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We present the development of a lab-on-chip system potentially able to determine specific miRNA levels that enable a differential diagnosis between ischemic and hemorrhagic stroke, through the specialization of CMOS Image Sensors. In particular, the system allows investigations on the photoluminescence of samples of biological liquid to be analyzed (plasma, lysate, biological fluid) following the capture of the specific miRNA by an antisense set of ad hoc designed Peptide Nucleic Acids (PNA) that confers the biological specificity and sensitivity. The CMOS Image Sensor-biochip is modified with a first PNA that captures the target miRNA. A second PNA bringing a fluorescent tag binds the target miRNA enabling detection of the 3-component complex by the CMOS.

Nodal, a member of the TGF-β superfamily, is a potent embryonic morphogen also implicated in tumor progression. As for other TGF-βs, it triggers the signaling functions through the interaction with the extracellular domains of type I and type II serine/threonine kinase receptors and with the co-receptor Cripto. Recently, we reported the molecular models of Nodal in complex with its type I receptors (ALK4 and ALK7) as well as with Cripto, as obtained by homologymodeling and docking simulations. From such models, potential binding epitopes have been identified. To validate such hypotheses, a series of mutated Nodal fragments have been synthesized. These peptide analogs encompass residues 44–67 of the Nodal protein, corresponding to the prehelix loop and the H3 helix, and reproduce the wild-type sequence or bear some modifications to evaluate the hot-spot role of modified residues in the receptor binding. Here,we showthe structural characterization in solution by CD and NMR of the Nodal peptides and the measurement of binding affinity toward Cripto by surface plasmon resonance. Data collected by both conformational analyses and bindingmeasurements suggest a role for Y58 of Nodal in the recognition with Cripto and confirm that previously reported for E49 and E50. Surface plasmon resonance binding assays with recombinant proteins show that Nodal interacts in vitro also with ALK7 and ALK4 and preliminary data, generated using the Nodal synthetic fragments, suggest that Y58 of Nodal may also be involved in the recognition with these protein partners.

Peptides corresponding to retinoblastoma protein (RB) fragment 649-654 (LFYKKV) were tested for their ability to recognize the LXCXE sequence motif in human papilloma virus type 16E7 protein (HPV-16E7) encompassing E7 residues 21-26 (DLYCYE) and an identical motif in human insulin comprising insulin B-chain residues 16-21 (YLVCGE), respectively. Interaction between these complementary peptide sequences was observed by several approaches, including direct and competitive ELISA as well as affinity chromatography. Moreover, we demonstrated that immobilized RB649-654 displays specific recognition properties towards full-length insulin. Hence, this study provides a first experimental support for the previously anticipated complex formation between insulin and RB.

Item does not contain fulltext IL-32 is a multifaceted cytokine with a role in infections, autoimmune diseases, and cancer, and it exerts diverse functions, including aggravation of inflammation and inhibition of virus propagation. We previously identified IL-32 as a critical regulator of endothelial cell (EC) functions, and we now reveal that IL-32 also possesses angiogenic properties. The hyperproliferative ECs of human pulmonary arterial hypertension and glioblastoma multiforme exhibited a markedly increased abundance of IL-32, and, significantly, the cytokine colocalized with integrin alphaVbeta3. Vascular endothelial growth factor (VEGF) receptor blockade, which resulted in EC hyperproliferation, increased IL-32 three-fold. Small interfering RNA-mediated silencing of IL-32 negated the 58% proliferation of ECs that occurred within 24 h in scrambled-transfected controls. Reduction of IL-32 neither affected apoptosis (insignificant changes in Bak-1, Bcl-2, Bcl-xL, lactate dehydrogenase, annexin V, and propidium iodide) nor VEGF or TGF-beta levels, but siIL-32-transfected adult and neonatal ECs produced up to 61% less NO, IL-8, and matrix metalloproteinase-9, and up to 3-fold more activin A and endostatin. In coculture-based angiogenesis assays, IL-32gamma dose-dependently increased tube formation up to 3-fold; an alphaVbeta3 inhibitor prevented this activity and reduced IL-32gamma-induced IL-8 by 85%. In matrigel plugs loaded with IL-32gamma, VEGF, or vehicle and injected into live mice, we observed the anticipated VEGF-induced increase in neocapillarization (8-fold versus vehicle), but unexpectedly, IL-32gamma was equally angiogenic. A second signal such as IFN-gamma was required to render cells responsive to exogenous IL-32gamma; importantly, this was confirmed using a completely synthetic preparation of IL-32gamma. In summary, we add angiogenic properties that are mediated by integrin alphaVbeta3 but VEGF-independent to the portfolio of IL-32, implicating a role for this versatile cytokine in pulmonary arterial hypertension and neoplastic diseases.

