Aim of this study is the identification of an appropriate internal reference gene to quantifygene transcripts isolated from Rhodobacter (R.) sphaeroides cells grown in presence of highconcentrations of cobalt ions. RNA was isolated using a commercial kit protocol ad-hocmodified. Several primer pairs were used to perform reverse transcription PCR and real-timePCR to assess the suitable internal reference gene whose expression is not affected by cobaltions, identified with the gene rsp0154.This finding can be of definite help in the investigation of the response to heavy metals ofthe chosen strain, a potential candidate for environmental applications.

Making forecasts about biodiversity and giving support to policy relies increasingly on large collections of data held electronically, and on substantial computational capability and capacity to analyse, model, simulate and predict using such data. However, the physically distributed nature of data resources and of expertise in advanced analytical tools creates many challenges for the modern scientist. Across the wider biological sciences, presenting such capabilities on the Internet (as ``Web services'') and using scientific workflow systems to compose them for particular tasks is a practical way to carry out robust ``in silico'' science. However, use of this approach in biodiversity science and ecology has thus far been quite limited.

Much biodiversity data is collected worldwide, but it remains challenging to assemble the scattered knowledge for assessing biodiversity status and trends. The concept of Essential Biodiversity Variables (EBVs) was introduced to structure biodiversity monitoring globally, and to harmonize and standardize biodiversity data from disparate sources to capture a minimum set of critical variables required to study, report and manage biodiversity change. Here, we assess the challenges of a 'Big Data' approach to building global EBV data products across taxa and spatiotemporal scales, focusing on species distribution and abundance. The majority of currently available data on species distributions derives from incidentally reported observations or from surveys where presence-only or presence-absence data are sampled repeatedly with standardized protocols. Most abundance data come from opportunistic population counts or from population time series using standardized protocols (e.g. repeated surveys of the same population from single or multiple sites). Enormous complexity exists in integrating these heterogeneous, multi-source data sets across space, time, taxa and different sampling methods. Integration of such data into global EBV data products requires correcting biases introduced by imperfect detection and varying sampling effort, dealing with different spatial resolution and extents, harmonizing measurement units from different data sources or sampling methods, applying statistical tools and models for spatial inter- or extrapolation, and quantifying sources of uncertainty and errors in data and models. To support the development of EBVs by the Group on Earth Observations Biodiversity Observation Network (GEO BON), we identify 11 key workflow steps that will operationalize the process of building EBV data products within and across research infrastructures worldwide. These workflow steps take multiple sequential activities into account, including identification and aggregation of various raw data sources, data quality control, taxonomic name matching and statistical modelling of integrated data. We illustrate these steps with concrete examples from existing citizen science and professional monitoring projects, including eBird, the Tropical Ecology Assessment and Monitoring network, the Living Planet Index and the Baltic Sea zooplankton monitoring. The identified workflow steps are applicable to both terrestrial and aquatic systems and a broad range of spatial, temporal and taxonomic scales. They depend on clear, findable and accessible metadata, and we provide an overview of current data and metadata standards. Several challenges remain to be solved for building global EBV data products: (i) developing tools and models for combining heterogeneous, multi-source data sets and filling data gaps in geographic, temporal and taxonomic coverage, (ii) integrating emerging methods and technologies for data collection such as citizen science, sensor networks, D

The response of the carotenoidless Rhodobacter sphaeroides mutant R26 to chromate stress under photosynthetic conditions is investigated by biochemical and spectroscopic measurements, proteomic analysis and cell imaging. Cell cultures were found able to reduce chromate within 3-4 days. Chromate induces marked changes in the cellular dimension and morphology, as revealed by atomic force microscopy, along with compositional changes in the cell wall revealed by infrared spectroscopy. These effects are accompanied by significant changes in the level of several proteins: 15 proteins were found up-regulated and 15 down-regulated. The protein content found in chromate exposed cells is in good agreement with the biochemical, spectroscopic and microscopic results. Moreover at the present stage no specific chromate-reductase could be found in the soluble proteome, indicating that detoxification of the pollutant proceeds via aspecific reductants.

