Engineering Saccharomyces cerevisiae to release 3-Mercaptohexan-1-ol during fermentation through overexpression of an S. cerevisiae Gene, STR3, for improvement of wine aroma

Sulfur-containing aroma compounds are key contributors to the flavor of a diverse range of foods and beverages. The tropical fruit characters of Vitis vinifera L. cv. Sauvignon blanc wines are attributed to the presence of the aromatic thiols 3-mercaptohexan-1-ol (3MH), 3-mercaptohexan-1-ol-acetate, and 4-mercapto-4-methylpentan-2-one (4MMP). These volatile thiols are found in small amounts in grape juice and are formed from nonvolatile cysteinylated precursors during fermentation. In this study, we overexpressed a Saccharomyces cerevisiae gene, STR3, which led to an increase in 3MH release during fermentation of a V. vinifera L. cv. Sauvignon blanc juice. Characterization of the enzymatic properties of Str3p confirmed it to be a pyridoxal-5'-phosphate-dependent cystathionine β-lyase, and we demonstrated that this enzyme was able to cleave the cysteinylated precursors of 3MH and 4MMP to release the free thiols. These data provide direct evidence for a yeast enzyme able to release aromatic thiols in vitro that can be applied in the development of self-cloned yeast to enhance wine flavor.     

Profiling signatures of ovarian cancer tumour suppression using 2D-DIGE and 2D-LC-MS/MS with tandem mass tagging

Epithelial ovarian cancer (EOC) is the most common form of gynaecological malignancy in the developed world and has a poor prognosis due to its late detection. Identifying molecular markers of the disease may provide novel approaches to screening and could enable targeted treatment and the design of novel therapies. Although blood is recognized as a highly important source of disease-related biomarkers, the complexity and dynamic range of protein abundance in body fluids has hampered proteomic biomarker discovery and alternative approaches using cell models may be more successful. Herein, we have utilized two cellular models of EOC, where transfer of normal chromosome 18 material into the EOC cell lines TOV-112D and TOV-21G induced in vitro and in vivo suppression of their tumourigenic phenotype. A combination of quantitative two-dimensional difference gel electrophoresis (2D-DIGE) and two-dimensional-liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) with tandem mass tagging (TMT) was employed to examine the whole cell, secreted and crude membrane proteomes of the parental and hybrid cell models to identify differentially expressed proteins as potential markers of tumour suppression. Protein changes of interest were confirmed by immunoblotting in additional hybrid and revertant cell lines where incorporated chromosome 18 material was lost. One candidate marker was also tested in sera from a set of ovarian cancer cases and controls. We have identified a list of promising candidate biomarkers for further testing and functional characterization.     

Exploiting clinical trial data drastically narrows the window of possible solutions to the problem of clinical adaptation of a multiscale cancer model

The development of computational models for simulating tumor growth and response to treatment has gained significant momentum during the last few decades. At the dawn of the era of personalized medicine, providing insight into complex mechanisms involved in cancer and contributing to patient-specific therapy optimization constitute particularly inspiring pursuits. The in silico oncology community is facing the great challenge of effectively translating simulation models into clinical practice, which presupposes a thorough sensitivity analysis, adaptation and validation process based on real clinical data. In this paper, the behavior of a clinically-oriented, multiscale model of solid tumor response to chemotherapy is investigated, using the paradigm of nephroblastoma response to preoperative chemotherapy in the context of the SIOP/GPOH clinical trial. A sorting of the model's parameters according to the magnitude of their effect on the output has unveiled the relative importance of the corresponding biological mechanisms; major impact on the result of therapy is credited to the oxygenation and nutrient availability status of the tumor and the balance between the symmetric and asymmetric modes of stem cell division. The effect of a number of parameter combinations on the extent of chemotherapy-induced tumor shrinkage and on the tumor's growth rate are discussed. A real clinical case of nephroblastoma has served as a proof of principle study case, demonstrating the basics of an ongoing clinical adaptation and validation process. By using clinical data in conjunction with plausible values of model parameters, an excellent fit of the model to the available medical data of the selected nephroblastoma case has been achieved, in terms of both volume reduction and histological constitution of the tumor. In this context, the exploitation of multiscale clinical data drastically narrows the window of possible solutions to the clinical adaptation problem.     

The axonal guidance receptor neogenin promotes acute inflammation

Neuronal guidance proteins (NGP) were originally described in the context of axonal growth and migration. Yet recent work has demonstrated that NGPs also serve as guidance cues for immune competent cells. A crucial target receptor for NGPs during embryonic development is the neogenin receptor, however its role during acute inflammation is unknown. We report here that neogenin is abundantly expressed outside the nervous system and that animals with endogenous repression of neogenin (Neo1(-/-)) demonstrate attenuated changes of acute inflammation. Studies using functional inhibition of neogenin resulted in a significant attenuation of inflammatory peritonitis. In studies employing bone marrow chimeric animals we found the hematopoietic presence of Neo1(-/-) to be responsible for the attenuated inflammatory response. Taken together our studies suggest that the guidance receptor neogenin holds crucial importance for the propagation of an acute inflammatory response and further define mechanisms shared between the nervous and the immune system.     

