this mushroom can exert interesting biological activities. Aqueous (CW and HW) and alkaline (K5) extracts containing polysaccharides were prepared from this mushroom, and a ß-D-glucan was purified. This polymer was analysed by GC-MS and NMR spectrometry, showing a linear chain composed of ß-D-Glcp (1¿3)-linked. The six main signals in the 13C-NMR spectrum were assigned by comparison to reported data. The aqueous (CW, HW) extracts stimulated the expression of IL-1ß, TNF-a, and COX-2 by THP-1 macrophages, while the alkaline (K5) extract did not show any effect. However, when the extracts were added to the cells in the presence of LPS, K5 showed the highest inhibition of the pro-inflammatory genes expression. This inhibitory effect was also observed for the purified ß-(1¿3)-D-glucan, that seems to be the most potent anti-inflammatory compound present in the polysaccharide extracts of C. militaris. In vivo, ß-(1¿3)-D-glucan also inhibited significantly the inflammatory phase of formalin-induced nociceptive response, and, in addition, it reduced the migration of total leukocytes but not the neutrophils induced by LPS. In conclusion, this study clearly demonstrates the anti-inflammatory effect of ß-(1¿3)-D-glucan.
Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3
Gong, B. ; Shin, M. ; Sun, J. ; Jung, C.H. ; Bolt, E.L. ; Oost, J. van der; Kim, J.S. - \ 2014
Proceedings of the National Academy of Sciences of the United States of America 111 (2014)46. - ISSN 0027-8424 - p. 16359 - 16364.
bacterial immune-system - in-vitro reconstitution - escherichia-coli - antiviral defense - adaptive immunity - crystal-structure - structural basis - rna - complex - cascade
Mobile genetic elements in bacteria are neutralized by a system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas systems use a “Cascade” ribonucleoprotein complex to guide RNA specifically to complementary sequence in invader double-stranded DNA (dsDNA), a process called “interference.” After target recognition by Cascade, formation of an R-loop triggers recruitment of a Cas3 nuclease-helicase, completing the interference process by destroying the invader dsDNA. To elucidate the molecular mechanism of CRISPR interference, we analyzed crystal structures of Cas3 from the bacterium Thermobaculum terrenum, with and without a bound ATP analog. The structures reveal a histidine-aspartate (HD)-type nuclease domain fused to superfamily-2 (SF2) helicase domains and a distinct C-terminal domain. Binding of ATP analog at the interface of the SF2 helicase RecA-like domains rearranges a motif V with implications for the enzyme mechanism. The HD-nucleolytic site contains two metal ions that are positioned at the end of a proposed nucleic acid-binding tunnel running through the SF2 helicase structure. This structural alignment suggests a mechanism for 3' to 5' nucleolytic processing of the displaced strand of invader DNA that is coordinated with ATP-dependent 3' to 5' translocation of Cas3 along DNA. In agreement with biochemical studies, the presented Cas3 structures reveal important mechanistic details on the neutralization of genetic invaders by type I CRISPR-Cas systems.
Sel1L is indispensable for mammalian endoplasmic reticulum-associated degradation, endoplasmic reticulum homeostasis, and survival
Sun, S. ; Shi, Guojun ; Han, X. ; Francisco, A. ; Ji, Y. ; Kersten, A.H. - \ 2014
Proceedings of the National Academy of Sciences of the United States of America 111 (2014)5. - ISSN 0027-8424 - p. E582 - E591.
unfolded-protein response - er-associated degradation - misfolded glycoproteins - stress granules - caenorhabditis-elegans - complex - cell - dislocation - substrate - retrotranslocation
Suppressor/Enhancer of Lin-12-like (Sel1L) is an adaptor protein for the E3 ligase hydroxymethylglutaryl reductase degradation protein 1 (Hrd1) involved in endoplasmic reticulum-associated degradation (ERAD). Sel1L’s physiological importance in mammalian ERAD, however, remains to be established. Here, using the inducible Sel1L knockout mouse and cell models, we show that Sel1L is indispensable for Hrd1 stability, ER homeostasis, and survival. Acute loss of Sel1L leads to premature death in adult mice within 3 wk with profound pancreatic atrophy. Contrary to current belief, our data show that mammalian Sel1L is required for Hrd1 stability and ERAD function both in vitro and in vivo. Sel1L deficiency disturbs ER homeostasis, activates ER stress, attenuates translation, and promotes cell death. Serendipitously, using a biochemical approach coupled with mass spectrometry, we found that Sel1L deficiency causes the aggregation of both small and large ribosomal subunits. Thus, Sel1L is an indispensable component of the mammalian Hrd1 ERAD complex and ER homeostasis, which is essential for protein translation, pancreatic function, and cellular and organismal survival.
