Suono e Spettacolo. Athanasius Kircher, un percorso nelle Immagini sonore.

The Society of Jesus made great propaganda efforts throughout the seventeenth century and chose the images and the play as a privileged means to communicate and persuade. Athanasius Kircher, a key figure of the seventeenth century, he decided to dominate the wild nature of sound through Phonurgia Nova, which includes a gallery of powerful symbolic images for Baroque aesthetics. The essay, through the grant of the images from the Library of the Department of Mathematics "Guido Castelnuovo" Sapienza University of Rome, aims to understand, through the pictures offered by Kircher, the sound phenomenon and the spectacle that this produces. In Phonurgia Nova a process of dramatization sound effects takes place, often through machines and "visions" applied to the theatrical reality, as experimental and astonishing environment beloved in baroque. Kircher illustrates the sound through explanatory figures, so to dominate the sound through the eyes. Sound is seen, admired and represented: its spectacle not only takes place through the implementation of sound machines or the "wonders" applied to the theater, but even through images, creating create a sense of wonder in in the erudite person of the seventeenth century

Optimizing a kinase assay for IKK.beta. on an HTS station

Using a commercially available time-resolved fluorescence resonance energy transfer (TR-FRET)—based assay for IKKβ, the authors have automated the assay procedure on a high-throughput screening station to carry out screening campaigns on multiwell plates. They have determined the Z' factor and optimized volumes, times, and time-resolved fluorescence parameters. They have also compared 2 kinases with different fusion tags, the influence of different enzyme/substrate ratios and of DMSO presence at different concentration. The authors found that glutathione S-transferase (GST)—fused IKKβ shows better signal-to-noise (S/N) ratios over the poly-histidine-tagged variant. The substrate can be used at 50 nM with optimal performances when the enzyme is used at 2 nM. DMSO at 0.2% and 1% only slightly affects the S/N ratio, whereas when used at 2%, the final concentration deriving from a 50-fold dilution from a 5-mM stock solution in pure solvent, S/N undergoes a decrease of about 15%. Under the optimized conditions, the assay Z' factor calculated over 192 data points has an optimized value of 0.881 and allows the testing of 94 molecules in quadruplicate in 140 min

The ribosomal protein L2 interacts with the RNA polymerase alpha subunit and acts as a transcription modulator in Escherichia coli.

Identification of interacting proteins in stable complexes is essential to understand the mechanisms that regulate cellular processes at the molecular level. Transcription initiation in prokaryotes requires coordinated protein-protein and protein-DNA interactions that often involve one or more transcription factors in addition to RNA polymerase (RNAP) subunits. The RNAP alpha subunit (RNAPalpha) is a key regulatory element in gene transcription and functions through direct interaction with other proteins to control all stages of this process. A clear description of the RNAPalpha protein partners should greatly increase our understanding of transcription modulation. A functional proteomics approach was employed to investigate protein components that specifically interact with RNAPalpha. A tagged form of Escherichia coli RNAPalpha was used as bait to determine the molecular partners of this subunit in a whole-cell extract. Among other interacting proteins, 50S ribosomal protein L2 (RPL2) was clearly identified by mass spectrometry. The direct interaction between RNAPalpha and RPL2 was confirmed both in vivo and in vitro by performing coimmunoprecipitation and bacterial two-hybrid experiments. The functional role of this interaction was also investigated in the presence of a ribosomal promoter by using a beta-galactosidase gene reporter assay. The results clearly demonstrated that RPL2 was able to increase beta-galactosidase expression only in the presence of a specific ribosomal promoter, whereas it was inactive when it was assayed with an unrelated promoter. Interestingly, other ribosomal proteins (L1, L3, L20, and L27) did not have any effect on rRNA expression. The findings reported here strongly suggest that in addition to its role in ribosome assembly the highly conserved RPL2 protein plays a specific and direct role in regulation of transcription

Discovery of Small Peptide Antagonists of PED/PEA15-D4α Interaction from Simplified Combinatorial Libraries

