A pipeline for targeted metagenomics of environmental bacteria.

BackgroundMetagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted. The combination of fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) has the potential to enrich taxonomically well-defined clades for genomic analyses.MethodsCells hybridized with a taxon-specific FISH probe are enriched based on their fluorescence signal via flow cytometric cell sorting. A recently developed FISH procedure, the hybridization chain reaction (HCR)-FISH, provides the high signal intensities required for flow cytometric sorting while maintaining the integrity of the cellular DNA for subsequent genome sequencing. Sorted cells are subjected to shotgun sequencing, resulting in targeted metagenomes of low diversity.ResultsPure cultures of different taxonomic groups were used to (1) adapt and optimize the HCR-FISH protocol and (2) assess the effects of various cell fixation methods on both the signal intensity for cell sorting and the quality of subsequent genome amplification and sequencing. Best results were obtained for ethanol-fixed cells in terms of both HCR-FISH signal intensity and genome assembly quality. Our newly developed pipeline was successfully applied to a marine plankton sample from the North Sea yielding good quality metagenome assembled genomes from a yet uncultivated flavobacterial clade.ConclusionsWith the developed pipeline, targeted metagenomes at various taxonomic levels can be efficiently retrieved from environmental samples. The resulting metagenome assembled genomes allow for the description of yet uncharacterized microbial clades. Video abstract

The Imagined and Real Jerusalem in Art and Architecture

In The Imagined and Real Jerusalem in Art and Architecture specialists in various fields of art history, from Early Christian times to the present, discuss in depth a series of Western artworks, artefacts, and buildings, which question the visualization of Jerusalem

Internal friction and its thermal evolution on 304 L stainless steel films

Internal friction has been measured between 300 and 760K on 304 L stainless steel (SS) using a vibrating reed device. The 0.6 µm thick samples were deposited with ion beam sputtering technique on (100) silicon substrate. It has been shown that the damping level is considerably reduced by annealing between 500 and 760K. The calculated activation energy and the reaction order, using the Johnson-Mehl-Avrami (J-M-H) kinetic enable us to assume that the observed mechanism is closely related to microstructural rearrangements located at grain boundaries.Internal friction has been measured between 300 and 760K on 304 L stainless steel (SS) using a vibrating reed device. The 0.6 µm thick samples were deposited with ion beam sputtering technique on (100) silicon substrate. It has been shown that the damping level is considerably reduced by annealing between 500 and 760K. The calculated activation energy and the reaction order, using the Johnson-Mehl-Avrami (J-M-H) kinetic enable us to assume that the observed mechanism is closely related to microstructural rearrangements located at grain boundaries

A Genetic Screen Identifies New Steps In Oocyte Maturation That Enhance Proteostasis In The Immortal Germ Lineage

Somatic cells age and die, but the germ-cell lineage is immortal. In Caenorhabditis elegans, germline immortality involves proteostasis renewal at the beginning of each new generation, when oocyte maturation signals from sperm trigger the clearance of carbonylated proteins and protein aggregates. Here, we explore the cell biology of this proteostasis renewal in the context of a whole-genome RNAi screen. Oocyte maturation signals are known to trigger protein-aggregate removal via lysosome acidification. Our findings suggest that lysosomes are acidified as a consequence of changes in endoplasmic reticulum activity that permit assembly of the lysosomal V-ATPase, which in turn allows lysosomes to clear the aggregates via microautophagy. We define two functions for mitochondria, both of which appear to be independent of ATP generation. Many genes from the screen also regulate lysosome acidification and age-dependent protein aggregation in the soma, suggesting a fundamental mechanistic link between proteostasis renewal in the germline and somatic longevity

Mutations within the hepatitis C virus genotype 1b E2-PePHD domain do not correlate with treatment outcome.

The hepatitis C virus (HCV) envelope protein 2 (E2) interacts in vitro with the interferon alpha (IFN-alpha)-inducible double-stranded RNA-activated protein kinase, suggesting a possible mechanism by which HCV may evade the antiviral effects of IFN-alpha. Variability in the part of the HCV E2 gene encoding the carboxy-terminal part of the protein, which includes the interaction domain (E2-PePHD), was explored in 25 patients infected with HCV genotype 1b and receiving IFN-alpha therapy. PCR products were generated and sequenced for 15 patients with a sustained response and for 10 patients with no virological response after treatment with IFN-alpha and ribavirin. PePHD amino acid sequences were obtained for isolates from serum collected before and during treatment, after 2 months in responders, and after 6 months in nonresponders. Quasispecies analysis of the pretreatment PePHD region was performed for isolates from patients displaying amino acid substitutions in this domain on direct sequencing. The E2-PePHD sequence was highly conserved in both resistant and susceptible genotype 1b strains and was identical to the prototype HCV type J sequence. No significant emergence of PePHD mutants during therapy was observed in our clonal analysis, and sporadic mutations and treatment outcomes were not found to be correlated. The PePHD sequence before or during treatment cannot be used to predict reliably the outcome of treatment in HCV type 1b-infected patients

X-Ray synchronotron study of phase transforms in illite clays to extract information on sigillata manufacturing processes.

