Enzyme production by filamentous fungi: analysis of the secretome of Trichoderma reesei grown on unconventional carbon source

<p>Abstract</p> <p>Background</p> <p>Spent hydrolysates from bioethanolic fermentation processes based on agricultural residues have potential as an abundant and inexpensive source of pentose sugars and acids that could serve as nutrients for industrial enzyme-producing microorganisms, especially filamentous fungi. However, the enzyme mixtures produced in such media are poorly defined. In this study, the secretome of <it>Trichoderma reesei </it>Rut C-30 grown either on a spent hydrolysate model medium (SHMM) or on a lactose-based standard medium (LBSM) was explored using proteomics.</p> <p>Results</p> <p>Our results show that both the SHMM and LBSM serve as excellent growth media for <it>T. reesei </it>Rut C-30. In total, 52 protein spots on 2-D gels were identified by using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization liquid chromatography tandem mass spectrometry (ESI-LC MS/MS). As expected, a considerable number of the identified proteins were related to the degradation of lignocellulosic biomass. The enzyme production profiles in the two media were similar, but β-glucosidase and β-galactosidase were only produced in LBSM. The main cellobiohydrolases (Cel7A/Cel6A) and endoglucanases (Cel7B/Cel5A) were identified in both media and the cellobiohydrolases, i.e. Cel7A and Cel6A, were the most abundant cellulolytic enzymes. Moreover, both media can also serve as a potent inducer of xylanolytic enzymes. Several key enzymes involved in sugar assimilation and regulation of cellulase formation were identified, and were found to be differentially expressed in the two growth media.</p> <p>Conclusions</p> <p>This study not only provides a catalogue of the prevalent proteins secreted by <it>T. reesei </it>in the two media, but the results also suggest that production of hydrolytic enzymes using unconventional carbon sources, such as components in spent hydrolysates, deserves further attention in the future.</p

Oxidation state governs structural transitions in peroxiredoxin II that correlate with cell cycle arrest and recovery

Inactivation of eukaryotic 2-Cys peroxiredoxins (Prxs) by hyperoxidation has been proposed to promote accumulation of hydrogen peroxide (H2O2) for redox-dependent signaling events. We examined the oxidation and oligomeric states of PrxI and -II in epithelial cells during mitogenic signaling and in response to fluxes of H2O2. During normal mitogenic signaling, hyperoxidation of PrxI and -II was not detected. In contrast, H2O2-dependent cell cycle arrest was correlated with hyperoxidation of PrxII, which resulted in quantitative recruitment of ∼66- and ∼140-kD PrxII complexes into large filamentous oligomers. Expression of cyclin D1 and cell proliferation did not resume until PrxII-SO2H was reduced and native PrxII complexes were regenerated. Ectopic expression of PrxI or -II increased Prx-SO2H levels in response to oxidant exposure and failed to protect cells from arrest. We propose a model in which Prxs function as peroxide dosimeters in subcellular processes that involve redox cycling, with hyperoxidation controlling structural transitions that alert cells of perturbations in peroxide homeostasis

Independent Ion Migration in Suspensions of Strongly Interacting Charged Colloidal Spheres

We report on sytematic measurements of the low frequency conductivity in aequous supensions of highly charged colloidal spheres. System preparation in a closed tubing system results in precisely controlled number densities between 1E16/m3 and 1E19/m^3 (packing fractions between 1E-7 and 1E-2) and electrolyte concentrations between 1E-7 and 1E-3 mol/l. Due to long ranged Coulomb repulsion some of the systems show a pronounced fluid or crystalline order. Under deionized conditions we find s to depend linearily on the packing fraction with no detectable influence of the phase transitions. Further at constant packing fraction s increases sublinearily with increasing number of dissociable surface groups N. As a function of c the conductivity shows pronounced differences depending on the kind of electrolyte used. We propose a simple yet powerful model based on independent migration of all species present and additivity of the respective conductivity contributions. It takes account of small ion macro-ion interactions in terms of an effectivly transported charge. The model successfully describes our qualitatively complex experimental observations. It further facilitates quantitative estimates of conductivity over a wide range of particle and experimental parameters.Comment: 32 pages, 17 figures, 2 tables, Accepted by Physical Review

Slow-Speed Supernovae from the Palomar Transient Factory: Two Channels

Since the discovery of the unusual prototype SN 2002cx, the eponymous class of low-velocity, hydrogen-poor supernovae has grown to include at most another two dozen members identified from several heterogeneous surveys, in some cases ambiguously. Here we present the results of a systematic study of 1077 hydrogen-poor supernovae discovered by the Palomar Transient Factory, leading to nine new members of this peculiar class. Moreover we find there are two distinct subclasses based on their spectroscopic, photometric, and host galaxy properties: The "SN 2002cx-like" supernovae tend to be in later-type or more irregular hosts, have more varied and generally dimmer luminosities, have longer rise times, and lack a Ti II trough when compared to the "SN 2002es-like" supernovae. None of our objects show helium, and we counter a previous claim of two such events. We also find that these transients comprise 5.6+17-3.7% (90% confidence) of all SNe Ia, lower compared to earlier estimates. Combining our objects with the literature sample, we propose that these subclasses have two distinct physical origins.Comment: 49 pages, 36 figures, submitted to Ap