Porocercospora seminalis gen. et comb. nov., the causal organism of buffalograss false smut
Amaradasa, B.S. ; Madrid, H. ; Groenewald, J.Z. ; Crous, P.W. ; Amundsen, K. - \ 2014
Mycologia 106 (2014)1. - ISSN 0027-5514 - p. 77 - 85.
ribosomal dna - mycosphaerella - cochliobolus - cercospora - complex
False smut caused by Cercospora seminalis is an important disease of buffalograss (Buchloë dactyloides) affecting seed production. The pathogen prevents normal caryopsis development and causes considerable yield loss and reduced seed germination. The current taxonomic placement of the false-smut causal pathogen in the genus Cercospora is incorrect based on its morphological characteristics and DNA phylogeny. In the present study the phylogenetic position of C. seminalis is clarified based on DNA sequence analysis of three loci namely the internal transcribed spacer (ITS) region, partial nuclear ribosomal large subunit (LSU) and partial sequences of the RNA polymerase II second largest subunit (RPB2). A collection of C. seminalis isolates was made from buffalograss sites near Lincoln, Nebraska. DNA sequence data indicated that Cercospora seminalis is phylogenetically close to but distinct from species of Bipolaris and Curvularia (Pleosporaceae, Pleosporales). Cercospora seminalis morphologically had unique characteristics, namely densely aggregated and repeatedly branched conidiophores arising from a brown stroma, monotretic conidiogenous cells with inconspicuous loci, and scolecosporous conidia with distosepta, and thickened, darkened hila. Porocercospora is introduced as a new genus to accommodate the buffalograss false-smut pathogen.
Live imaging of baculovirus infection of midgut epithelium cells: a functional assay of per os infectivity factors (PIFs)
Mu, J.F. ; Lent, J.W.M. van; Smagghe, G. ; Wang, Q. ; Chen Xinwen, ; Vlak, J.M. ; Oers, M.M. van - \ 2014
Journal of General Virology 95 (2014)Pt 1. - ISSN 0022-1317 - p. 2531 - 2539.
occlusion-derived virus - heliothis-virescens larvae - nucleopolyhedrovirus - binding - complex - replication - proteins - surface - genes - p74
The occlusion-derived viruses (ODVs) of baculoviruses are responsible for oral infection of insect hosts, whereas budded viruses (BVs) are responsible for the systemic infection within the host. The ODV membrane proteins play crucial roles in mediating virus entry into midgut epithelium cells to initiate infection and are important factors in host range determination. For Autographa californica multiple nucleopolyhedrovirus (AcMNPV), seven conserved ODV membrane proteins have been shown to be essential for oral infectivity and are called per os infectivity factors (PIFs). Information on the function of the individual PIF proteins in virus entry is limited, partly due to the lack of a good in vitro system for monitoring ODV entry. Here, we constructed a baculovirus with an enhanced green fluorescent protein (EGFP) fused to the nucleocapsid to monitor the entry of virus into primary midgut epithelium cells ex vivo by confocal fluorescence microscopy. The EGFP-labeled virus showed the same BV virulence and ODV infectivity as wild type virus. The ability to bind and enter host cells was then visualized for wild type AcMNPVs and viruses with mutations in P74 (PIF0), PIF1 or PIF2, showing that P74 is required for ODV binding, while PIF1 and PIF2 play important roles in entry of ODV after binding to midgut cells. This is the first live imaging of ODV entry into midgut cells and complements the genetic and biochemical evidence for the role of PIFs in the oral infection process.
Defining, researching and struggling for water justice: some conceptual building blocks for research and action
Zwarteveen, M.Z. ; Boelens, R.A. - \ 2014
Water International 39 (2014)2. - ISSN 0250-8060 - p. 143 - 158.
political ecology - irrigation - scale - governance - efficiency - services - strategy - complex
This article provides a framework for understanding water problems as problems of justice. Drawing on wider (environmental) justice approaches, informed by interdisciplinary ontologies that define water as simultaneously natural (material) and social, and based on an explicit acceptance of water problems as always contested, the article posits that water justice is embedded and specific to historical and socio-cultural contexts. Water justice includes but transcends questions of distribution to include those of cultural recognition and political participation, and is intimately linked to the integrity of ecosystems. Justice requires the creative building of bridges and alliances across differences.
