Field phenomics:will it enable crop improvement?

Field phenomics has been identified as a promising enabling technology to assist plant breeders with the development of improved cultivars for farmers. Yet, despite much investment, there are few examples demonstrating the application of phenomics within a plant breeding program. We review recent progress in field phenomics and highlight the importance of targeting breeders’ needs, rather than perceived technology needs, through developing and enhancing partnerships between phenomics researchers and plant breeders

Tourism destination modelling: building a sustainable planning tool for Australian tourism destinations

The Ningaloo Destination Model is a tourism planning tool for the Ningaloo Coast region of Western Australia that assesses the economic, social and environmental impacts of different planning decisions and events. This report describes the features of the tourism destination model, and analyses its application in the region and to other parts of Australia. Destination modelling integrates a number of research methodologies developed through past STCRC projects (on visitor spending and characteristics, social impacts and economic impacts), secondary data and ecological research. The key to this process is a model development technique that uses scenario planning methodologies to facilitate stakeholder engagement and conceptual modelling techniques to facilitate research integration. The report describes the methodologies used for model development and for data collection, provides two case studies demonstrating outputs, and explores applications of the Ningaloo Destination Model to the region, to other locations and to other sectors. The Ningaloo Destination Model provides estimates of the impacts of plans and events related to tourism in four dimensions: tourism specific; economy; social; and environmental (both resource use and ecological). These outputs are explored through two case studies: a nodal coastal development; and a large resort development. The model can be used for four broad (oftentimes overlapping) categories of assessment: operational planning and decision making for specific organisations and groups (such as local government or agencies that manage land or sea use), regional planning, participatory planning and collaborations and to assist monitoring and evaluation.The Ningaloo Destination Model will be available to the general public (in a limited format) through websites; to agencies through a desktop version; and through integration into a larger model of the region being developed by the CSIRO. Destination modelling is relevant for other tourism destinations and a process for the rapid and cost-effective application of destination modelling is feasible. While much of the data is available, a broader roll-out would require benchmarking of water, electricity and waste data, and developing a wireframe for all tourism destinations. Making destination modelling tools broadly available would significantly broaden the impacts considered in tourism planning and lead to enhancement of desirable effects of tourism development, and early mitigation of negative impacts across Australia. The techniques developed for destination modelling were also found to be applicable to other sectors

Sequestration of Polo kinase to microtubules by phosphopriming-independent binding to Map205 is relieved by phosphorylation at a CDK site in mitosis

The conserved Polo kinase controls multiple events in mitosis and cytokinesis. Although Polo-like kinases are regulated by phosphorylation and proteolysis, control of subcellular localization plays a major role in coordinating their mitotic functions. This is achieved largely by the Polo-Box Domain, which binds prephosphorylated targets. However, it remains unclear whether and how Polo might interact with partner proteins when priming mitotic kinases are inactive. Here we show that Polo associates with microtubules in interphase and cytokinesis, through a strong interaction with the microtubule-associated protein Map205. Surprisingly, this interaction does not require priming phosphorylation of Map205, and the Polo-Box Domain of Polo is required but not sufficient for this interaction. Moreover, phosphorylation of Map205 at a CDK site relieves this interaction. Map205 can stabilize Polo and inhibit its cellular activity in vivo. In syncytial embryos, the centrosome defects observed in polo hypomorphs are enhanced by overexpression of Map205 and suppressed by its deletion. We propose that Map205-dependent targeting of Polo to microtubules provides a stable reservoir of Polo that can be rapidly mobilized by the activity of Cdk1 at mitotic entry

Spatiotemporal proteomic profiling of the pro-inflammatory response to lipopolysaccharide in the THP-1 human leukaemia cell line.

Protein localisation and translocation between intracellular compartments underlie almost all physiological processes. The hyperLOPIT proteomics platform combines mass spectrometry with state-of-the-art machine learning to map the subcellular location of thousands of proteins simultaneously. We combine global proteome analysis with hyperLOPIT in a fully Bayesian framework to elucidate spatiotemporal proteomic changes during a lipopolysaccharide (LPS)-induced inflammatory response. We report a highly dynamic proteome in terms of both protein abundance and subcellular localisation, with alterations in the interferon response, endo-lysosomal system, plasma membrane reorganisation and cell migration. Proteins not previously associated with an LPS response were found to relocalise upon stimulation, the functional consequences of which are still unclear. By quantifying proteome-wide uncertainty through Bayesian modelling, a necessary role for protein relocalisation and the importance of taking a holistic overview of the LPS-driven immune response has been revealed. The data are showcased as an interactive application freely available for the scientific community

Development of novel methods for non-canonical myeloma protein analysis with an innovative adaptation of immunofixation electrophoresis, native top-down mass spectrometry, and middle-down de novo sequencing

