ELUCIDATING THE BIOCHEMICAL WIZARDRY OF TRITERPENE METABOLISM IN \u3ci\u3eBOTROYCOCCUS BRAUNII\u3c/i\u3e

B. braunii is a green alga that has attracted attention as a potential renewable fuel source due to its high oil content and the archeological record of its unique contribution to oil and coal shales. Three extant chemotypes of B. braunii have been described, namely race A, race B, and race L, which accumulate alkadienes and alkatrienes, botryococcene and squalene and their methylated derivatives, and lycopadiene, respectively. The methylated triterpenes, particularly botryococcenes, produced by race B can be efficiently converted to high quality combustible fuels and other petrochemicals; however, botryococcene biosynthesis has remained enigmatic. It has been suggested that botryococcene biosynthesis could resemble that of squalene, arising from an initial condensation of two molecules of farnesyl diphosphate (FPP) to form pre-squalene diphosphate (PSPP), which then undergoes a reductive rearrangement to form squalene, or in an alternative reductive rearrangement, botryococcene. Based on the proposed similarities, we predicted that a botryococcene synthase would resemble squalene synthase and hence, isolated squalene synthase-like genes from B. braunii race B. While B. braunii does harbor at least one typical squalene synthase, none of the other three squalene synthase-like (SSL) genes encode for botryococcene biosynthesis directly. SSL-1 catalyzes the biosynthesis of PSPP and SSL-2 the biosynthesis of bisfarnesyl ether and to a lesser extent squalene, while SSL-3 does not appear able to directly utilize FPP as a substrate. However, when SSL-1 is combined with either SSL-2 or SSL-3, in vivo and in vitro, robust squalene or botryococcene biosynthesis was observed, respectively. These findings were unexpected because squalene synthase, an ancient and likely progenitor to the other Botryococcus triterpene synthases, catalyzes a two-step reaction within a single enzyme unit without intermediate release, yet in B. braunii, these activities appear to have separated and evolved inter-dependently for specialized triterpene production. Expression of various configurations of the SSL genes in TN-7 yeast demonstrates that botryococcene can be efficiently produced in a heterologous host. Additionally, three triterpene methyltransferase (TMTs) were isolated which efficiently catalyze the transfer of a methyl group from S-adenosyl methionine (SAM) to either squalene (TMT-1 and TMT-2) or botryococcene (TMT-3) in vivo and in vitro. Co-expression of the various TMT genes with either squalene synthase or botryococcene synthase in TN-7 yeast resulted in the accumulation of C31 and C32 methyl derivatives of squalene or botryococcene, demonstrating their potential for heterologous production. The methylation sites were determined by NMR spectroscopy to be identical to C31 and C32 methyl-derivatives of squalene or botryococcene observed in B. braunii race B. Expression studies of various heterologous squalene synthase genes in S. cerevisiae corroborated an earlier but surprising observation reported in the literature. While the squalene synthase gene of S. cerevisiae was able to complement an erg9 (squalene synthase) knockout in yeast, squalene synthase genes from plants and animals were not. Chemical profiles revealed that squalene accumulated to significant levels in yeast expressing the squalene synthase of plant, animal, or S. cerevisiae. This suggested that it was not the ability of these heterologous synthase enzymes to produce squalene, but their inability to feed squalene into the native sterol biosynthetic pathway that prevented them from restoring normal ergosterol biosynthesis in S. cerevisiae. By examining the ability of chimera squalene synthase enzymes to complement the erg9 mutation, a discrete sequence of amino acids near the C-terminus of the enzyme was identified which is necessary and sufficient for allowing any squalene synthase to restore normal sterol metabolism

Draft Nuclear Genome Sequence of the Liquid Hydrocarbon-Accumulating Green Microalga Botryococcus braunii Race B (Showa).