Modulation of flavonoid metabolites in Arabidopsis thaliana through overexpression of the MYB75 transcription factor: role of kaempferol-3,7-dirhamnoside in resistance to the specialist insect herbivore Pieris brassicae
Onkokesung, N. ; Reichelt, M. ; Doorn, A. van; Schuurink, R.C. ; Loon, J.J.A. van; Dicke, M. - \ 2014
Journal of Experimental Botany 65 (2014)8. - ISSN 0022-0957 - p. 2203 - 2217.
plant-responses - anthocyanin accumulation - lepidopteran herbivores - coexpression analysis - biosynthetic-pathway - indole-glucosinolate - functional genomics - signaling pathways - defense responses - complex
Anthocyanins and flavonols are secondary metabolites that can function in plant defence against herbivores. In Arabidopsis thaliana, anthocyanin and flavonol biosynthesis are regulated by MYB transcription factors. Overexpression of MYB75 (oxMYB75) in Arabidopsis results in increasing anthocyanin and flavonol levels which enhances plant resistance to generalist caterpillars. However, how these metabolites affect specialist herbivores has remained unknown. Performance of a specialist aphid (Brevicoryne brassicae) was unaffected after feeding on oxMYB75 plants, whereas a specialist caterpillar (Pieris brassicae) gained significantly higher body mass when feeding on this plant. An increase in anthocyanin and total flavonol glycoside levels correlated negatively with the body mass of caterpillars fed on oxMYB75 plants. However, a significant reduction of kaempferol-3,7-dirhamnoside (KRR) corresponded to an increased susceptibility of oxMYB75 plants to caterpillar feeding. Pieris brassicae caterpillars also grew less on an artificial diet containing KRR or on oxMYB75 plants that were exogenously treated with KRR, supporting KRR's function in direct defence against this specialist caterpillar. The results show that enhancing the activity of the anthocyanin pathway in oxMYB75 plants results in re-channelling of quercetin/kaempferol metabolites which has a negative effect on the accumulation of KRR, a novel defensive metabolite against a specialist caterpillar.
The leucine-rich repeat receptor kinase BIR2 is a negative regulator of BAK1 in plant immunity
Halter, T. ; Imkampe, J. ; Mazzotta, S. ; Wierzba, M. ; Postel, S. ; Bücherl, C.A. ; Kiefer, C. ; Stahl, M. ; Chinchilla, D. ; Wang, X. ; Nürnberger, T. ; Zipfel, C. ; Clouse, S. ; Borst, J.W. ; Boeren, S. ; Vries, S.C. de; Tax, F. ; Kemmerling, B. - \ 2014
Current Biology 24 (2014)2. - ISSN 0960-9822 - p. 134 - 143.
innate immunity - cell-death - necrotrophic pathogens - molecular-patterns - protein-kinase - arabidopsis - complex - bri1 - perception - activation
Background Transmembrane leucine-rich repeat (LRR) receptors are commonly used innate immune receptors in plants and animals but can also sense endogenous signals to regulate development. BAK1 is a plant LRR-receptor-like kinase (RLK) that interacts with several ligand-binding LRR-RLKs to positively regulate their functions. BAK1 is involved in brassinosteroid-dependent growth and development, innate immunity, and cell-death control by interacting with the brassinosteroid receptor BRI1, immune receptors, such as FLS2 and EFR, and the small receptor kinase BIR1, respectively. Results Identification of in vivo BAK1 complex partners by LC/ESI-MS/MS uncovered two novel BAK1-interacting RLKs, BIR2 and BIR3. Phosphorylation studies revealed that BIR2 is unidirectionally phosphorylated by BAK1 and that the interaction between BAK1 and BIR2 is kinase-activity dependent. Functional analyses of bir2 mutants show differential impact on BAK1-regulated processes, such as hyperresponsiveness to pathogen-associated molecular patterns (PAMP), enhanced cell death, and resistance to bacterial pathogens, but have no effect on brassinosteroid-regulated growth. BIR2 interacts constitutively with BAK1, thereby preventing interaction with the ligand-binding LRR-RLK FLS2. PAMP perception leads to BIR2 release from the BAK1 complex and enables the recruitment of BAK1 into the FLS2 complex. Conclusions Our results provide evidence for a new regulatory mechanism for innate immune receptors with BIR2 acting as a negative regulator of PAMP-triggered immunity by limiting BAK1-receptor complex formation in the absence of ligands.
