Characterisation of novel biomass degradation enzymes from the genome of <i>Cellulomonas fimi</i>

Recent analyses of genome sequences belonging to cellulolytic bacteria have revealed many genes potentially coding for cellulosic biomass degradation enzymes. Annotation of these genes however, is based on few biochemically characterised examples. Here we present a simple strategy based on BioBricks for the rapid screening of candidate genes expressed in Escherichia coli. As proof of principle we identified over 70 putative biomass degrading genes from bacterium Cellulomonas fimi, expressing a subset of these in BioBrick format. Six novel genes showed activity in E. coli. Four interesting enzymes were characterised further. α-l-arabinofuranosidase AfsB, β-xylosidases BxyF and BxyH and multi-functional β-cellobiosidase/xylosidase XynF were partially purified to determine their optimum pH, temperature and kinetic parameters. One of these enzymes, BxyH, was unexpectedly found to be highly active at strong alkaline pH and at temperatures as high as 100 °C. This report demonstrates a simple method of quickly screening and characterising putative genes as BioBricks

Fob1 and Fob2 Proteins Are Virulence Determinants of Rhizopus oryzae via Facilitating Iron Uptake from Ferrioxamine.

Dialysis patients with chronic renal failure receiving deferoxamine for treating iron overload are uniquely predisposed for mucormycosis, which is most often caused by Rhizopus oryzae. Although the deferoxamine siderophore is not secreted by Mucorales, previous studies established that Rhizopus species utilize iron from ferrioxamine (iron-rich form of deferoxamine). Here we determined that the CBS domain proteins of Fob1 and Fob2 act as receptors on the cell surface of R. oryzae during iron uptake from ferrioxamine. Fob1 and Fob2 cell surface expression was induced in the presence of ferrioxamine and bound radiolabeled ferrioxamine. A R. oryzae strain with targeted reduced Fob1/Fob2 expression was impaired for iron uptake, germinating, and growing on medium with ferrioxamine as the sole source of iron. This strain also exhibited reduced virulence in a deferoxamine-treated, but not the diabetic ketoacidotic (DKA), mouse model of mucormycosis. The mechanism by which R. oryzae obtains iron from ferrioxamine involves the reductase/permease uptake system since the growth on ferrioxamine supplemented medium is associated with elevated reductase activity and the use of the ferrous chelator bathophenanthroline disulfonate abrogates iron uptake and growth on medium supplemented with ferrioxamine as a sole source of iron. Finally, R. oryzae mutants with reduced copies of the high affinity iron permease (FTR1) or with decreased FTR1 expression had an impaired iron uptake from ferrioxamine in vitro and reduced virulence in the deferoxamine-treated mouse model of mucormycosis. These two receptors appear to be conserved in Mucorales, and can be the subject of future novel therapy to maintain the use of deferoxamine for treating iron-overload

Engineering a catabolic pathway in plants for the degradation of 1,2-dichloroethane

Plants are increasingly being employed to clean up environmental pollutants such as heavy metals; however, a major limitation of phytoremediation is the inability of plants to mineralize most organic pollutants. A key component of organic pollutants is halogenated aliphatic compounds that include 1,2-dichloroethane (1,2-DCA). Although plants lack the enzymatic activity required to metabolize this compound, two bacterial enzymes, haloalkane dehalogenase (DhlA) and haloacid dehalogenase (DhlB) from the bacterium Xanthobacter autotrophicus GJ10, have the ability to dehalogenate a range of halogenated aliphatics, including 1,2-DCA. We have engineered the dhlA and dhlB genes into tobacco (Nicotiana tabacum ‘Xanthi’) plants and used 1,2-DCA as a model substrate to demonstrate the ability of the transgenic tobacco to remediate a range of halogenated, aliphatic hydrocarbons. DhlA converts 1,2-DCA to 2-chloroethanol, which is then metabolized to the phytotoxic 2-chloroacetaldehyde, then chloroacetic acid, by endogenous plant alcohol dehydrogenase and aldehyde dehydrogenase activities, respectively. Chloroacetic acid is dehalogenated by DhlB to produce the glyoxylate cycle intermediate glycolate. Plants expressing only DhlA produced phytotoxic levels of chlorinated intermediates and died, while plants expressing DhlA together with DhlB thrived at levels of 1,2-DCA that were toxic to DhlA-expressing plants. This represents a significant advance in the development of a low-cost phytoremediation approach toward the clean-up of halogenated organic pollutants from contaminated soil and groundwater

