Glutamine Phosphoribosylpyrophosphate Amidotransferase-independent Phosphoribosyl Amine Synthesis from Ribose 5-Phosphate and Glutamine or Asparagine

Phosphoribosylamine (PRA) is the first intermediate in the common pathway to purines and thiamine and is generated in bacteria by glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (EC 2.4.2.14) from PRPP and glutamine. Genetic data have indicated that multiple, non-PRPP amidotransferase mechanisms exist to generate PRA sufficient for thiamine but not purine synthesis. Here we describe the purification and identification of an activity (present in both Escherichia coli and Salmonella enterica) that synthesizes PRA from ribose 5-phosphate and glutamine/asparagine. A purification resulting in greater than a 625-fold increase in specific activity identified 8 candidate proteins. Of the candidates, overexpression of AphA (EC 3.1.3.2), a periplasmic class B nonspecific acid phosphatase, significantly increased activity in partially purified extracts. Native purification of AphA to >95% homogeneity determined that the periplasmic L-asparaginase II, AnsB (EC 3.5.1.1), co-purified with AphA and was also necessary for PRA formation. The potential physiological relevance of AphA and AnsB in contributing to thiamine biosynthesis in vivo is discussed

Do neighbourhood environmental perceptions affect practices?

In this paper, we examine how environmental practices related to public transit and urban green space use are influenced by perceptions of local level environmental change, neighbourhood inhabitation, and socio-demographic factors. The analysis shows that perceptions of change and neighbourhood inhabitation offer better explanations for changing local environmental practices than socio-demographic orientations. We contribute to social practice theory by drawing attention to the interplay of environmental perceptions and neighbourhood inhabitation as factors that facilitate changing environmental practices. By gaining insight into the relationship between perceptions of change and environmental practices, we thereby learn how sustainability goals, such as those embodied by SDG11, can be translated into social practices at the community level.Dans cet article, nous examinons comment les pratiques environnementales liées au transport en commun et à l’utilisation des espaces verts urbains sont influencées par les perceptions du changement environnemental au niveau local, l’habitation des quartiers et les facteurs sociodémographiques. L’analyse montre que les perceptions du changement et de l’habitat du quartier offrent de meilleures explications pour l’évolution des pratiques environnementales locales que les orientations sociodémographiques. Nous contribuons è la théorie de la pratique sociale en attirant l’attention sur l’interaction des perceptions environnementales et de l’habitation du quartier en tant que facteurs qui facilitent l’évolution des pratiques environnementales. En acquérant un aperçu de la relation entre les perceptions du changement et les pratiques environnementales, nous apprenons ainsi comment les objectifs de durabilité, tels que ceux incarnées par ODD (Agenda 2030 du développement durable), peuvent être traduits en pratiques sociales au niveau communautaire

U-Pb SHRIMP zircon dating of Grenvillian metamorphism in Western Sierras Pampeanas (Argentina) : correlation with the Arequipa-Antofalla craton and constraints on the extent of the Precordillera Terrane