Massive activation of archaeal defense genes during viral infection
Quax, T.E.F. ; Voet, M. ; Sismeiro, O. ; Dillies, M.A. ; Jagla, B. ; Coppée, J.Y. ; Sezonov, G. ; Forterre, P. ; Oost, J. van der; Lavigne, R. ; Prangishvili, D. - \ 2013
Journal of Virology 87 (2013)15. - ISSN 0022-538X - p. 8419 - 8428.
differential expression analysis - turreted icosahedral virus - toxin-antitoxin systems - sulfolobus-solfataricus - cell-division - host interactions - crispr rnas - dna - prokaryotes - complex
Archaeal viruses display unusually high genetic and morphological diversity. Studies of these viruses proved to be instrumental for the expansion of knowledge on viral diversity and evolution. The Sulfolobus islandicus rod-shaped virus 2 (SIRV2) is a model to study virus-host interactions in Archaea. It is a lytic virus that exploits a unique egress mechanism based on the formation of remarkable pyramidal structures on the host cell envelope. Using whole-transcriptome sequencing, we present here a global map defining host and viral gene expression during the infection cycle of SIRV2 in its hyperthermophilic host S. islandicus LAL14/1. This information was used, in combination with a yeast two-hybrid analysis of SIRV2 protein interactions, to advance current understanding of viral gene functions. As a consequence of SIRV2 infection, transcription of more than one-third of S. islandicus genes was differentially regulated. While expression of genes involved in cell division decreased, those genes playing a role in antiviral defense were activated on a large scale. Expression of genes belonging to toxin-antitoxin and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems was specifically pronounced. The observed different degree of activation of various CRISPR-Cas systems highlights the specialized functions they perform. The information on individual gene expression and activation of antiviral defense systems is expected to aid future studies aimed at detailed understanding of the functions and interplay of these systems in vivo
Alternative treatments for indoor residual spraying for malaria control in a village with pyrethroid- and DDT-resistant vectors in The Gambia
Tangena, J.A.A. ; Adiamoh, M. ; Alessandro, U. D'; Jarju, L. ; Jawara, M. ; Jeffries, D. ; Malik, N. ; Nwakanma, D. ; Kaur, H. ; Takken, W. ; Lindsay, S.W. ; Pinder, M. - \ 2013
PLoS ONE 8 (2013)9. - ISSN 1932-6203
insecticide-treated nets - anopheles-gambiae - pirimiphos-methyl - molecular-forms - area - complex - susceptibility - identification - protection - mozambique
Background: Malaria vector control is threatened by resistance to pyrethroids, the only class of insecticides used for treating bed nets. The second major vector control method is indoor residual spraying with pyrethroids or the organochloride DDT. However, resistance to pyrethroids frequently confers resistance to DDT. Therefore, alternative insecticides are urgently needed. Methodology/Principal Findings: Insecticide resistance and the efficacy of indoor residual spraying with different insecticides was determined in a Gambian village. Resistance of local vectors to pyrethroids and DDT was high (31% and 46% mortality, respectively) while resistance to bendiocarb and pirimiphos methyl was low (88% and 100% mortality, respectively). The vectors were predominantly Anopheles gambiae s.s. with 94% of them having the putative resistant genotype kdr 1014F. Four groups of eight residential compounds were each sprayed with either (1) bendiocarb, a carbamate, (2) DDT, an organochlorine, (3) microencapsulated pirimiphos methyl, an organophosphate, or (4) left unsprayed. All insecticides tested showed high residual activity up to five months after application. Mosquito house entry, estimated by light traps, was similar in all houses with metal roofs, but was significantly less in IRS houses with thatched roofs (p=0.02). Residents participating in focus group discussions indicated that IRS was considered a necessary nuisance and also may decrease the use of long-lasting insecticidal nets. Conclusion/Significance: Bendiocarb and microencapsulated pirimiphos methyl are viable alternatives for indoor residual spraying where resistance to pyrethroids and DDT is high and may assist in the management of pyrethroid resistance.
Communication between L-galactono-¿-lactone dehydrogenase and cytochrome c.