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds

Regional trends in soil acidification and exchangeable metal concentrations in relation to acid deposition rates

The deposition of high levels of reactive nitrogen (N) and sulphur (S), or the legacy of that deposition, remain among the world's most important environmental problems. Although regional impacts of acid deposition in aquatic ecosystems have been well documented, quantitative evidence of wide-scale impacts on terrestrial ecosystems is not common. In this study we analysed surface and subsoil chemistry of 68 acid grassland sites across the UK along a gradient of acid deposition, and statistically related the concentrations of exchangeable soil metals (1 M KCl extraction) to a range of potential drivers. The deposition of N, S or acid deposition was the primary correlate for 8 of 13 exchangeable metals measured in the topsoil and 5 of 14 exchangeable metals in the subsoil. In particular, exchangeable aluminium and lead both show increased levels above a soil pH threshold of about 4.5, strongly related to the deposition flux of acid compound

Dose response of the 16p11.2 distal copy number variant on intracranial volume and basal ganglia.

Carriers of large recurrent copy number variants (CNVs) have a higher risk of developing neurodevelopmental disorders. The 16p11.2 distal CNV predisposes carriers to e.g., autism spectrum disorder and schizophrenia. We compared subcortical brain volumes of 12 16p11.2 distal deletion and 12 duplication carriers to 6882 non-carriers from the large-scale brain Magnetic Resonance Imaging collaboration, ENIGMA-CNV. After stringent CNV calling procedures, and standardized FreeSurfer image analysis, we found negative dose-response associations with copy number on intracranial volume and on regional caudate, pallidum and putamen volumes (β = -0.71 to -1.37; P &lt; 0.0005). In an independent sample, consistent results were obtained, with significant effects in the pallidum (β = -0.95, P = 0.0042). The two data sets combined showed significant negative dose-response for the accumbens, caudate, pallidum, putamen and ICV (P = 0.0032, 8.9 × 10-6, 1.7 × 10-9, 3.5 × 10-12 and 1.0 × 10-4, respectively). Full scale IQ was lower in both deletion and duplication carriers compared to non-carriers. This is the first brain MRI study of the impact of the 16p11.2 distal CNV, and we demonstrate a specific effect on subcortical brain structures, suggesting a neuropathological pattern underlying the neurodevelopmental syndromes

Mechanochemical Polarization of Contiguous Cell Walls Shapes Plant Pavement Cells.

The epidermis of aerial plant organs is thought to be limiting for growth, because it acts as a continuous load-bearing layer, resisting tension. Leaf epidermis contains jigsaw puzzle piece-shaped pavement cells whose shape has been proposed to be a result of subcellular variations in expansion rate that induce local buckling events. Paradoxically, such local compressive buckling should not occur given the tensile stresses across the epidermis. Using computational modeling, we show that the simplest scenario to explain pavement cell shapes within an epidermis under tension must involve mechanical wall heterogeneities across and along the anticlinal pavement cell walls between adjacent cells. Combining genetics, atomic force microscopy, and immunolabeling, we demonstrate that contiguous cell walls indeed exhibit hybrid mechanochemical properties. Such biochemical wall heterogeneities precede wall bending. Altogether, this provides a possible mechanism for the generation of complex plant cell shapes

Stellar Characterization and Radius Inflation of Hyades M Dwarf Stars From the APOGEE Survey

We present a spectroscopic analysis of a sample of 48 M dwarf stars ($0.2 M_{\odot}< M < 0.6 M_{\odot}$) from the Hyades open cluster using high-resolution H-band spectra from the SDSS/APOGEE survey. Our methodology adopts spectrum synthesis with LTE MARCS model atmospheres, along with the APOGEE DR17 line list, to determine effective temperatures, surface gravities, metallicities, and projected rotational velocities. The median metallicity obtained for the Hyades M dwarfs is [M/H]= 0.09$\pm$0.03 dex, indicating a small internal uncertainty and good agreement with optical results for Hyades red-giants. Overall, the median radii are larger than predicted by stellar models by 1.6$\pm$2.3\% and 2.4$\pm$2.3\%, relative to a MIST and DARTMOUTH isochrone, respectively. We emphasize, however, that these isochrones are different and the fractional radius inflation for the fully- and partially-convective regimes have distinct behaviors depending on the isochrone. Using a MIST isochrone there is no evidence of radius inflation for the fully convective stars, while for the partially convective M-dwarfs the radii are inflated by 2.7$\pm$2.1\%, which is in agreement with predictions from models that include magnetic fields. For the partially-convective stars, rapid-rotators present on average higher inflation levels than slow-rotators. The comparison with SPOTS isochrone models indicates that the derived M dwarf radii can be explained by accounting for stellar spots in the photosphere of the stars, with 76\% of the studied M dwarfs having up to 20\% spot coverage, and the most inflated stars with $\sim$20 -- 40\% spot coverage.Comment: Accepted for publication by The Astrophysical Journal (ApJ