The Sierras Pampeanas of Argentina, the largest outcrop of pre-Andean crystalline basement in southern South America, resulted from plate interactions along the proto-Andean margin of Gondwana, from as early as Mesoproterozoic to Late Paleozoic times (e.g., Ramos, 2004, and references therein). Two discrete Paleozoic orogenic belts have been recognized: the Early Cambrian Pampean belt in the eastern sierras, and the Ordovician Famatinian belt, which partially overprints it to the west (e.g., Rapela et al., 1998). In the Western Sierras Pampeanas, Mesoproterozoic igneous rocks (ca. 1.0–1.2 Ga) have been recognized in the Sierra de Pie de Palo (Fig. 1) (McDonough et al., 1993 M.R. McDonough, V.A. Ramos, C.E. Isachsen, S.A. Bowring and G.I. Vujovich, Edades preliminares de circones del basamento de la Sierra de Pie de Palo, Sierras Pampeanas occidentales de San Juán: sus implicancias para el supercontinente proterozoico de Rodinia, 12° Cong. Geol. Argentino, Actas vol. 3 (1993), pp. 340–342.McDonough et al., 1993, Pankhurst and Rapela, 1998 and Vujovich et al., 2004) that are time-coincident with the Grenvillian orogeny of eastern and northeastern North America (e.g., Rivers, 1997 and Corrievau and van Breemen, 2000). These Grenvillian-age rocks have been considered to be the easternmost exposure of basement to the Precordillera Terrane, a supposed Laurentian continental block accreted to Gondwana during the Famatinian orogeny (Thomas and Astini, 2003, and references therein). However, the boundaries of this Grenvillian belt are still poorly defined, and its alleged allochthoneity has been challenged (Galindo et al., 2004). Moreover, most of the Grenvillian ages so far determined relate to igneous protoliths, and there is no conclusive evidence for a Grenvillian orogenic belt, other than inferred from petrographic evidence alone (Casquet et al., 2001). We provide here the first evidence, based on U–Pb SHRIMP zircon dating at Sierra de Maz, for a Grenville-age granulite facies metamorphism, leading to the conclusion that a continuous mobile belt existed throughout the proto-Andean margin of Gondwana in Grenvillian times

Bulk and single-molecule analysis of a bacterial DNA2-like helicase-nuclease reveals a single-stranded DNA looping motor

DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5' to 3' ssDNA translocase and 5' to 3' helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of ∼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.Wellcome Trust [100401/Z/12/Z to M.D.]; EuropeanResearch Council [681299 to F.M.-H.]; Spanish Min-istry of Economy and Competitiveness [BFU2017-83794-PAEI/FEDER, UE to F.M.-H.]; Comunidad de MadridTec4Bio [S2018/NMT-4443 to F.M.-H.]; NanoBioCancer[Y2018/BIO-4747 to F.M.-H.]. Funding for open accesscharge: Wellcome Trust [100401/Z/12/Z].Peer reviewe

Interstitial Fractionalization and Spherical Crystallography

Finding the ground states of identical particles packed on spheres has relevance for stabilizing emulsions and a venerable history in the literature of theoretical physics and mathematics. Theory and experiment have confirmed that defects such as disclinations and dislocations are an intrinsic part of the ground state. Here we discuss the remarkable behavior of vacancies and interstitials in spherical crystals. The strain fields of isolated disclinations forced in by the spherical topology literally rip interstitials and vacancies apart, typically into dislocation fragments that combine with the disclinations to create small grain boundary scars. The fractionation is often into three charge-neutral dislocations, although dislocation pairs can be created as well. We use a powerful, freely available computer program to explore interstitial fractionalization in some detail, for a variety of power law pair potentials. We investigate the dependence on initial conditions and the final state energies, and compare the position dependence of interstitial energies with the predictions of continuum elastic theory on the sphere. The theory predicts that, before fragmentation, interstitials are repelled from 5-fold disclinations and vacancies are attracted. We also use vacancies and interstitials to study low energy states in the vicinity of "magic numbers" that accommodate regular icosadeltahedral tessellations.Comment: 21 pages, 9 figure

Fate of Biological Control Introductions: Monitoring an Australian Fungal Pathogen of Grasshoppers in North America

