HybriChip: An antigen microarray based screening tool, designed for a high-throughput production platform of mouse derived monoclonal antibodies

The incapacity to generate a sufficient number of mAbs to conduct large-scale analyses remains one of the most serious limitations in the field of functional genomics. Current methods of mAb production are very labour intensive and disappointingly low throughput, with a per capita production level of about 20 antigens per year. Here we present a modified hybridoma production method incorporating a novel screening assay which can be scaled up to generate antibodies in sufficient quantities for proteomics-scale analysis. Our method circumvents previous obstacles to increasing the throughput level of mAb production, in two ways. First, by immunizing a single mouse with multiple target antigens, we dramatically reduce the number of tissue culture operations normally necessary for performing multiple fusions simultaneously using only one antigen per animal. This minimises tissue culture operations and results in hybridomas that secrete antibodies specifically recognizing each of the target antigens. Second, we developed a novel antigen microarray assay (HybriChip) to screen supernatants generated by large-scale production. In this assay, an antigen chip is generated by coating an aminosilane treated slide with a single target antigen. Hybridoma culture supernatants from a fusion are consolidated and spotted as a microarray onto the antigen chip. After probing with a suitable fluorescently labelled secondary antibody, positive hybridomas are identified in a microarray scanner. The isotype of the bound antibody can be concomitantly determined by probing the antigen chip with mixtures of isotype-specific secondary antibodies, such as Cy5 -conjugated anti-mouse IgM and Cy3-conjugated antimouse pan-IgG (recognizing all mouse IgG isotypes). Different antigen chips can simultaneously be spotted in parallel with the same hybridoma culture supernatants, allowing rapid automated assay of multiple antibodies against many target antigens

Control of lysosomal biogenesis and Notch-dependent tissue patterning by components of the TFEB-V-ATPase axis in <i>Drosophila melanogaster</i>

<p>In vertebrates, TFEB (transcription factor EB) and MITF (microphthalmia-associated transcription factor) family of basic Helix-Loop-Helix (bHLH) transcription factors regulates both lysosomal function and organ development. However, it is not clear whether these 2 processes are interconnected. Here, we show that Mitf, the single TFEB and MITF ortholog in <i>Drosophila</i>, controls expression of vacuolar-type H⁺-ATPase pump (V-ATPase) subunits. Remarkably, we also find that expression of <i>Vha16-1</i> and <i>Vha13</i>, encoding 2 key components of V-ATPase, is patterned in the wing imaginal disc. In particular, <i>Vha16-1</i> expression follows differentiation of proneural regions of the disc. These regions, which will form sensory organs in the adult, appear to possess a distinctive endolysosomal compartment and Notch (N) localization. Modulation of Mitf activity in the disc in vivo alters endolysosomal function and disrupts proneural patterning. Similar to our findings in <i>Drosophila</i>, in human breast epithelial cells we observe that impairment of the <i>Vha16-1</i> human ortholog <i>ATP6V0C</i> changes the size and function of the endolysosomal compartment and that depletion of TFEB reduces ligand-independent N signaling activity. Our data suggest that lysosomal-associated functions regulated by the TFEB-V-ATPase axis might play a conserved role in shaping cell fate.</p

A DNA-binding-site landscape and regulatory network analysis for NAC transcription factors in Arabidopsis thaliana.

Target gene identification for transcription factors is a prerequisite for the systems wide understanding of organismal behaviour. NAM-ATAF1/2-CUC2 (NAC) transcription factors are amongst the largest transcription factor families in plants, yet limited data exist from unbiased approaches to resolve the DNA-binding preferences of individual members. Here, we present a TF-target gene identification workflow based on the integration of novel protein binding microarray data with gene expression and multi-species promoter sequence conservation to identify the DNA-binding specificities and the gene regulatory networks of 12 NAC transcription factors. Our data offer specific single-base resolution fingerprints for most TFs studied and indicate that NAC DNA-binding specificities might be predicted from their DNA-binding domain's sequence. The developed methodology, including the application of complementary functional genomics filters, makes it possible to translate, for each TF, protein binding microarray data into a set of high-quality target genes. With this approach, we confirm NAC target genes reported from independent in vivo analyses. We emphasize that candidate target gene sets together with the workflow associated with functional modules offer a strong resource to unravel the regulatory potential of NAC genes and that this workflow could be used to study other families of transcription factors

Systematic Discovery of New Recognition Peptides Mediating Protein Interaction Networks