Chromate is a highly soluble and toxic non-essential oxyanion for most organisms. A number of chromate resistant bacteria have been investigated and diverse resistance mechanisms were found [1].We are investigating the potentialities of the photosynthetic facultative bacterium Rhodobacter sphaeroides, known for its ability to tolerate high concentrations of several heavy metal ions [2] and bioaccumulate some of them, such as nickel and cobalt [3, 4], in the bioremediation of chromate polluted sites. Employing an interdisciplinary approach, the response to chromate stress was investigated by combining biochemical and spectroscopic measurements, proteomic characterization and cell imaging.An efficient resistance mechanism to chromate is suggested both by the high EC50 value and the lag-phase lengthening induced at concentrations above 0.05 mM. R. sphaeroides is also able to reduce chromate to the less toxic and soluble form Cr(III) with reductase activity preferentially associated with the protein soluble fraction. Chromate effect on soluble enzymes was investigated by a proteomic approach: soluble protein expression profiles of cells exposed to chromate were compared with those of untreated control cells through two-dimensional gel electrophoresis analysis. Upon exposure to chromate at least 30 soluble proteins were differentially expressed. The wide variety of differentially expressed proteins suggests that different metabolic pathways are involved as response to chromate exposure. The accompanying physiological response to Cr(VI) exposure included marked changes in cellular morphology as revealed by atomic force microscopy.

A wide range of biopeptides potentially able to lower blood pressure through inhibition of the angiotensin-I converting enzyme (ACE) is produced in fermented foods by proteolytic starter cultures. This work applies a procedure based on recombinant DNA technologies for the synthesis and expression of three ACE-inhibitory peptides using a probiotic cell factory. ACE-inhibitory genes and their pro-active precursors were designed, synthesized by PCR, and cloned in Escherichia coli; after which, they were cloned into the pAM1 E. coli-bifidobacteria shuttle vector. After E. coli transformation, constructs carrying the six recombinant clones were electrotransferred into the Bifidobacterium pseudocatenulatum M115 probiotic strain. Interestingly, five of the six constructs proved to be stable. Their expression was confirmed by reverse transcription PCR. Furthermore, transformed strains displayed ACE-inhibitory activity linearly correlated to increasing amounts of cell-free cellular lysates. In particular, 50 ?g of lysates from constructs pAM1-Pro-BP3 and pAM1-BP2 showed a 50% higher ACE-inhibitory activity than that of the controls. As a comparison, addition of 50 ng of Pro-BP1 and Pro-BP3 synthetic peptides to 50 ?g of cell-free extracts of B. pseudocatenulatum M115 wild-type strain showed an average of 67% of ACE inhibition; this allowed estimating the amount of the peptides produced by the transformants. Engineering of bifidobacteria for the production of biopeptides is envisioned as a promising cell factory model system.

The objective of this study was to develop a new synbiotic beverage evaluating the ability of some bifidobacteria strains to grow in this beverage which was fortified with whey proteins up to 20 g L-1, and enriched with 10 g L-1 of prebiotic inulin or resistant starch. The ability of Bifidobacterium strains to survive for 30 days at 4°C was evaluated in two synbiotic whey protein fortified beverages formulated with 2% of whey proteins and 1% of inulin or resistant starch. Microbial growth was significantly affected by the whey protein amount as well as by the kind of prebiotic fibre. Resistant starch promoted the growth of the Bifidobacterium pseudocatenulatum strain and its viability under cold storage, also conferring higher sensory scores. The development of this new functional beverage will allow to carry out in vivo trials in order to validate its pre- and probiotic effects.