Generation of mice with a conditional Foxp2 null allele

Disruptions of the human FOXP2 gene cause problems with articulation of complex speech sounds, accompanied by impairment in many aspects of language ability. The FOXP2/Foxp2 transcription factor is highly similar in humans and mice, and shows a complex conserved expression pattern, with high levels in neuronal subpopulations of the cortex, striatum, thalamus, and cerebellum. In the present study we generated mice in which loxP sites flank exons 12–14 of Foxp2; these exons encode the DNA-binding motif, a key functional domain. We demonstrate that early global Cre-mediated recombination yields a null allele, as shown by loss of the loxP-flanked exons at the RNA level and an absence of Foxp2 protein. Homozygous null mice display severe motor impairment, cerebellar abnormalities and early postnatal lethality, consistent with other Foxp2 mutants. When crossed to transgenic lines expressing Cre protein in a spatially and/or temporally controlled manner, these conditional mice will provide new insights into the contributions of Foxp2 to distinct neural circuits, and allow dissection of roles during development and in the mature brain. genesis 45:440–446, 2007. Published 2007 Wiley-Liss, Inc

Flat-H Redundant Frangible Joint Design Evolution 2018: Feasibility Study Conclusions

This paper reports results of an investigation into developing a single failure tolerant pyrotechnic linear separation system which features completely redundant explosive trains suitable for human spaceflight. It is a follow up to Flat-H Redundant Frangible Joint Design Evolution 2017 and Flat-H Redundant Frangible Joint Evolution. The paper chronicles the history of the redundant frangible joint development program including testing, analysis, and design improvements from 2014 to the present culminating in a successful proof-of-concept prototype. The paper describes work done to address debris control and containment of combustion products. A performance optimization strategy is presented along with optimization results. Additionally a novel containment manifold design is presented with test results

Investigation of pulsed laser induced dewetting in nanoscopic metal films

Hydrodynamic pattern formation (PF) and dewetting resulting from pulsed laser induced melting of nanoscopic metal films have been used to create spatially ordered metal nanoparticle arrays with monomodal size distribution on SiO_{\text{2}}/Si substrates. PF was investigated for film thickness h\leq7 nm < laser absorption depth \sim11 nm and different sets of laser parameters, including energy density E and the irradiation time, as measured by the number of pulses n. PF was only observed to occur for E\geq E_{m}, where E_{m} denotes the h-dependent threshold energy required to melt the film. Even at such small length scales, theoretical predictions for E_{m} obtained from a continuum-level lumped parameter heat transfer model for the film temperature, coupled with the 1-D transient heat equation for the substrate phase, were consistent with experimental observations provided that the thickness dependence of the reflectivity of the metal-substrate bilayer was incorporated into the analysis. The spacing between the nanoparticles and the particle diameter were found to increase as h^{2} and h^{5/3} respectively, which is consistent with the predictions of the thin film hydrodynamic (TFH) dewetting theory. These results suggest that fast thermal processing can lead to novel pattern formation, including quenching of a wide range of length scales and morphologies.Comment: 36 pages, 11 figures, 1 tabl

Experimental admixture among geographically disjunct populations of an invasive plant yields a global mosaic of reproductive incompatibility and heterosis