Alpha helical structures in the leader sequence of human GLUD2 glutamate dehydrogenase responsible for mitochondrial import

Human glutamate dehydrogenase (hGDH) exists in two highly homologous isoforms with a distinct regulatory and tissue expression profile: a housekeeping hGDH1 isoprotein encoded by the GLUD1 gene and an hGDH2 isoenzyme encoded by the GLUD2 gene. There is evidence that both isoenzymes are synthesized as pro-enzymes containing a 53 amino acid long N-terminal leader peptide that is cleaved upon translocation into the mitochondria. However, this GDH signal peptide is substantially larger than that of most nuclear DNA-encoded mitochondrial proteins, the leader sequence of which typically contains 17-35 amino acids and they often form a single amphipathic α-helix. To decode the structural elements that are essential for the mitochondrial targeting of human GDHs, we performed secondary structure analyses of their leader sequence. These analyses predicted, with 82% accuracy, that both leader peptides are positively charged and that they form two to three α-helices, separated by intermediate loops. The first α-helix of hGDH2 is strongly amphipathic, displaying both a positively charged surface and a hydrophobic plane. We then constructed GLUD2-EGFP deletion mutants and used them to transfect three mammalian cell lines (HEK293, COS 7 and SHSY-5Y). Confocal laser scanning microscopy, following co-transfection with pDsRed2-Mito mitochondrial targeting vector, revealed that deletion of the entire leader sequence prevented the enzyme from entering the mitochondria, resulting in its retention in the cytoplasm. Deletion of the first strongly amphipathic α-helix only was also sufficient to prevent the mitochondrial localization of the truncated protein. Moreover, truncated leader sequences, retaining the second and/or the third putative α-helix, failed to restore the mitochondrial import of hGDH2. As such, the first N-terminal alpha helical structure is crucial for the mitochondrial import of hGDH2 and these findings may have implications in understanding the evolutionary mechanisms that led to the large mitochondrial targeting signals of human GDHs.     

Relationships between nitrogen,dry matter accumulation and glucosinolates in <Emphasis Type="Italic">Eruca sativa</Emphasis> Mills. The applicability of the critical NO<Subscript>3</Subscript>-N levels approach

Background and Aims

Rocket salad (Eruca sativa Mills) is one of the major leafy vegetables produced worldwide and has been characterized as a rich source of chemoprotective glucosinolates (GSL). The relationship between N fertilization and the resulting plant biomass and N status with GSL quantity and quality in rocket leaves was examined.

Methods

A pot experiment was conducted, applying ten different N-rates and destructive sampling was carried out 15, 30 and 45 days after transplanting (DAT). The Mitscherlich equation was used to establish NO₃-N critical levels at each growth stage and as an indicator of N demand for relative maximum dry matter accumulation and glucosinolate content and composition was determined.

Results

Glucosinolate content was significantly influenced by N rate, growth stage and their interaction. Different GSL types showed dissimilar responses to N fertilization: aliphatic GSLs were significantly reduced under increased N rates whereas indole GSL showed the reverse. Under excess N fertilization (>1.04 g/plant), dry matter accumulation remained constant, NO₃-N was significantly increased and total GSL content was significantly reduced, factors that could lead to an anticipated product quality decline.

Conclusions

The application of the critical NO₃-N level approach used to identify optimal N fertilization rates for plant growth could serve as means to obtain optimized GSL content in the edible plant parts.

     

Discovery and Characterization of a Thermostable and Highly Halotolerant GH5 Cellulase from an Icelandic Hot Spring Isolate

With the ultimate goal of identifying robust cellulases for industrial biocatalytic conversions, we have isolated and characterized a new thermostable and very halotolerant GH5 cellulase. This new enzyme, termed CelDZ1, was identified by bioinformatic analysis from the genome of a polysaccharide-enrichment culture isolate, initiated from material collected from an Icelandic hot spring. Biochemical characterization of CelDZ1 revealed that it is a glycoside hydrolase with optimal activity at 70°C and pH 5.0 that exhibits good thermostability, high halotolerance at near-saturating salt concentrations, and resistance towards metal ions and other denaturing agents. X-ray crystallography of the new enzyme showed that CelDZ1 is the first reported cellulase structure that lacks the defined sugar-binding 2 subsite and revealed structural features which provide potential explanations of its biochemical characteristics.     

Biotechnology Towards Energy Crops

New crops are gradually establishing along with cultivation systems to reduce reliance on depleting fossil fuel reserves and sustain better adaptation to climate change. These biological assets could be efficiently exploited as bioenergy feedstocks. Bioenergy crops are versatile renewable sources with the potential to alternatively contribute on a daily basis towards the coverage of modern society’s energy demands. Biotechnology may facilitate the breeding of elite energy crop genotypes, better suited for bio-processing and subsequent use that will improve efficiency, further reduce costs, and enhance the environmental benefits of biofuels. Innovative molecular techniques may improve a broad range of important features including biomass yield, product quality and resistance to biotic factors like pests or microbial diseases or environmental cues such as drought, salinity, freezing injury or heat shock. The current review intends to assess the capacity of biotechnological applications to develop a beneficial bioenergy pipeline extending from feedstock development to sustainable biofuel production and provide examples of the current state of the art on future energy crops.