Botryococcus braunii has long been known as a prodigious producer of liquid hydrocarbon oils that can be converted into combustion engine fuels. This draft genome for the B race of B. braunii will allow researchers to unravel important hydrocarbon biosynthetic pathways and identify possible regulatory networks controlling this unusual metabolism

Draft Nuclear Genome Sequence of the Liquid Hydrocarbon–Accumulating Green Microalga \u3cem\u3eBotryococcus braunii\u3c/em\u3e Race B (Showa)

Botryococcus braunii has long been known as a prodigious producer of liquid hydrocarbon oils that can be converted into combustion engine fuels. This draft genome for the B race of B. braunii will allow researchers to unravel important hydrocarbon biosynthetic pathways and identify possible regulatory networks controlling this unusual metabolism

Wireless Stimulation of Antennal Muscles in Freely Flying Hawkmoths Leads to Flight Path Changes

Insect antennae are sensory organs involved in a variety of behaviors, sensing many different stimulus modalities. As mechanosensors, they are crucial for flight control in the hawkmoth Manduca sexta. One of their roles is to mediate compensatory reflexes of the abdomen in response to rotations of the body in the pitch axis. Abdominal motions, in turn, are a component of the steering mechanism for flying insects. Using a radio controlled, programmable, miniature stimulator, we show that ultra-low-current electrical stimulation of antennal muscles in freely-flying hawkmoths leads to repeatable, transient changes in the animals' pitch angle, as well as less predictable changes in flight speed and flight altitude. We postulate that by deflecting the antennae we indirectly stimulate mechanoreceptors at the base, which drive compensatory reflexes leading to changes in pitch attitude.United States. Defense Advanced Research Projects Agenc

'To live and die [for] Dixie': Irish civilians and the Confederate States of America

Around 20,000 Irishmen served in the Confederate army in the Civil War. As a result, they left behind, in various Southern towns and cities, large numbers of friends, family, and community leaders. As with native-born Confederates, Irish civilian support was crucial to Irish participation in the Confederate military effort. Also, Irish civilians served in various supporting roles: in factories and hospitals, on railroads and diplomatic missions, and as boosters for the cause. They also, however, suffered in bombardments, sieges, and the blockade. Usually poorer than their native neighbours, they could not afford to become 'refugees' and move away from the centres of conflict. This essay, based on research from manuscript collections, contemporary newspapers, British Consular records, and Federal military records, will examine the role of Irish civilians in the Confederacy, and assess the role this activity had on their integration into Southern communities. It will also look at Irish civilians in the defeat of the Confederacy, particularly when they came under Union occupation. Initial research shows that Irish civilians were not as upset as other whites in the South about Union victory. They welcomed a return to normalcy, and often 'collaborated' with Union authorities. Also, Irish desertion rates in the Confederate army were particularly high, and I will attempt to gauge whether Irish civilians played a role in this. All of the research in this paper will thus be put in the context of the Drew Gilpin Faust/Gary Gallagher debate on the influence of the Confederate homefront on military performance. By studying the Irish civilian experience one can assess how strong the Confederate national experiment was. Was it a nation without a nationalism

Whole-genome sequencing for prediction of Mycobacterium tuberculosis drug susceptibility and resistance : a retrospective cohort study