Most biological processes involve permanent and temporary interactions between different proteins: protein complexes often play key roles in human diseases and, as a consequence, molecules that prevent protein-protein interactions can be potential new therapeutic agents to treat diseases. Here, we describe a simplified approach by which small synthetic peptide libraries were screened to identify the inhibitors of the complex between phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes15 (PED/PEA15) and D4α, a functional domain of the phospholipase D1, that is involved in the molecular mechanisms of insulin resistance occurring in type 2 diabetes. By using an enzyme-linked immunosorbent assay (ELISA)-based screening, performed on a fully automated platform, we analyzed two simplified peptide libraries in a positional scanning format. This screening led to the identification of small peptides able to inhibit PED/PEA15-D4α interaction. The selection of inhibitors was carried out employing combined competitive and direct experiments, through ELISA and surface plasmon resonance techniques, providing peptides with IC50 values in the micromolar range. Our results showed that the protein complex PED/PEA15-D4α is susceptible to peptides having H-donor groups and aromatic rings on specific positions. These small sequences can be considered as promising scaffolds that could be converted into higher-affinity inhibitor compounds. By a simplified approach small synthetic peptide libraries were screened to identify inhibitors of the complex between PED/PEA15 and D4α that is involved in molecular mechanisms of insulin resistance in type 2 diabetes. Small peptide sequences having H-donor groups and aromatic rings on specific positions are able to inhibit the complex and are promising scaffolds that could be converted into higher-affinity inhibitor compounds. © 2011 John Wiley & Sons A/S

Inhibition of the AIF/CypA complex protects against intrinsic death pathways induced by oxidative stress

Delayed neuronal cell death largely contributes to the progressive infarct development and associated functional impairments after cerebral ischemia or brain trauma. Previous studies exposed a key role for the interaction of the mitochondrial protein apoptosis-inducing factor (AIF) and cytosolic cyclophilin A (CypA) in pathways of programmed cell death in neurons in vitro and in vivo. These studies suggested that pro-apoptotic activities of AIF, such as its translocation to the nucleus and subsequent DNA degradation, depend on the physical interaction of AIF with CypA. Hence, this protein complex may represent a new pharmacological target for inhibiting the lethal action of AIF on the brain tissue. In this study, we show that the AIF amino-acid residues 370-394 mediate the protein complex formation of AIF with CypA. The synthetic AIF(370-394) peptide inhibited AIF/CypA complex formation in vitro by binding CypA with a KD of 12 μM. Further, the peptide exerted pronounced neuroprotective effects in a model of glutamate-induced oxidative stress in cultured HT-22 cells. In this model system of AIF-dependent cell death, the AIF(370-394) peptide preserved mitochondrial integrity, as detected by measurements of the mitochondrial membrane potential and quantification of mitochondrial fragmentation. Further, the AIF(370-394) peptide inhibited perinuclear accumulation of fragmented mitochondria, mitochondrial release of AIF to the nucleus and glutamate-induced cell death to a similar extent as CypA-siRNA. These data indicate that the targeting of the AIF-CypA axis is an effective strategy of neuroprotection. © 2014 Macmillan Publishers Limited

Discovery of small peptide antagonists of PED/PEA15-D4alpha interaction from simplified combinatorial libraries

Most biological processes involve permanent and temporary interactions between different proteins: protein complexes often play key roles in human diseases and, as a consequence, molecules that prevent protein-protein interactions can be potential new therapeutic agents to treat diseases. Here, we describe a simplified approach by which small synthetic peptide libraries were screened to identify the inhibitors of the complex between phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes15 (PED/PEA15) and D4??, a functional domain of the phospholipase D1, that is involved in the molecular mechanisms of insulin resistance occurring in type 2 diabetes. By using an enzyme-linked immunosorbent assay (ELISA)-based screening, performed on a fully automated platform, we analyzed two simplified peptide libraries in a positional scanning format. This screening led to the identification of small peptides able to inhibit PED/PEA15-D4?? interaction. The selection of inhibitors was carried out employing combined competitive and direct experiments, through ELISA and surface plasmon resonance techniques, providing peptides with IC 50 values in the micromolar range. Our results showed that the protein complex PED/PEA15-D4?? is susceptible to peptides having H-donor groups and aromatic rings on specific positions. These small sequences can be considered as promising scaffolds that could be converted into higher-affinity inhibitor compounds. By a simplified approach small synthetic peptide libraries were screened to identify inhibitors of the complex between PED/PEA15 and D4?? that is involved in molecular mechanisms of insulin resistance in type 2 diabetes. Small peptide sequences having H-donor groups and aromatic rings on specific positions are able to inhibit the complex and are promising scaffolds that could be converted into higher-affinity inhibitor compound