1. Invasive species have the ability to rapidly adapt in the new regions where they are introduced. Classic evolutionary theory predicts that the accumulation of genetic differences over time in allopatric isolation may lead to reproductive incompatibilities resulting in decreases in reproductive success and, eventually, to speciation. However, experimental evidence for this theoretical prediction in the context of invasive species is lacking. We aimed to test for the potential of allopatry to determine reproductive success of invasive plants, by experimentally admixing genotypes from six different native and non‐native regions of Centaurea solstitialis, an invasive forb for which preliminary studies have detected some degree of reproductive isolation between one native and non‐native region. 2. We grew plants under common garden conditions and outcrossed individuals originating from different source populations in the native and introduced range to evaluate reproductive success in terms of seed to ovule ratio produced. We also assessed geographical and genetic isolation among C. solstitialis regions as a potential driving factor of reproductive success. 3. Experimental admixture generated mixed fitness effects, including significant increases, decreases and no differences in reproductive success as compared to crosses within population (control). Centaurea solstitialis invasive populations in the Americas generated preponderantly negative fitness interactions, regardless of the pollen source, suggesting selection against immigrants and reinforcement. Other non‐native populations (Australia) as well as individuals from the native range of Spain demonstrated an increase in fitness for between‐region crosses, indicating inbreeding. These differences show an asymmetrical response to inter‐regional gene flow, but no evidence of isolation by distance. 4. Synthesis. The speed of adaptation and the accumulation of reproductive incompatibilities among allopatric populations of invasive species might be more rapid than previously assumed. Our data show a global mosaic of reproductive outputs, showcasing an array of evolutionary processes unfolding during colonization at large biogeographical scales

Experimental admixture among geographically disjunct populations of an invasive plant yields a global mosaic of reproductive incompatibility and heterosis

Invasive species have the ability to rapidly adapt in the new regions where they are introduced. Classic evolutionary theory predicts that the accumulation of genetic differences over time in allopatric isolation may lead to reproductive incompatibilities resulting in decreases in reproductive success and, eventually, to speciation. However, experimental evidence for this theoretical prediction in the context of invasive species is lacking. We aimed to test for the potential of allopatry to determine reproductive success of invasive plants, by experimentally admixing genotypes from six different native and non-native regions of Centaurea solstitialis, an invasive forb for which preliminary studies have detected some degree of reproductive isolation between one native and non-native region. We grew plants under common garden conditions and outcrossed individuals originating from different source populations in the native and introduced range to evaluate reproductive success in terms of seed to ovule ratio produced. We also assessed geographical and genetic isolation among C. solstitialis regions as a potential driving factor of reproductive success. Experimental admixture generated mixed fitness effects, including significant increases, decreases and no differences in reproductive success as compared to crosses within population (control). Centaurea solstitialis invasive populations in the Americas generated preponderantly negative fitness interactions, regardless of the pollen source, suggesting selection against immigrants and reinforcement. Other non-native populations (Australia) as well as individuals from the native range of Spain demonstrated an increase in fitness for between-region crosses, indicating inbreeding. These differences show an asymmetrical response to inter-regional gene flow, but no evidence of isolation by distance. Synthesis. The speed of adaptation and the accumulation of reproductive incompatibilities among allopatric populations of invasive species might be more rapid than previously assumed. Our data show a global mosaic of reproductive outputs, showcasing an array of evolutionary processes unfolding during colonization at large biogeographical scales.Fil: Irimia, Ramona E.. Eberhard Karls Universität Tübingen; Alemania. Universidad de Coimbra; PortugalFil: Hierro, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias de la Tierra y Ambientales de La Pampa. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de La Pampa; ArgentinaFil: Branco, Soraia. Universidad de Coimbra; PortugalFil: Sotes, Gastón Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias de la Tierra y Ambientales de La Pampa. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de La Pampa; Argentina. Universidad de Chile; ChileFil: Cavieres, Lohengrin A.. Universidad de Chile; Chile. Universidad de Concepción; ChileFil: Eren, Ozkan. Adnan Menderes Universitesi; TurquíaFil: Lortie, Christopher J.. University of York; Reino UnidoFil: French, Kristine. No especifíca;Fil: Callaway, Ragan M.. University of Montana; Estados UnidosFil: Montesinos, Daniel. Universidad de Coimbra; Portuga