BACKGROUND : Diagnosing drug-resistance remains an obstacle to the elimination of tuberculosis. Phenotypic drugsusceptibility testing is slow and expensive, and commercial genotypic assays screen only common resistancedetermining mutations. We used whole-genome sequencing to characterise common and rare mutations predicting drug resistance, or consistency with susceptibility, for all fi rst-line and second-line drugs for tuberculosis. METHODS : Between Sept 1, 2010, and Dec 1, 2013, we sequenced a training set of 2099 Mycobacterium tuberculosis genomes. For 23 candidate genes identifi ed from the drug-resistance scientifi c literature, we algorithmically characterised genetic mutations as not conferring resistance (benign), resistance determinants, or uncharacterised. We then assessed the ability of these characterisations to predict phenotypic drug-susceptibility testing for an independent validation set of 1552 genomes. We sought mutations under similar selection pressure to those characterised as resistance determinants outside candidate genes to account for residual phenotypic resistance. FINDINGS : We characterised 120 training-set mutations as resistance determining, and 772 as benign. With these mutations, we could predict 89·2% of the validation-set phenotypes with a mean 92·3% sensitivity (95% CI 90·7–93·7) and 98·4% specifi city (98·1–98·7). 10·8% of validation-set phenotypes could not be predicted because uncharacterised mutations were present. With an in-silico comparison, characterised resistance determinants had higher sensitivity than the mutations from three line-probe assays (85·1% vs 81·6%). No additional resistance determinants were identifi ed among mutations under selection pressure in non-candidate genes. INTERPRETATION : A broad catalogue of genetic mutations enable data from whole-genome sequencing to be used clinically to predict drug resistance, drug susceptibility, or to identify drug phenotypes that cannot yet be genetically predicted. This approach could be integrated into routine diagnostic workfl ows, phasing out phenotypic drugsusceptibility testing while reporting drug resistance early.Wellcome Trust, National Institute of Health Research, Medical Research Council, and the European Union.http://www.thelancet.com/infectionhb201

Excitonic and charge transfer interactions in tetracene stacked and T-shaped dimers

International audienceExtended quantum chemical calculations were performed for the tetracene dimer to provide benchmark results, analyze the excimer survival process, and explore the possibility of using long-range-corrected (LC) time-dependent (TD) second-order density functional tight-biding (DFTB2) for this system. Ground- and first-excited-states optimized geometries, vertical excitations at relevant minima, and intermonomer displacement potential energy curves (PECs) were calculated for these purposes. Ground-state geometries were optimized with the scaled-opposite-spin (SOS) second-order Møller-Plesset perturbation theory (MP2) and LC-DFT (density functional theory) and LC-DFTB2 levels. Excited-state geometries were optimized with SOS-ADC(2) (algebraic diagrammatic construction to second-order) and the time-dependent approaches for the latter two methods. Vertical excitations and PECs were compared to multireference configuration interaction DFT (DFT/MRCI). All methods predict the lowest-energy S0 conformer to have monomers parallel and rotated relative to each other and the lowest S1 conformer to be of a displaced-stacked type. LC-DFTB2, however, presents some relevant differences regarding other conformers for S0. Despite some state-order inversions, an overall good agreement between methods was observed in the spectral shape, state character, and PECs. Nevertheless, DFT/MRCI predicts that the S1 state should acquire a doubly excited-state character relevant to the excimer survival process and, therefore, cannot be completely described by the single reference methods used in this work. PECs also revealed an interesting relation between dissociation energies and the intermonomer charge-transfer interactions for some states

Analysis of Aerosol Spreading in a German Inter City Express (ICE) Train Carriage

This paper focuses on the propagation of aerosols in a rolling stock passenger compartment. Extensive measurements were carried out in our stationary test vehicle DIRK, an ICE 2 rail car, operated in a climate chamber. It is shown that the propagation of aerosols only occurs for a distance of a few seat rows. Furthermore, the maximum percentage of particles exhaled by a passenger and inhaled by another passenger is less than 0.35%. A mouth-nose-protection (surgical mask) at the aerosol source reduces this value to a maximum of 0.25%. Moreover, the use of a mouth-nose-cover reduced the propagation lengths. Here, only the effect of the mask at the source was considered, a further reduction of inhaled aerosols will be achieved when the receivers also wear masks. It is concluded that, for this type of passenger coach, the indirect propagation of aerosols, i.e., via the HVAC system, is nearly irrelevant compared to the direct propagation from one passenger to another. However, there is a non-zero aerosol transport via the HVAC system, resulting in local inhaled particles far away from the source of around 0.015–0.026%, which is more than one order of magnitude lower than on the most highly contaminated seats