Genome walking is a molecular procedure for the direct identification of nucleotide sequences from purified genomes. The only requirement is the availability of a known nucleotide sequence from which to start. Several genome walking methods have been developed in the last 20 years, with continuous improvements added to the first basic strategies, including the recent coupling with next generation sequencing technologies. This review focuses on the use of genome walking strategies in several aspects of the study of eukaryotic genomes. In a first part, the analysis of the numerous strategies available is reported. The technical aspects involved in genome walking are particularly intriguing, also because they represent the synthesis of the talent, the fantasy and the intelligence of several scientists. Applications in which genome walking can be employed are systematically examined in the second part of the review, showing the large potentiality of this technique, including not only the simple identification of nucleotide sequences but also the analysis of large collections of mutants obtained from the insertion of DNA of viral origin, transposons and transfer DNA (T-DNA) constructs. The enormous amount of data obtained indicates that genome walking, with its large range of applicability, multiplicity of strategies and recent developments, will continue to have much to offer for the rapid identification of unknown sequences in several fields of genomic research.

PlantPIs is a web querying system for a database collection of plant protease inhibitors data. Protease inhibitors in plants are naturally occurring proteins that inhibit the function of endogenous and exogenous proteases. In this paper the design and development of a web framework providing a clear and very flexible way of querying plant protease inhibitors data is reported. The web resource is based on a relational database, containing data of plants protease inhibitors publicly accessible, and a graphical user interface providing all the necessary browsing tools, including a data exporting function. PlantPIs contains information extracted principally from MEROPS database, filtered, annotated and compared with data stored in other protein and gene public databases, using both automated techniques and domain expert evaluations. The data are organized to allow a flexible and easy way to access stored information. The database is accessible at http://www.plantpis.ba.itb.cnr.it/.

Cells of the carotenoidless strain R-26.1 of Rhodobacter sphaeroides were grown in the presence of a high concentration (5 mM) of cobalt ions. The photosynthetic intracytoplasmic membranes were isolated and investigated by proteomic analysis using non-denaturating blue native electrophoresis in combination with LC-ESI-MS/MS. Comparison with intracytoplasmic membranes of cells grown under control conditions showed a change in the relative amount of proteins belonging to the photosynthetic apparatus, with net downregulation of light-harvesting complexes and increased concentration of the nude reaction center (RC), as well as upregulation of enzymes related to chemoorganotrophy. These effects represent possible bacterial adaptation so as to retrieve energy for metabolic processes from sources alternative to less efficient photosynthesis. The influence of cobalt on the photochemistry of the RC in cell extracts was also investigated by charge recombination. The kinetics of the charge recombination reaction was found to be slower in extracts from cells exposed to Co(2+), indicating that the reorganization of the photosynthetic apparatus also involves its photochemical core.

Essential biodiversity variables (EBVs) have been proposed by the Group on Earth Observations Biodiversity Observation Network (GEO BON) to identify a minimum set of essential measurements that are required for studying, monitoring and reporting biodiversity and ecosystem change. Despite the initial conceptualisation, however, the practical implementation of EBVs remains challenging. There is much discussion about the concept and implementation of EBVs: which variables are meaningful; which data are needed and available; at which spatial, temporal and topical scales can EBVs be calculated; and how sensitive are EBVs to variations in underlying data? To advance scientific progress in implementing EBVs we propose that both scientists and research infrastructure operators need to cooperate globally to serve and process the essential large datasets for calculating EBVs. We introduce GLOBIS-B (GLOBal Infrastructures for Supporting Biodiversity research), a global cooperation funded by the Horizon 2020 research and innovation framework programme of the European Commission. The main aim of GLOBIS-B is to bring together biodiversity scientists, global research infrastructure operators and legal interoperability experts to identify the research needs and infrastructure services underpinning the concept of EBVs. The project will facilitate the multi-lateral cooperation of biodiversity research infrastructures worldwide and identify the required primary data, analysis tools, methodologies and legal and technical bottlenecks to develop an agenda for research and infrastructure development to compute EBVs. This requires development of standards, protocols and workflows that are 'self-documenting' and openly shared to allow the discovery and analysis of data across large spatial extents and different temporal resolutions. The interoperability of existing biodiversity research infrastructures will be crucial for integrating the necessary biodiversity data to calculate EBVs, and to advance our ability to assess progress towards the Aichi targets for 2020 of the Convention on Biological Diversity (CBD).