Invasive and adherent bacterial pathogens co-Opt host clathrin for infection

Infection by the bacterium Listeria monocytogenes depends on host cell clathrin. To determine whether this requirement is widespread, we analyzed infection models using diverse bacteria. We demonstrated that bacteria that enter cells following binding to cellular receptors (termed "zippering" bacteria) invade in a clathrin-dependent manner. In contrast, bacteria that inject effector proteins into host cells in order to gain entry (termed "triggering" bacteria) invade in a clathrin-independent manner. Strikingly, enteropathogenic Escherichia coli (EPEC) required clathrin to form actin-rich pedestals in host cells beneath adhering bacteria, even though this pathogen remains extracellular. Furthermore, clathrin accumulation preceded the actin rearrangements necessary for Listeria entry. These data provide evidence for a clathrin-based entry pathway allowing internalization of large objects (bacteria and ligand-coated beads) and used by "zippering" bacteria as part of a general mechanism to invade host mammalian cells. We also revealed a nonendocytic role for clathrin required for extracellular EPEC infections.     

Genetic and physical characterisation of the locus controlling columnar habit in apple (Malus × domestica Borkh.)

A better understanding of the genetic control of tree architecture would potentially allow improved tailoring of newly bred apple cultivars in terms of field management aspects, such as planting density, pruning, pest control and disease protection. It would also have an indirect impact on yield and fruit quality. The Columnar (Co) locus strongly suppresses lateral branch elongation and is the most important genetic locus influencing tree architecture in apple. Co has previously been mapped on apple linkage group (LG) 10. In order to obtain fine mapping of Co, both genetically and physically, we have phenotypically analysed and screened three adult segregating experimental populations, with a total of 301 F₁ plants, and one substantial 3-year old population of 1,250 F₁ plants with newly developed simple sequence repeat (SSR) markers, based on the ‘Golden delicious’ apple genome sequence now available. Co was found to co-segregate with SSR marker Co04R12 and was confined in a region of 0.56 cM between SSR markers Co04R11 and Co04R13, corresponding to 393 kb on the ‘Golden delicious’ genome sequence. In this region, 36 genes were predicted, including at least seven sequences potentially belonging to genes that could be considered candidates for involvement in control of shoot development. Our results provide highly reliable, virtually co-segregating markers that will facilitate apple breeding aimed at modifications of the tree habit and lay the foundations for the cloning of Co.     

Nucleophosmin mutations alter its nucleolar localization by impairing G-quadruplex binding at ribosomal DNA

Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and export, centrosome duplication and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia. Mutations at the C-terminal domain led to variant proteins that aberrantly and stably translocate to the cytoplasm. We have previously shown that NPM1 C-terminal domain binds with high affinity G-quadruplex DNA. Here, we investigate the structural determinants of NPM1 nucleolar localization. We show that NPM1 interacts with several G-quadruplex regions found in ribosomal DNA, both in vitro and in vivo. Furthermore, the most common leukemic NPM1 variant completely loses this activity. This is the consequence of G-quadruplex–binding domain destabilization, as mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplex–binding activity and nucleolar localization. Finally, we show that treatment of cells with a G-quadruplex selective ligand results in wild-type NPM1 dislocation from nucleoli into nucleoplasm. In conclusion, this work establishes a direct correlation between NPM1 G-quadruplex binding at rDNA and its nucleolar localization, which is impaired in the acute myeloid leukemia-associated protein variants.     

Fast Two-Dimensional Bubble Analysis of Biopolymer Filamentous Networks Pore Size from Confocal Microscopy Thin Data Stacks

The average pore size ξ₀ of filamentous networks assembled from biological macromolecules is one of the most important physical parameters affecting their biological functions. Modern optical methods, such as confocal microscopy, can noninvasively image such networks, but extracting a quantitative estimate of ξ₀ is a nontrivial task. We present here a fast and simple method based on a two-dimensional bubble approach, which works by analyzing one by one the (thresholded) images of a series of three-dimensional thin data stacks. No skeletonization or reconstruction of the full geometry of the entire network is required. The method was validated by using many isotropic in silico generated networks of different structures, morphologies, and concentrations. For each type of network, the method provides accurate estimates (a few percent) of the average and the standard deviation of the three-dimensional distribution of the pore sizes, defined as the diameters of the largest spheres that can be fit into the pore zones of the entire gel volume. When applied to the analysis of real confocal microscopy images taken on fibrin gels, the method provides an estimate of ξ₀ consistent with results from elastic light scattering data.     

Structure of an engineered intein reveals thiazoline ring and provides mechanistic insight

We have engineered an intein which spontaneously and reversibly forms a thiazoline ring at the native N-terminal Lys-Cys splice junction. We identified conditions to stablize the thiazoline ring and provided the first crystallographic evidence, at 1.54 Å resolution, for its existence at an intein active site. The finding bolsters evidence for a tetrahedral oxythiazolidine splicing intermediate. In addition, the pivotal mutation maps to a highly conserved B-block threonine, which is now seen to play a causative role not only in ground-state destabilization of the scissile N-terminal peptide bond, but also in steering the tetrahedral intermediate toward thioester formation, giving new insight into the splicing mechanism. We demonstrated the stability of the thiazoline ring at neutral pH as well as sensitivity to hydrolytic ring opening under acidic conditions. A pH cycling strategy to control N-terminal cleavage is proposed, which may be of interest for biotechnological applications requiring a splicing activity switch, such as for protein recovery in bioprocessing.     

