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Background Pichia pastoris is widely used as a production platform for heterologous proteins and model organism for organelle proliferation. Without a published genome sequence available, strain and process development relied mainly on analogies to other, well studied yeasts like Saccharomyces cerevisiae . Results To investigate specific features of growth and protein secretion, we have sequenced the 9.4 Mb genome of the type strain DSMZ 70382 and analyzed the secretome and the sugar transporters. The computationally predicted secretome consists of 88 ORFs. When grown on glucose, only 20 proteins were actually secreted at detectable levels. These data highlight one major feature of P. pastoris , namely the low contamination of heterologous proteins with host cell protein, when applying glucose based expression systems. Putative sugar transporters were identified and compared to those of related yeast species. The genome comprises 2 homologs to S. cerevisiae low affinity transporters and 2 to high affinity transporters of other Crabtree negative yeasts. Contrary to other yeasts, P. pastoris possesses 4 H + /glycerol transporters. Conclusion This work highlights significant advantages of using the P. pastoris system with glucose based expression and fermentation strategies. As only few proteins and no proteases are actually secreted on glucose, it becomes evident that cell lysis is the relevant cause of proteolytic degradation of secreted proteins. The endowment with hexose transporters, dominantly of the high affinity type, limits glucose uptake rates and thus overflow metabolism as observed in S. cerevisiae . The presence of 4 genes for glycerol transporters explains the high specific growth rates on this substrate and underlines the suitability of a glycerol/glucose based fermentation strategy. Furthermore, we present an open access web based genome browser http://www.pichiagenome.org .

The first genome sequences of the important yeast protein production host Pichia pastoris have been released into the public domain this spring. In order to provide the scientific community easy and versatile access to the sequence, two web-sites have been installed as a resource for genomic sequence, gene and protein information for P. pastoris : A GBrowse based genome browser was set up at http://www.pichiagenome.org and a genome portal with gene annotation and browsing functionality at http://bioinformatics.psb.ugent.be/webtools/bogas . Both websites are offering information on gene annotation and function, regulation and structure. In addition, a WiKi based platform allows all users to create additional information on genes, proteins, physiology and other items of P. pastoris research, so that the Pichia community can benefit from exchange of knowledge, data and materials.

The rheological behaviour of a gluten plasticized with glycerol has been studied in oscillatory shear. The mixing operation in a Haake batch mixer leads to a maximum torque for a level of specific energy (500–600 kJ/kg) and temperature (50–60 °C) quite independent of mixing conditions (rotor speed, mixing time, filling ratio). The gluten/glycerol dough behaves as a classical gluten/water dough, with a storage modulus higher than the loss modulus over the frequency range under study. A temperature increase induces a decrease of moduli, but the material is not thermorheologically simple. Glycerol has a plasticizing effect, which can be classically described by an exponential dependence. Mixing conditions influence the viscoelastic properties of the material, mainly through the specific mechanical energy input (to 2000 kJ/kg) and temperature increase (to 80 °C). Above 50 °C, specific mechanical energy highly increases the complex modulus. The aggregation of proteins, as evidenced by size-exclusion chromatography measurements, occurs later as the dough temperature reaches 70 °C. The nature of network interactions and the respective influence of hydrophobic and disulphide contribution is discussed. A general expression is proposed for describing the viscous behaviour of a gluten/glycerol mix, which could seem simplistic for such a complex rheological behaviour, but would remain sufficient for modelling the flow behaviour in a twin screw extruder.

Fusion power plants require ELM-free, detached operation to prevent divertor damage and erosion. The separatrix operational space (SepOS) is proposed as a tool for identifying access to the type-I ELM-free quasi-continuous exhaust regime. In this work, we recast the SepOS framework using simple parameters and present dedicated ASDEX Upgrade discharges to demonstrate how to interpret its results. Analyzing an extended ASDEX Upgrade database consisting of 6688 individual measurements, we show that SepOS accurately describes how the H-mode boundary varies with plasma current and magnetic field strength. We then introduce a normalized SepOS framework and LH minimum scaling and show that normalized boundaries across multiple machines are nearly identical, suggesting that the normalized SepOS can be used to translate results between different machines. The LH minimum density predicted by SepOS is found to closely match an experimentally determined multi-machine scaling, which provides a further indirect validation of SepOS across multiple devices. Finally, we demonstrate how SepOS can be used predictively, identifying a viable QCE operational point for SPARC, at a separatrix density of 4e20/m3, a separatrix temperature of 156eV and an alpha-t of 0.7 - a value solidly within the QCE operational space on ASDEX Upgrade. This demonstrates how SepOS provides a concise, intuitive method for scoping ELM-free operation on next-step devices.

Effective regeneration of bone defects often presents significant challenges, particularly in patients with decreased tissue regeneration capacity due to extensive trauma, disease, and/or advanced age. A number of studies have focused on enhancing bone regeneration by applying mesenchymal stromal cells (MSCs) or MSC-based bone tissue engineering strategies. However, translation of these approaches from basic research findings to clinical use has been hampered by the limited understanding of MSC therapeutic actions and complexities, as well as costs related to the manufacturing, regulatory approval, and clinical use of living cells and engineered tissues. More recently, a shift from the view of MSCs directly contributing to tissue regeneration toward appreciating MSCs as \"cell factories\" that secrete a variety of bioactive molecules and extracellular vesicles with trophic and immunomodulatory activities has steered research into new MSC-based, \"cell-free\" therapeutic modalities. The current review recapitulates recent developments, challenges, and future perspectives of these various MSC-based bone tissue engineering and regeneration strategies.