Hervas, M. ; Bashir, Q. ; Leferink, N.G.H. ; Ferreira, P. ; Moreno-Beltran, J.B. ; Westphal, A.H. ; Diaz Moreno, I. ; Medina, M. ; La Rosa, M.A. De; Ubbink, M. ; Navarro, J.A. ; Berkel, W.J.H. van - \ 2013
FEBS Journal 280 (2013)8. - ISSN 1742-464X - p. 1830 - 1840.
electron-transfer - nmr-spectroscopy - vitamin-c - yeast iso-1-cytochrome-c - arabidopsis-thaliana - protein interactions - kinetic-analysis - viologen analog - complex - plastocyanin
l-galactono-1,4-lactone dehydrogenase (GALDH) catalyzes the terminal step of vitamin C biosynthesis in plant mitochondria. Here we investigated the communication between Arabidopsis thaliana GALDH and its natural electron acceptor cytochrome c (Cc). Using laser-generated radicals we observed the formation and stabilization of the GALDH semiquinone anionic species (GALDHSQ ). GALDHSQ oxidation by Cc exhibited a nonlinear dependence on Cc concentration consistent with a kinetic mechanism involving protein-partner association to form a transient bimolecular complex prior to the electron transfer step. Oxidation of GALDHSQ by Cc was significantly impaired at high ionic strength, revealing the existence of attractive charge-charge interactions between the two reactants. Isothermal titration calorimetry showed that GALDH weakly interacts with both oxidized and reduced Cc. Chemical shift perturbations for (1) H and (15) N nuclei of Cc, arising from the interactions with unlabeled GALDH, were used to map the interacting surface of Cc. For Arabidopsis Cc and yeast Cc, similar residues are involved in the interaction with GALDH. These residues are confined to a single surface surrounding the heme edge. The range of chemical shift perturbations for the physiological Arabidopsis Cc-GALDH complex is larger than that of the non-physiological yeast Cc-GALDH complex, indicating that the former complex is more specific. In summary, the results point to a relatively low affinity GALDH-Cc interaction, similar for all partner redox states, involving protein-protein dynamic motions. Evidence is also provided that Cc utilizes a conserved surface surrounding the heme edge for the interaction with GALDH and other redox partners.
Bacterial Diversity in Meconium of Preterm Neonates and Evolution of Their Fecal Microbiota during the First Month of Life
Moles, L. ; Gómez, M. ; Heilig, G.H.J. ; Bustos, G. ; Fuentes Enriquez de Salamanca, S. ; Vos, W.M. de; Fernandez, L. ; Rodriguez, J.M. ; Jimenez, E. - \ 2013
PLoS ONE 8 (2013)6. - ISSN 1932-6203
16s ribosomal-rna - birth-weight infants - intestinal microbiota - necrotizing enterocolitis - amniotic-fluid - colonization - gut - pcr - communities - complex
The establishment and succession of bacterial communities in infants may have a profound impact in their health, but information about the composition of meconium microbiota and its evolution in hospitalized preterm infants is scarce. In this context, the objective of this work was to characterize the microbiota of meconium and fecal samples obtained during the first 3 weeks of life from 14 donors using culture and molecular techniques, including DGGE and the Human Intestinal Tract Chip (HITChip) analysis of 16S rRNA amplicons. Culture techniques offer a quantification of cultivable bacteria and allow further study of the isolate, while molecular techniques provide deeper information on bacterial diversity. Culture and HITChip results were very similar but the former showed lower sensitivity. Inter-individual differences were detected in the microbiota profiles although the meconium microbiota was peculiar and distinct from that of fecal samples. Bacilli and other Firmicutes were the main bacteria groups detected in meconium while Proteobacteria dominated in the fecal samples. Culture technique showed that Staphylococcus predominated in meconium and that Enterococcus, together with Gram-negative bacteria such as Escherichia coli, Escherichia fergusonii, Klebsiella pneumoniae and Serratia marcescens, was more abundant in fecal samples. In addition, HITChip results showed the prevalence of bacteria related to Lactobacillus plantarum and Streptococcus mitis in meconium samples whereas those related to Enterococcus, Escherichia coli, Klebsiella pneumoniae and Yersinia predominated in the 3(rd) week feces. This study highlights that spontaneously-released meconium of preterm neonates contains a specific microbiota that differs from that of feces obtained after the first week of life. Our findings indicate that the presence of Serratia was strongly associated with a higher degree of immaturity and other hospital-related parameters, including antibiotherapy and mechanical ventilation
The human leukocyte antigen-presented ligandome of B lymphocytes
Hassan, C. ; Kester, M.G.D. ; Ru, A.H. ; Hombrink, P. ; Drijfhout, J.W. ; Nijveen, H. ; Leunissen, J.A.M. ; Heemskerk, M.H.M. ; Falkenburg, J.H.F. ; Veelen, P.A. van - \ 2013
Molecular and Cellular Proteomics 12 (2013). - ISSN 1535-9476 - p. 1829 - 1843.
