A versatile toolkit for high throughput functional genomics with Trichoderma reesei

<p>Abstract</p> <p>Background</p> <p>The ascomycete fungus, <it>Trichoderma reesei </it>(anamorph of <it>Hypocrea jecorina</it>), represents a biotechnological workhorse and is currently one of the most proficient cellulase producers. While strain improvement was traditionally accomplished by random mutagenesis, a detailed understanding of cellulase regulation can only be gained using recombinant technologies.</p> <p>Results</p> <p>Aiming at high efficiency and high throughput methods, we present here a construction kit for gene knock out in <it>T. reesei</it>. We provide a primer database for gene deletion using the <it>pyr4, amdS </it>and <it>hph </it>selection markers. For high throughput generation of gene knock outs, we constructed vectors using yeast mediated recombination and then transformed a <it>T. reesei </it>strain deficient in non-homologous end joining (NHEJ) by spore electroporation. This NHEJ-defect was subsequently removed by crossing of mutants with a sexually competent strain derived from the parental strain, QM9414.</p> <p>Conclusions</p> <p>Using this strategy and the materials provided, high throughput gene deletion in <it>T. reesei </it>becomes feasible. Moreover, with the application of sexual development, the NHEJ-defect can be removed efficiently and without the need for additional selection markers. The same advantages apply for the construction of multiple mutants by crossing of strains with different gene deletions, which is now possible with considerably less hands-on time and minimal screening effort compared to a transformation approach. Consequently this toolkit can considerably boost research towards efficient exploitation of the resources of <it>T. reesei </it>for cellulase expression and hence second generation biofuel production.</p

Translating environments

Far from being inert materials activated by human ingenuity, natural resources come to be made and unmade through ongoing processes of translation, through which they acquire new potentialities and meanings. In this introduction, we review the key concept of translation for anthropology and explore some of its multiple analytical possibilities in the context of human-environment relations. Based on insights offered by the articles in this collection, we propose a twofold definition of environments as both translating subjects and objects of translation. In grounding our analytical definition, we focus on the enactment of material transformations (as the result of both relations of mutual determination with humans and processes of objectification of the environment), the implications of incommensurability and erasure in processes of (attempted) translation, and the indeterminacy that accompanies (re)configurations of materials, relations and values

Use of human perivascular stem cells for bone regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering(1-3). PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines(1,2). In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)(8). FSDs are bicortical and are stabilized with a polyethylene bar and K-wires(4). The FSD described is also a critical size defect, which does not significantly heal on its own(4). In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models

Pericytic mimicry in well-differentiated liposarcoma / atypical lipomatous tumor

Pericytes are modified smooth muscle cells that closely enwrap small blood vessels, regulating and supporting the microvasculature through direct endothelial contact. Pericytes demonstrate a distinct immunohistochemical profile, including expression of αSMA (Smooth Muscle Actin), CD146, PDGFRβ (Platelet Derived Growth Factor Receptor β), and RGS5 (regulator of G-protein signaling 5). Previously, pericyte related antigens have been observed to be present among a group of soft tissue tumors with a perivascular growth pattern, including glomus tumor, myopericytoma and angioleiomyoma. Similarly, malignant tumor cells have been shown to pericyte-like immunoprofile when present in a perivascular location, seen in malignant melanoma, glioblastoma, and adenocarcinoma. Here, we examine well-differentiated liposarcoma specimens, which showed some element of perivascular areas with the appearance of smooth muscle (N=7 tumors). Immunohistochemical staining was performed for pericyte antigens, including αSMA, CD146, PDGFRβ, and RGS5. Results showed consistent pericytic marker expression among liposarcoma tumor cells within a perivascular distribution. MDM2 immunohistochemistry and fluorescence in situ hybridization for MDM2 revealed that these perivascular cells were of tumor origin (7/7 tumors), while double immunohistochemical detection for CD31/CD146 ruled out an endothelial cell contribution. These findings further support the concept of pericytic mimicry, already established in diverse malignancies, and its presence in well-differentiated liposarcoma. The extent to which pericytic mimicry has prognostic significance in liposarcoma is as yet unknown

Influence of laser polarization on collective electron dynamics in ultraintense laser-foil interactions

The collective response of electrons in an ultrathin foil target irradiated by an ultraintense laser pulse is investigated experimentally and via 3D particle-in-cell simulations. It is shown that if the target is sufficiently thin that the laser induces significant radiation pressure, but not thin enough to become relativistically transparent to the laser light, the resulting relativistic electron beam is elliptical, with the major axis of the ellipse directed along the laser polarization axis. When the target thickness is decreased such that it becomes relativistically transparent early in the interaction with the laser pulse, diffraction of the transmitted laser light occurs through a so called 'relativistic plasma aperture', inducing structure in the spatial-intensity profile of the beam of energetic electrons. It is shown that the electron beam profile can be modified by variation of the target thickness and degree of ellipticity in the laser polarization

Towards optical polarization control of laser-driven proton acceleration in foils undergoing relativistic transparency

Control of the collective response of plasma particles to intense laser light is intrinsic to relativistic optics, the development of compact laser-driven particle and radiation sources, as well as investigations of some laboratory astrophysics phenomena. We recently demonstrated that a relativistic plasma aperture produced in an ultra-thin foil at the focus of intense laser radiation can induce diffraction, enabling polarization-based control of the collective motion of plasma electrons. Here we show that under these conditions the electron dynamics are mapped into the beam of protons accelerated via strong charge-separation-induced electrostatic fields. It is demonstrated experimentally and numerically via 3D particle-in-cell simulations that the degree of ellipticity of the laser polarization strongly influences the spatial-intensity distribution of the beam of multi-MeV protons. The influence on both sheath accelerated and radiation pressure accelerated protons is investigated. This approach opens up new routes to control laser-driven ion sources

Phylogeographic analysis reveals multiple international transmission events have driven the global emergence of Escherichia coli O157:H7

This work was supported by: Scotland by Food Standards Scotland [Grant Number FS102029] and University of Aberdeen; New Zealand, Institute of Environmental Science and Research; Canada, the Public Health Agency of Canada; United States, United States Department of AgriculturePeer reviewedPostprin

Design of Experiments for Screening

The aim of this paper is to review methods of designing screening experiments, ranging from designs originally developed for physical experiments to those especially tailored to experiments on numerical models. The strengths and weaknesses of the various designs for screening variables in numerical models are discussed. First, classes of factorial designs for experiments to estimate main effects and interactions through a linear statistical model are described, specifically regular and nonregular fractional factorial designs, supersaturated designs and systematic fractional replicate designs. Generic issues of aliasing, bias and cancellation of factorial effects are discussed. Second, group screening experiments are considered including factorial group screening and sequential bifurcation. Third, random sampling plans are discussed including Latin hypercube sampling and sampling plans to estimate elementary effects. Fourth, a variety of modelling methods commonly employed with screening designs are briefly described. Finally, a novel study demonstrates six screening methods on two frequently-used exemplars, and their performances are compared