Dwarfism and low insulin-like growth factor-1 due to dopamine depletion in Pts-/- mice rescued by feeding neurotransmitter precursors and H4-biopterin

The tetrahydrobiopterin (BH4) cofactor is essential for the biosynthesis of catecholamines and serotonin and for nitric-oxide synthase (NOS). Alterations in BH4 metabolism are observed in various neurological and psychiatric diseases, and mutations in one of the human metabolic genes causes hyperphenylalaninemia and/or monoamine neurotransmitter deficiency. We report on a knockout mouse for the Pts gene, which codes for a BH4-biosynthetic enzyme. Homozygous Pts-/- mice developed with normal morphology but died after birth. Upon daily oral administration of BH4 and neurotransmitter precursors the Pts-/- mice eventually survived. However, at sexual maturity (6 weeks) the mice had only one-third of the normal body weight and were sexually immature. Biochemical analysis revealed no hyperphenylalaninemia, normal brain NOS activity, and almost normal serotonin levels, but brain dopamine was 3% of normal. Low dopamine leads to impaired food consumption as reflected by the severe growth deficiency and a 7-fold reduced serum insulin-like growth factor-1 (IGF-1). This is the first link shown between 6-pyruvoyltetrahydropterin synthase- or BH4-biosynthetic activity and IGF-1.     

High‐density molecular characterization and association mapping in Ethiopian durum wheat landraces reveals high diversity and potential for wheat breeding

Durum wheat (Triticum turgidum subsp. durum) is a key crop worldwide, and yet, its improvement and adaptation to emerging environmental threats is made difficult by the limited amount of allelic variation included in its elite pool. New allelic diversity may provide novel loci to international crop breeding through quantitative trait loci (QTL) mapping in unexplored material. Here, we report the extensive molecular and phenotypic characterization of hundreds of Ethiopian durum wheat landraces and several Ethiopian improved lines. We test 81 587 markers scoring 30 155 single nucleotide polymorphisms and use them to survey the diversity, structure, and genome‐specific variation in the panel. We show the uniqueness of Ethiopian germplasm using a siding collection of Mediterranean durum wheat accessions. We phenotype the Ethiopian panel for ten agronomic traits in two highly diversified Ethiopian environments for two consecutive years and use this information to conduct a genome‐wide association study. We identify several loci underpinning agronomic traits of interest, both confirming loci already reported and describing new promising genomic regions. These loci may be efficiently targeted with molecular markers already available to conduct marker‐assisted selection in Ethiopian and international wheat. We show that Ethiopian durum wheat represents an important and mostly unexplored source of durum wheat diversity. The panel analysed in this study allows the accumulation of QTL mapping experiments, providing the initial step for a quantitative, methodical exploitation of untapped diversity in producing a better wheat.     

Direct amplification of new cellulase genes from woodland soil purified DNA

Eight genes encoding cellulolytic enzymes were obtained by direct PCR amplification of genomic DNA recovered from woodland soil samples. The direct amplifications were carried out by using primers designed from available online cellulase nucleotide sequences. The isolated genes were all different from each other and homologous to endo-β-1,4-glucanases of Bacillus subtilis. The cellulases were functionally expressed in Escherichia coli and tested on soluble substrate at 37 and 60 °C, showing different cellulolytic activities. Among these, the enzyme renamed CelWS6 exhibited good activity at higher temperatures. Further analysis of CelWS6 showed a high performance in acid environments (between pH 4.0 and 6.0) and at elevated temperatures with its maximum activity at pH 5.0 and 50 °C. At the optimum pH, it was very stable since more than 80 % of its original activity was maintained after an incubation of 120 min at 60 °C. Because the cellulases had different cellulolytic activities, but similar amino acid sequences, it was possible to assess the relationship between sequence and protein function.     

Three-dimensional soft tissue preservation revealed in the skin of a non-avian dinosaur

The most commonly preserved soft tissues associated with ornithischian dinosaurs are skin remains. The apparent resistance of hadrosaur skin to decay, and its abundance in the fossil record relative to that of other tetrapods, has been attributed to factors such as thickness and composition. Here we report additional intrinsic factors within hadrosaur skin: 3D-preserved eumelanin-bearing bodies, dermal cells and blood vessel fragments in an organic matrix composed of protein fossilization products. The skin is much thinner than that of living mammals of similar size. It is likely that the preservation of hadrosaur skin is related to the arrangement of the layers composing it.     

Seasonal variations in food web dynamics of floodplain lakes with contrasting hydrological connectivity in the Southern Gulf of Mexico

Hydrobiologia - Floodplains are some of the most productive and diverse ecosystems on Earth. The Usumacinta River Basin, in the Southern Gulf of Mexico, hosts several floodplain lakes, whose...