Many aspects of cell signalling, trafficking, and targeting are governed by interactions between globular protein domains and short peptide segments. These domains often bind multiple peptides that share a common sequence pattern, or “linear motif” (e.g., SH3 binding to PxxP). Many domains are known, though comparatively few linear motifs have been discovered. Their short length (three to eight residues), and the fact that they often reside in disordered regions in proteins makes them difficult to detect through sequence comparison or experiment. Nevertheless, each new motif provides critical molecular details of how interaction networks are constructed, and can explain how one protein is able to bind to very different partners. Here we show that binding motifs can be detected using data from genome-scale interaction studies, and thus avoid the normally slow discovery process. Our approach based on motif over-representation in non-homologous sequences, rediscovers known motifs and predicts dozens of others. Direct binding experiments reveal that two predicted motifs are indeed protein-binding modules: a DxxDxxxD protein phosphatase 1 binding motif with a K (D) of 22 μM and a VxxxRxYS motif that binds Translin with a K (D) of 43 μM. We estimate that there are dozens or even hundreds of linear motifs yet to be discovered that will give molecular insight into protein networks and greatly illuminate cellular processes

SWIPE: a bolometric polarimeter for the Large-Scale Polarization Explorer

The balloon-borne LSPE mission is optimized to measure the linear polarization of the Cosmic Microwave Background at large angular scales. The Short Wavelength Instrument for the Polarization Explorer (SWIPE) is composed of 3 arrays of multi-mode bolometers cooled at 0.3K, with optical components and filters cryogenically cooled below 4K to reduce the background on the detectors. Polarimetry is achieved by means of large rotating half-wave plates and wire-grid polarizers in front of the arrays. The polarization modulator is the first component of the optical chain, reducing significantly the effect of instrumental polarization. In SWIPE we trade angular resolution for sensitivity. The diameter of the entrance pupil of the refractive telescope is 45 cm, while the field optics is optimized to collect tens of modes for each detector, thus boosting the absorbed power. This approach results in a FWHM resolution of 1.8, 1.5, 1.2 degrees at 95, 145, 245 GHz respectively. The expected performance of the three channels is limited by photon noise, resulting in a final sensitivity around 0.1-0.2 uK per beam, for a 13 days survey covering 25% of the sky.Comment: In press. Copyright 2012 Society of Photo-Optical Instrumentation Engineers. One print or electronic copy may be made for personal use only. Systematic reproduction and distribution, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper are prohibite

Water planning and management in the Cornia river plain by means of the gis-integrated freewat platform

Coastal aquifer, Cornia valley, Tuscany, Managed Aquifer Recharge, FREEWAT platform

Using flood water in Managed Aquifer Recharge schemes as a solution for groundwater management in the Cornia valley (Italy)

The lower Cornia valley aquifer system (Tuscany, Italy) provides the only source of water for drinking, irrigation, industrial purposes and it also contributes to the water needs of the nearby Elba island. Since 60 years, intensive exploitation of groundwater resulted in consistent head lowering and water balance deficit, causing subsidence, reduction of groundwater dependent ecosystems, and salinization of freshwater resources. Rebalancing the water budget of the hydrologic system is the main objective of the LIFE REWAT project (sustainable WATer management in the lower Cornia valley through demand REduction, aquifer Recharge and river REstoration; http://www.liferewat.eu). Here, five demonstration measures (river restoration; Managed Aquifer Recharge; reuse of treated wastewater for irrigation; high irrigation efficiency scheme; leakage management in water distribution systems) are set in place for promoting water resource management, along with capacity building and participatory actions. A pilot Managed Aquifer Recharge (MAR) infiltration basin for using flood-water was designed and set in operation in Suvereto, testing the new-issued Italian regulation on artificial recharge of aquifers (DM 100/2016). The infiltration basin is located at a pre-existing topographical low near the Cornia River. The river, having intermittent flow, provides the recharge water during high flow periods, including floods, and when discharge is above the minimum ecological flow. The infiltration basin is set in a groundwater recharge area where the aquifer is constituted by gravel and sands. A preliminary project and an executive one were prepared and discussed with the relevant authorities, following one-year long monthly monitoring of surface- and ground-water. The project was supported by a groundwater flow modelling-based approach using the FREEWAT platform (www.freewat.eu). The facility consists of the following elements: i) intake work on the River Cornia; ii) the inlet structure control system, managed by quality (mass spectrometer defining surface water spectral signature) and level probes, and allowing pumping into the facility at predefined head and chemical quality thresholds; iii) a sedimentation basin; iv) the infiltration area (less than 1 ha large); v) the operational monitoring system, based on a network of piezometers where both continuous data (head, T, EC, DO) are gathered and discrete measurements/sampling performed. The cost of construction of the plant is about 300000 C well below the cost of a surface water reservoir for a similar storage. Depending on the climatic conditions, the estimated volume of diverted surface water may vary between 300000 m3/year and 2 Mm3/year. Being the facility a pilot one, diverted water discharge ranges between 20 to 50 l/s. Minimal site development and modification was required, resulting in a no-impact water-work, while providing ecosystem benefits by reconnecting and inundating former abandoned riverbeds. The effectiveness of such pilot may demonstrate the potential for Flood-MAR schemes to increase water availability in scarcity prone areas