The technique of sigillata really began in central Italy during the first century B. C. with the development of red vitrified slips obtained through vitrification of a clay preparation. These ceramics, usually decorated with raised motifs and standardised shapes, quickly took over as semi luxury crockery. Given this success, this technique quickly extended to the entire Italian peninsula and then to the Mediterranean coast. From the very start of our era, great centres of production were set up in the south of Gaul. The aspect of sigillata comes from the nature and the texture of its slip. Studies have shown that sigillata slips of quality were obtained from a non calcareous clay while the local calcareous clay was used for the bodies. During firing the slips are vitrified and get a specific microstructure containing hematite and nanometric corundum crystals [1]. An investigation of the clays surrounding La Graufesenque site started and it seems that only the Trias levels are chemically compatible with the composition of antique slips. Apart from the in depth study of the mineralogical nature of these clays realized at a geological Laboratory, we have studied the structural transformations as a function of temperature of two of these clays, chosen for the quality of vitrification in the firing temperature range of sigillata [1030-1080°C]. The main difference between the chemical composition of these two clays is the amount of Mg (2.4 % and 4.5 % in oxide weight). Time-resolved measurements were made at Daresbury (station 2.3) up to 1100oC in oxidizing conditions. An abrupt increase of the hematite cell was observed around 850°C. Above 1000°C, the hematite peaks get sharper which indicate an increase of coherence length (Fig. 1). A spinel phase with cell parameter close to MgAl2O3 was detected from this temperature. As for the hematite, its coherence length increases with the temperature but also during the beginning of the cooling. For the clay sample with the smaller amount of Mg, a corundum phase with very small coherence length was detected above 1000°C. Slips were prepared from the last clay by modern potters and firing at 1050°C in oxidizing atmosphere. A mineral quantitative analysis performed using the Rietveld method revealed that the amount of spinel phase is very high while the corundum contributes to a small part of crystal phases. It is the inverse in the antique slip where the amount of Mg in oxide weight is around 1%. It is clear that the amount of Mg plays a key role in the corundum/spinel competition and that the present slips contain too much Mg. Two questions arise: (i) As the Trias levels are quite heterogeneous is it possible to find clay with less Mg? and (ii) Did the gallo-roman potters eliminated a great part of Mg during the slip preparation process? We discuss the merits of these two alternative hypotheses

Elastic-strain distribution in metallic film-polymer substrate composites

Synchrotron x-ray radiation was used for in situ strain measurements during uniaxial tests on polymer substrates coated by a metallic gold film 400 nm thick deposited without interlayer or surface treatment. X-ray diffraction allowed capturing both components elastic strains and determining how these were partitioned between the metallic film and the polymeric substrate. For strains below 0.8%, deformation is continuous through the metal-polymer interface while above, the onset of plasticity in the metallic film induces a shift between film and substrate elastic strains

Synchrotron X-ray diffraction experiments with a prototype hybrid pixel detector

International audienceA prototype X-ray pixel area detector (XPAD3.1) has been used for X-ray diffraction experiments with synchrotron radiation. The characteristics of this detector are very attractive in terms of fast readout time, high dynamic range and high signal-to-noise ratio. The prototype XPAD3.1 enabled various diffraction experiments to be performed at different energies, sample-to-detector distances and detector angles with respect to the direct beam, yet it was necessary to perform corrections on the diffraction images according to the type of experiment. This paper is focused on calibration and correction procedures to obtain high-quality scientific results specifically developed in the context of three different experiments, namely mechanical characterization of nanostructured multilayers, elastic-plastic deformation of duplex steel and growth of carbon nanotubes

Effect of thermal treatments on the structure of MoNxOy thin films

Article in PressMoNxOy films were deposited on steel substrates by dc reactive magnetron sputtering. The depositions were carried out from a pure molybdenum target, varying the flow rate of reactive gases. X-ray diffraction (XRD) results revealed the occurrence of cubic MoNx and hexagonal (d-MoN) phases for the films with high nitrogen flow rates. The increase of oxygen content induces the decrease of the grain size of the molybdenum nitride crystallites. The thermal stability of a set of samples was studied in vacuum, for an annealing time of 1 h, for temperatures ranging from 500 to 800 C in 100 C steps. The results showed that pure molybdenum nitride films changed their structure from a meta-stable cubic MoN to hexagonal d-MoN and cubic g-Mo2N-type structures with increasing annealing temperatures. The samples with molybdenum nitride films evidenced a good thermal stability, but those with molybdenum oxynitride coatings showed a tendency to detach with the increase of the annealing temperature.Fundação para a Ciência e a Tecnologia (FCT) - POCTI/CTM/38086/200