minor histocompatibility antigen - versus-host-disease - t-cell responses - hla class-i - mass-spectrometry - peptide motifs - cancer-cells - identification - complex - epitopes
Peptides presented by human leukocyte antigen (HLA) molecules on the cell surface play a crucial role in adaptive immunology, mediating the communication between T cells and antigen presenting cells. Knowledge of these peptides is of pivotal importance in fundamental studies on T cell action, and in cellular immunotherapy and transplantation. In this study we present the in-depth identification and relative quantification of 14,500 peptide ligands constituting the HLA-ligandome of B-cells. This large number of identified ligands provides a general insight in the presented peptide repertoire and antigen presentation. Our uniquely large set of HLA-ligands allowed us to characterize in detail the peptides constituting the ligandome in terms of relative abundance, peptide length distribution, physicochemical properties, binding affinity to the HLA molecule and presence of post-translational modifications. The presented B-lymphocyte ligandome is shown to be a rich source of information by the presence of minor histocompatibility antigens, virus-derived epitopes and post-translationally modified HLA ligands and can be a good starting point to solve a wealth of specific immunological questions. These HLA ligands can form the basis for reversed immunology approaches to identify T cell epitopes, not based on in silico predictions, but based on the bona fide eluted HLA-ligandome.
The Arabidopsis exocyst subunit SEC3A is essential for embryo development and accumulates in transient puncta at the plasma membrane
Zhang, Y. ; Immink, G.H. ; Liu, C.M. ; Emons, A.M.C. ; Ketelaar, T. - \ 2013
New Phytologist 199 (2013)1. - ISSN 0028-646X - p. 74 - 88.
plant-cell growth - auxin transport - root hairs - complex - exocytosis - secretion - gene - expression - thaliana - yeast
The exocyst is a protein complex that is essential for polarized secretion in mammals and fungi. Although the exocyst is essential for plant development, its precise function has not been elucidated. We studied the role of exocyst subunit SEC3A in plant development and its subcellular localization. T-DNA insertional mutants were identified and complemented with a SEC3A-green fluorescent protein (GFP) fusion construct. SEC3A-GFP localization was determined using confocal microscopy. sec3a mutants are defective in the globular to heart stage transition in embryogenesis. SEC3A-GFP has similar cell plate localization to the other plant exocyst subunits. In interphase cells, SEC3A-GFP localizes to the cytoplasm and to the plasma membrane, where it forms immobile, punctate structures with discrete lifetimes of 2-40 s. These puncta are equally distributed over the cell surface of root epidermal cells and tip growing root hairs. The density of puncta does not decrease after growth termination of these cells, but decreases strongly when exocytosis is inhibited by treatment with brefeldin A. SEC3A does not appear to be involved in polarized secretion for cell expansion in tip growing root hairs. The landmark function performed by SEC3 in mammals and yeast is likely to be conserved in plants
The effect of linkage disequilibrium and family relationships on the reliability of genomic prediction
Wientjes, Y.C.J. ; Veerkamp, R.F. ; Calus, M.P.L. - \ 2013
Genetics 193 (2013)2. - ISSN 0016-6731 - p. 621 - 631.