Pulsed electromagnetic field (PEMF) therapy is a non-invasive treatment that delivers electric and magnetic fields to tissues via inductive coils inducing salutary effects. Previously PEMF was reported to accelerate gas transport in the liquid phase. Drawing analogies from electrical elements, we hypothesized that PEMF can modulate transmembrane potential of biological membranes. Given that mitochondria interact with gaseous molecules such as oxygen and nitric oxide (NO) and contain voltage-sensitive channels, this study focused on the effects of a single PEMF device with a low input-energy and a 1ms, 30 kHz sine wave duty cycle per pulse on mitochondrial function. Experiments were conducted using cell cultures, tissue homogenates, and isolated mitochondria. We assessed mitochondrial membrane potential, NO levels, and mitochondrial respiration. We found no evidence that PEMF restores mitochondrial respiration inhibited by NO, but it can be restored by exposure of mitochondria to blue light. Our findings show that PEMF selectively stimulates respiration linked to ATP-synthesis affecting less uncoupled respiration. This suggests that changes in ATP-synthesis underlies the primary beneficial effects observed here. The underlying mechanisms may include interaction of PEMF with mitochondrial transport systems or activity of mitochondrial complexes. The exact mechanisms still should be investigated.

The ‘brain regionalization genes’ Six3/6 , Otx , Pax2/5/8 , Gbx , and Hox1 are expressed in a similar fashion in the deuterostome, ecdysozoan, and the cephalopod brain, questioning whether this holds also true for the remaining Mollusca. We investigated developmental Gbx -expression in representatives of both molluscan sister groups, the Aculifera and Conchifera. Gbx is expressed in the posterior central nervous system of an aculiferan polyplacophoran and solenogaster but not in a conchiferan bivalve suggesting that Gbx , together with Six3/6 , Otx , Pax2/5/8 , and Hox1 , is involved in central nervous system regionalization as reported for other bilaterians. Gbx is, however, also expressed in the anterior central nervous system, i.e. the anlagen of the cerebral ganglia, in the solenogaster, a condition not reported for any other bilaterian so far. Strikingly, all Gbx- orthologs and the other ‘posterior brain regionalization genes’ such as Pax2/5/8 and Hox1 are expressed in the mantle that secretes shell(s) and spicules of mollusks (except cephalopods). In bivalves, the ancestral condition has even been lost, with Gbx and Pax2/5/8 not being expressed in the developing central nervous system anymore. This suggests an additional role in the formation of the molluscan shell field(s) and spicule-bearing cells, key features of mollusks.

Viticultural practices and landscape composition are the main drivers influencing biological pest control in vineyards. Predatory mites, mainly phytoseiid (Phytoseiidae) and tydeoid mites (Tydeidae), are important to control phytophagous mites (Tetranychidae and Eriophyidae) on vines. In the absence of arthropod prey, pollen is an important food source for predatory mites. In 32 paired vineyards located in Burgenland/Austria, we examined the effect of landscape composition, management type (organic/integrated), pesticide use, and cover crop diversity of the inter-row on the densities of phytoseiid, tydeoid, and phytophagous mites. In addition, we sampled pollen on vine leaves. Typhlodromus pyri Scheuten was the main phytoseiid mite species and Tydeus goetzi Schruft the main tydeoid species. Interestingly, the area-related acute pesticide toxicity loading was higher in organic than in integrated vineyards. The densities of phytoseiid and tydeoid mites was higher in integrated vineyards and in vineyards with spontaneous vegetation. Their population also profited from an increased viticultural area at the landscape scale. Eriophyoid mite densities were extremely low across all vineyards and spider mites were absent. Biological pest control of phytophagous mites benefits from less intensive pesticide use and spontaneous vegetation cover in vineyard inter-rows, which should be considered in agri-environmental schemes.

Background The aim of this study was to evaluate potential synergistic effects of a single, local application of human umbilical cord MSC-derived sEVs in combination with a low dose of recombinant human rhBMP-2 to promote the regeneration of a metaphyseal femoral defect in an osteoporotic rat model. Methods 6 weeks after induction of osteoporosis by bilateral ventral ovariectomy and administration of a special diet, a total of 64 rats underwent a distal femoral metaphyseal osteotomy using a manual Gigli wire saw. Defects were stabilized with an adapted Y-shaped mini-locking plate and were subsequently treated with alginate only, or alginate loaded with hUC-MSC-sEVs (2 × 10 9 ), rhBMP-2 (1.5 µg), or a combination of sEVs and rhBMP-2 ( n  = 16 for each group). 6 weeks post-surgery, femora were evaluated by µCT, descriptive histology, and biomechanical testing. Results Native radiographs and µCT analysis confirmed superior bony union with callus formation after treatment with hUC-MSC-sEVs in combination with a low dose of rhBMP-2. This finding was further substantiated by histology, showing robust defect consolidation 6 weeks after treatment. Torsion testing of the explanted femora revealed increased stiffness after application of both, rhBMP-2 alone, or in combination with sEVs, whereas torque was only significantly increased after treatment with rhBMP-2 together with sEVs. Conclusion The present study demonstrates that the co-application of hUC-MSC-sEVs can improve the efficacy of rhBMP-2 to promote the regeneration of osteoporotic bone defects.