In North America there are two generally recognized pathotypes (pathotypes 1 and 2) of the fungus Entomophaga grylli which show host-preferential infection of grasshopper subfamilies. Pathotype 3, discovered in Austra- lia,hasabroadergrasshopperhostrangeandwasconsidered to be a good biocontrol agent. Between 1989 and 1991 patho- type3wasintroducedattwofieldsitesinNorthDakota.Since resting spores are morphologically indistinguishable among pathotypes, we used pathotype-specific DNA probes to con- firm pathotype identification in E. grylli-infected grasshop- pers collected at the release sites in 1992, 1993, and 1994. In 1992, up to 23% of E. grylli-infected grasshoppers of the subfamilies Melanoplinae, Oedipodinae, and Gomphocerinae were infected by pathotype 3,with no infections \u3e1 km from the release sites. In 1993, pathotype 3 infections declined to 1.7%. In 1994 grasshopper populations were low and no pathotype3infectionswerefound.Thefrequencyofpathotype 3 infection has declined to levels where its long-term survival in North America is questionable. Analyses of biocontrol releases are critical to evaluating the environmental risks associatedwiththeseecologicalmanipulations,andmolecular probesarepowerfultoolsformonitoringbiocontrolreleases

Liquid sprays and flow studies in the direct-injection diesel engine under motored conditions

A two dimensional, implicit finite difference method of the control volume variety, a two equation model of turbulence, and a discrete droplet model were used to study the flow field, turbulence levels, fuel penetration, vaporization, and mixing in diesel engine environments. The model was also used to study the effects of engine speed, injection angle, spray cone angle, droplet distribution, and intake swirl angle on the flow field, spray penetration and vaporization, and turbulence in motored two-stroke diesel engines. It is shown that there are optimum conditions for injection, which depend on droplet distribution, swirl, spray cone angle, and injection angle. The optimum conditions result in good spray penetration and vaporization and in good fuel mixing. The calculation presented clearly indicates that internal combustion engine models can be used to assess, at least qualitatively, the effects of injection characteristics and engine operating conditions on the flow field and on the spray penetration and vaporization in diesel engines

Human HELB is a processive motor protein that catalyzes RPA clearance from single-stranded DNA

Human DNA helicase B (HELB) is a poorly characterized helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single-molecule approaches to characterize the biochemical activities of HELB protein with a particular focus on its interactions with Replication Protein A (RPA) and RPA–single-stranded DNA (ssDNA) filaments. HELB is a monomeric protein that binds tightly to ssDNA with a site size of ∼20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5′ to 3′ direction accompanied by the formation of DNA loops. HELB also displays classical helicase activity, but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA, which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from ssDNA. This activity, which can allow other proteins access to ssDNA intermediates despite their shielding by RPA, may underpin the diverse roles of HELB in cellular DNA transactions.[Significance] Single-stranded DNA (ssDNA) is a key intermediate in many cellular DNA transactions, including DNA replication, repair, and recombination. Nascent ssDNA is rapidly bound by the Replication Protein A (RPA) complex, forming a nucleoprotein filament that both stabilizes ssDNA and mediates downstream processing events. Paradoxically, however, the very high affinity of RPA for ssDNA may block the recruitment of further factors. In this work, we show that RPA–ssDNA nucleoprotein filaments are specifically targeted by the human HELB helicase. Recruitment of HELB by RPA–ssDNA activates HELB translocation activity, leading to processive removal of upstream RPA complexes. This RPA clearance activity may underpin the diverse roles of HELB in replication and recombination.Work in the laboratory of M.S.D. was supported by an Elizabeth Blackwell Early Career Fellowship from the University of Bristol (to O.J.W.) and Wellcome Trust Investigator Grant 100401/Z/12/Z (to M.S.D.). Work in the laboratory of E.A. was supported by NIH Grants GM130746 (to E.A.) and GM133967 (to E.A.). F.M.-H. acknowledges support from the European Research Council under European Union Horizon 2020 Research and Innovation Program Grant Agreement 681299. Work in the laboratory of F.M.-H. was also supported by Spanish Ministry of Science and Innovation Grants BFU2017-83794-P (AEI/FEDER, UE; to F.M.-H.) and PID2020-112998GB-100 (AEI/10.13039/501100011033; to F.M.-H.) and Comunidad de Madrid Grants Tec4-Bio–S2018/NMT-4443 (to F.M.-H.) and NanoBioCancer–Y2018/BIO-4747 (to F.M.-H.)