2 research outputs found
Method for Noninvasive Analysis of Proteins and Small Molecules from Ancient Objects
Proteins and small molecules from
ancient objects and cultural
heritage can provide key information and contribute to study the context
of objects and artists. However, all present-day protocols and strategies
for the analysis of ancient samples are often invasive and require
microsampling. Here, we present a new method for the noninvasive analysis
of proteins and small molecules: the technique uses a special ethyl-vinyl
acetate film functionalized with strong cation/anion exchange and
C<sub>8</sub> resins, for interacting with both proteins and small
molecules present on the surface of the objects, followed by LC–MS/MS
analysis. The new method was fully validated for the determination
of both proteins and small molecules on several types of supports,
showing excellent analytical performances such as, for example, <i>R</i><sup>2</sup> of the calibration curve of 0.98 and 0.99
for proteins and small molecules, low but very repeatable recoveries,
particularly adequate for investigations on precious ancient samples
that must not be altered by the analytical procedure. ESEM images
and LED multispectral imaging confirmed that no damages or alterations
occurred onto the support surfaces and no residues were left from
the extractive film. Finally, the new method was applied for the characterization
of the binders of a historical fresco of the XVI century from the
Flemish painter Paul Brill and of a recently discovered fresco from
Isidoro Bianchi (XVII century). Moreover the method was employed for
the identification of the colorant used by Pietro Gallo (XIV century)
on a wood panel. The method here reported can be easily applied to
any other research on ancient precious objects and cultural heritage,
since it does not require microsampling and the proteins/small molecules
extraction can be performed directly <i>in situ</i>, leaving
the object unchanged and intact
DataSheet_1_Wiskott-Aldrich syndrome protein interacts and inhibits diacylglycerol kinase alpha promoting IL-2 induction.docx
BackgroundPhosphorylation of diacylglycerol by diacylglycerol-kinases represents a major inhibitory event constraining T cell activation upon antigen engagement. Efficient TCR signalling requires the inhibition of the alpha isoform of diacylglycerol kinase, DGKα, by an unidentified signalling pathway triggered by the protein adaptor SAP. We previously demonstrated that, in SAP absence, excessive DGKα activity makes the T cells resistant to restimulation-induced cell death (RICD), an apoptotic program counteracting excessive T cell clonal expansion.ResultsHerein, we report that the Wiskott-Aldrich syndrome protein (WASp) inhibits DGKα through a specific interaction of the DGKα recoverin homology domain with the WH1 domain of WASp. Indeed, WASp is necessary and sufficient for DGKα inhibition, and this WASp function is independent of ARP2/3 activity. The adaptor protein NCK-1 and the small G protein CDC42 connect WASp-mediated DGKα inhibition to SAP and the TCR signalosome. In primary human T cells, this new signalling pathway is necessary for a full response in terms of IL-2 production, while minimally affecting TCR signalling and restimulation-induced cell death. Conversely, in T cells made resistant to RICD by SAP silencing, the enhanced DAG signalling due to DGKα inhibition is sufficient to restore apoptosis sensitivity.ConclusionWe discover a novel signalling pathway where, upon strong TCR activation, the complex between WASp and DGKα blocks DGKα activity, allowing a full cytokine response.</p