Francesca De Leo

Genome walking in eukaryotes

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.

Cystatins, Serpins and other Families of Protease Inhibitors in Plants

Plant protease inhibitors (PIs) are generally small proteins present in high concentrations in storage tissues (tubers and seeds), and to a lower level in leaves. Even if most of them are active against serine and cysteine proteases, PIs active against aspartic proteases and carboxypeptidases have also been identified. Inhibitors of serine proteases are further classifiable in several families on the basis of their structural features. They comprise the families known as Bowman-Birk, Kunitz, Potato I and Potato II, which are the subject of review articles included in this special issue. In the present article we aim to give an overview of other families of plant PIs, active either against serine proteases or other class of proteases, describing their distribution, activity and main structural characteristics.

Towards global interoperability for supporting biodiversity research on essential biodiversity variables (EBVs)

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).

Building essential biodiversity variables (EBVs) of species distribution and abundance at a global scale

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, DNA‐based techniques and satellite remote sensing, (iii) solving major technical issues related to data product structure, data storage, execution of workflows and the production process/cycle as well as approaching technical interoperability among research infrastructures, (iv) allowing semantic interoperability by developing and adopting standards and tools for capturing consistent data and metadata, and (v) ensuring legal interoperability by endorsing open data or data that are free from restrictions on use, modification and sharing. Addressing these challenges is critical for biodiversity research and for assessing progress towards conservation policy targets and sustainable development goals.

BioVeL: a virtual laboratory for data analysis and modelling in biodiversity science and ecology

BackgroundMaking 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.ResultsBioVeL is a virtual laboratory for data analysis and modelling in biodiversity science and ecology, freely accessible via the Internet. BioVeL includes functions for accessing and analysing data through curated Web services; for performing complex in silico analysis through exposure of R programs, workflows, and batch processing functions; for on-line collaboration through sharing of workflows and workflow runs; for experiment documentation through reproducibility and repeatability; and for computational support via seamless connections to supporting computing infrastructures. We developed and improved more than 60 Web services with significant potential in many different kinds of data analysis and modelling tasks. We composed reusable workflows using these Web services, also incorporating R programs. Deploying these tools into an easy-to-use and accessible ‘virtual laboratory’, free via the Internet, we applied the workflows in several diverse case studies. We opened the virtual laboratory for public use and through a programme of external engagement we actively encouraged scientists and third party application and tool developers to try out the services and contribute to the activity.ConclusionsOur work shows we can deliver an operational, scalable and flexible Internet-based virtual laboratory to meet new demands for data processing and analysis in biodiversity science and ecology. In particular, we have successfully integrated existing and popular tools and practices from different scientific disciplines to be used in biodiversity and ecological research.

Effect of Cobalt Ions on the Soluble Proteome of a Rhodobacter sphaeroides Carotenoidless Mutant

Rhodobacter sphaeroides strain R26.1 showed tolerance to Co2+ ions, up to 10 mM concentration. Interestingly the bacteriochlorophyll biosynthesis was found to decrease upon addition of such metal in the growth medium. Analysis of R. sphaeroides proteome from cells grown in control and in Co2+ enriched media was performed by two dimensional electrophoresis (2DE) followed by mass spectrometry. Proteome and functional analyses of differentially expressed proteins in cobalt response clearly highlighted the involvement of several metabolic pathways, and in particular of some enzymes involved in tetrapyrrole biosynthesis pathway.

One of the three proteinase inhibitor genes newly identified in the Brassica napus genome codes for an inhibitor of glutamyl endopeptidase

In germinating seeds, only the transcript of one gene, coding for a trypsin inhibitor, is detectable by Northern analysis. The other two genes are transcribed at basal levels detectable only by reverse transcription PCR. One of the other two genes (rti‐2) encodes a polypeptide with a glutamic residue in the P1 position, characteristic of glutamyl proteinase inhibitors. The recombinant RTI‐2 protein strongly inhibits (K i = 44 nM) a glutamyl proteinase from Streptomyces griseus.

PlantPIs – An Interactive Web Resource on Plant Protease Inhibitors

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/.

Assessment of an internal reference gene in Rhodobacter sphaeroides grown under cobalt exposure

Aim of this study is the identification of an appropriate internal reference gene to quantify gene transcripts isolated from Rhodobacter (R.) sphaeroides cells grown in presence of high concentrations of cobalt ions. RNA was isolated using a commercial kit protocol ad‐hoc modified. Several primer pairs were used to perform reverse transcription PCR and real‐time PCR to assess the suitable internal reference gene whose expression is not affected by cobalt ions, identified with the gene rsp0154.

Identification and characterization of protease inhibitors in Diplotaxis species

PCR analysis of the genomes of two wild Brassicaceae plants, Diplotaxis muralis and Diplotaxis tenuifolia, demonstrated the presence of several genes coding for potential protease inhibitors, classifiable within the mustard inhibitor family (MSI). This is a small family of plant protease inhibitors named after the mustard trypsin inhibitor MTI-2, the first protease inhibitor characterized in Brassicaceae. From identified sequences two recombinant inhibitors were expressed in Pichia pastoris. In comparison with MTI-2, they show a reduced activity against bovine trypsin. However, when tested against trypsin-like proteases present in the guts of Helicoverpa zea larvae, the Diplotaxis inhibitors and MTI-2 show similar activities, indicating that the usually adopted procedure of reporting activity of plant protease inhibitors against bovine trypsin may lead to wrong estimation of their effect on insect proteases. This issue is of particular relevance when planning the use of PI genes for developing insect resistant plants.