genetic-relationship information - chromosome substitution strains - breeding values - dairy-cows - pedigree information - relationship matrix - angus cattle - selection - accuracy - complex
Although the concept of genomic selection relies on linkage disequilibrium (LD) between quantitative trait loci and markers, reliability of genomic predictions is strongly influenced by family relationships. In this study, we investigated the effects of LD and family relationships on reliability of genomic predictions and the potential of deterministic formulas to predict reliability using population parameters in populations with complex family structures. Five groups of selection candidates were simulated by taking different information sources from the reference population into account: (1) allele frequencies, (2) LD pattern, (3) haplotypes, (4) haploid chromosomes, and (5) individuals from the reference population, thereby having real family relationships with reference individuals. Reliabilities were predicted using genomic relationships among 529 reference individuals and their relationships with selection candidates and with a deterministic formula where the number of effective chromosome segments (Me) was estimated based on genomic and additive relationship matrices for each scenario. At a heritability of 0.6, reliabilities based on genomic relationships were 0.002 ± 0.0001 (allele frequencies), 0.022 ± 0.001 (LD pattern), 0.018 ± 0.001 (haplotypes), 0.100 ± 0.008 (haploid chromosomes), and 0.318 ± 0.077 (family relationships). At a heritability of 0.1, relative differences among groups were similar. For all scenarios, reliabilities were similar to predictions with a deterministic formula using estimated Me. So, reliabilities can be predicted accurately using empirically estimated Me and level of relationship with reference individuals has a much higher effect on the reliability than linkage disequilibrium per se. Furthermore, accumulated length of shared haplotypes is more important in determining the reliability of genomic prediction than the individual shared haplotype length
Flightlessness affects cranial morphology in birds
Gussekloo, S.W.S. ; Cubo, J. - \ 2013
Zoology : Analysis of Complex Systems 116 (2013)2. - ISSN 0944-2006 - p. 75 - 84.
phylogenetic analysis - genome sequences - aves - evolution - palaeognathae - characters - hypothesis - palatinum - reveals - complex
Flightless birds belonging to phylogenetically distant clades share several morphological features in the pectoral and pelvic apparatus. There are indications that skull morphology is also influenced by flightlessness. In this study we used a large number of flightless species to test whether flightlessness in modern birds does indeed affect cranial morphology. Discriminant analyses and variation partitioning show evidence for a relationship between skull morphology and the flightless condition in birds. A possible explanation for the change in cranial morphology can be linked to the reduced selective force for light-weight skulls in flightless birds. This makes an increase in muscle mass, and therefore an enlargement of muscle insertion areas on the skull, possible. We also compared the ontogenetic trajectory of Gallus with the adult morphology of a sample of flightless species to see whether the apomorphic features characterizing the skull of flightless birds share the same developmental basis, which would indicate convergent evolution by parallelism. Skull morphology (expressed as principal component scores) of palaeognathous flightless birds (ratites) is dissimilar (higher scores) to juvenile stages of the chicken and therefore seem peramorphic (overdeveloped). Principal component scores of adult neognathous flightless birds fall within the range of chicken development, so no clear conclusions about the ontogenetic trajectories leading to their sturdier skull morphology could be drawn
Inferring patient to patient transmission of Mycobacterium tuberculosis from whole genome sequencing data
Bryant, J.M. ; Schürch, A.C. ; Deutekom, H. van; Harris, S.R. ; Beer, J.L. de; Jager, V.C.L. de; Kremer, K. ; Hijum, S.A.F.T. van; Siezen, R.J. ; Borgdorff, M. ; Bentley, S.D. ; Parkhill, J. ; Soolingen, D. van - \ 2013
Bmc Infectious Diseases 13 (2013)1. - ISSN 1471-2334
resistance - strains - mutations - evolution - epidemiology - complex - gene
BACKGROUND: Mycobacterium tuberculosis is characterised by limited genomic diversity, which makes the application of whole genome sequencing particularly attractive for clinical and epidemiological investigation. However, in order to confidently infer transmission events, an accurate knowledge of the rate of change in the genome over relevant timescales is required. METHODS: We attempted to estimate a molecular clock by sequencing 199 isolates from epidemiologically linked tuberculosis cases, collected in the Netherlands spanning almost 16 years. RESULTS: Multiple analyses support an average mutation rate of ~0.3 SNPs per genome per year. However, all analyses revealed a very high degree of variation around this mean, making the confirmation of links proposed by epidemiology, and inference of novel links, difficult. Despite this, in some cases, the phylogenetic context of other strains provided evidence supporting the confident exclusion of previously inferred epidemiological links. CONCLUSIONS: This in-depth analysis of the molecular clock revealed that it is slow and variable over short time scales, which limits its usefulness in transmission studies. However, the superior resolution of whole genome sequencing can provide the phylogenetic context to allow the confident exclusion of possible transmission events previously inferred via traditional DNA fingerprinting techniques and epidemiological cluster investigation. Despite the slow generation of variation even at the whole genome level we conclude that the investigation of tuberculosis transmission will benefit greatly from routine whole genome sequencing