OBJECTIVES: Multiple myeloma (MM) is a malignant plasma cell neoplasm, requiring the integration of clinical examination, laboratory and radiological investigations for diagnosis. Detection and isotypic identification of the monoclonal protein(s) and measurement of other relevant biomarkers in serum and urine are pivotal analyses. However, occasionally this approach fails to characterize complex protein signatures. Here we describe the development and application of next generation mass spectrometry (MS) techniques, and a novel adaptation of immunofixation, to interrogate non-canonical monoclonal immunoproteins. METHODS: Immunoprecipitation immunofixation (IP-IFE) was performed on a Sebia Hydrasys Scan2. Middle-down de novo sequencing and native MS were performed with multiple instruments (21T FT-ICR, Q Exactive HF, Orbitrap Fusion Lumos, and Orbitrap Eclipse). Post-acquisition data analysis was performed using Xcalibur Qual Browser, ProSight Lite, and TDValidator. RESULTS: We adapted a novel variation of immunofixation electrophoresis (IFE) with an antibody-specific immunosubtraction step, providing insight into the clonal signature of gamma-zone monoclonal immunoglobulin (M-protein) species. We developed and applied advanced mass spectrometric techniques such as middle-down de novo sequencing to attain in-depth characterization of the primary sequence of an M-protein. Quaternary structures of M-proteins were elucidated by native MS, revealing a previously unprecedented non-covalently associated hetero-tetrameric immunoglobulin. CONCLUSIONS: Next generation proteomic solutions offer great potential for characterizing complex protein structures and may eventually replace current electrophoretic approaches for the identification and quantification of M-proteins. They can also contribute to greater understanding of MM pathogenesis, enabling classification of patients into new subtypes, improved risk stratification and the potential to inform decisions on future personalized treatment modalities

The Use of On-line Capillary Electrophoresis/Electrospray Ionization with Detection via an Ion Trap Storage/Reflectron Time-of-flight Mass Spectrometer for Rapid Mutation-site Analysis of Hemoglobin Variants

Capillary electrophoresis/electrospray ionization using an ion trap storage/reflectron time-of-flight mass spectrometer detector (CE/ESI-IT/reTOF) is used to provide a rapid and sensitive method for analyzing structural variants in the hemoglobin (Hb) β-chain. The Hb α- and β-chains are separated and the β-chain is digested by trypsin. The digest is analyzed by CE/ESI-IT/reTOF where a comparison of the total ion electrophorograms and mass spectra of the mutant and normal hemoglobins (Hbs) can detect the presence of a mutation site. In addition, collision-induced dissociation in the vacuum interface — skimmer region can be used to pinpoint the identity of such a site. The unique capability of the CE/ESI-IT/reTOF system for accurately detecting fast separations with narrow peaks that may be under 1 s full width at half maximum is demonstrated. The speed of this system is essential for resolution of the large number of peaks that are separated in a short time duration using CE separations. © 1997 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35071/1/813_ftp.pd

Alkynyl Benzoxazines and Dihydroquinazolines as Cysteine Targeting Covalent Warheads and Their Application in Identification of Selective Irreversible Kinase Inhibitors.

With a resurgence in interest in covalent drugs, there is a need to identify new moieties capable of cysteine bond formation that are differentiated from commonly employed systems such as acrylamide. Herein, we report on the discovery of new alkynyl benzoxazine and dihydroquinazoline moieties capable of covalent reaction with cysteine. Their utility as alternative electrophilic warheads for chemical biological probes and drug molecules is demonstrated through site-selective protein modification and incorporation into kinase drug scaffolds. A potent covalent inhibitor of JAK3 kinase was identified with superior selectivity across the kinome and improvements in in vitro pharmacokinetic profile relative to the related acrylamide-based inhibitor. In addition, the use of a novel heterocycle as a cysteine reactive warhead is employed to target Cys788 in c-KIT, where acrylamide has previously failed to form covalent interactions. These new reactive and selective heterocyclic warheads supplement the current repertoire for cysteine covalent modification while avoiding some of the limitations generally associated with established moieties

Proteome Mapping of a Cyanobacterium Reveals Distinct Compartment Organization and Cell-Dispersed Metabolism.

Cyanobacteria are complex prokaryotes, incorporating a Gram-negative cell wall and internal thylakoid membranes (TMs). However, localization of proteins within cyanobacterial cells is poorly understood. Using subcellular fractionation and quantitative proteomics, we produced an extensive subcellular proteome map of an entire cyanobacterial cell, identifying ∼67% of proteins in Synechocystis sp. PCC 6803, ∼1000 more than previous studies. Assigned to six specific subcellular regions were 1,712 proteins. Proteins involved in energy conversion localized to TMs. The majority of transporters, with the exception of a TM-localized copper importer, resided in the plasma membrane (PM). Most metabolic enzymes were soluble, although numerous pathways terminated in the TM (notably those involved in peptidoglycan monomer, NADP+, heme, lipid, and carotenoid biosynthesis) or PM (specifically, those catalyzing lipopolysaccharide, molybdopterin, FAD, and phylloquinol biosynthesis). We also identified the proteins involved in the TM and PM electron transport chains. The majority of ribosomal proteins and enzymes synthesizing the storage compound polyhydroxybuyrate formed distinct clusters within the data, suggesting similar subcellular distributions to one another, as expected for proteins operating within multicomponent structures. Moreover, heterogeneity within membrane regions was observed, indicating further cellular complexity. Cyanobacterial TM protein localization was conserved in Arabidopsis (Arabidopsis thaliana) chloroplasts, suggesting similar proteome organization in more developed photosynthetic organisms. Successful application of this technique in Synechocystis suggests it could be applied to mapping the proteomes of other cyanobacteria and single-celled organisms. The organization of the cyanobacterial cell revealed here substantially aids our understanding of these environmentally and biotechnologically important organisms