Understanding the influence of a controlled spatial distribution of biological cues on cell activities can be useful to design ‘‘cell instructive” materials, able to control and guide the formation of engineered tissues in vivo and in vitro. To this purpose, biochemical and mechanical properties of the resulting biomaterial must be carefully designed and controlled. In this work, the effect of covalently immobilized RGD peptide gradients on poly(ethylene glycol) diacrylate hydrogels on cell behaviour was studied. We set up a mechanical device generating gradients based on a fluidic chamber. Cell response to RGD gradients with different slope (0.7, 1 and 2 mM cm1) was qualitatively and quantitatively assessed by evaluating cell adhesion and, in particular, cell migration, compared to cells seeded on hydrogels with uniform distribu- tion of RGD peptides. To evaluate the influence of RGD gradient and to exclude any concentration effect on cell response, all analyses were carried out in a specific region of the gradients which displayed the same average concentration of RGD (1.5 mM). Results suggest that cells recognize the RGD gradient and adhere onto it assuming a stretched shape. Moreover, cells tend to migrate in the direction of the gra- dient, as their speed is higher than that of cells migrating on hydrogels with a uniform distribution of RGD and increases by increasing RGD gradient steepness. This increment is due to an augmentation of bias speed component of the mean squared speed, that is, the drift of the cell population migrating on the anisotropic surface provided by the RGD gradient.

The protein Cripto is the founding member of the extra-cellular EGF-CFC growth factors, which are composed of two adjacent cysteine-rich domains: the EGF-like and the CFC. Members of the EGF-CFC family play key roles in embryonic development and are also implicated in tumourigenesis. Cripto is highly over-expressed in many tumours, while it is poorly detectable in normal tissues. Although both Cripto domains are involved in its tumourigenic activity, the CFC domain appears to play a crucial role. Indeed, through this domain, Cripto interferes with the onco-suppressive activity of Activins, either by blocking the Activin receptor ALK4 or by antagonising proteins of the TGF-beta family. We have undertaken the chemical synthesis and the structural characterisation of human CFC Cripto domain. Using a combined NMR and computational approach, supported by binding studies by SPR, we have investigated the molecular basis of the interaction between h-CFC and ALK4. Binding studies indicate that the synthetic h-CFC interacts with the ALK4 receptor with a K(D) in micro M range, whereas it does not recognise the ActRIIB receptor. The NMR study shows that the h-CFC overall topology is determined by the presence of three disulfide bridges and that residues H120 and W124 are located between the first strand and the first loop with the side chains externally exposed. A model of the CFC-ALK4 complex has also been obtained by molecular docking and shows that all residues indicated by prior mutagenesis studies can contribute to the ALK4-CFC interaction at the protein-protein interface.

PED (Phosphoprotein Enriched in Diabetes) is an ubiquitously expressed 15 kDa cytosolic protein with recognized multiple functions. It has been demonstrated that PED has broad anti-apoptotic properties1,2 and, by altering insulin secretion, has a proven implication in diabetes3. In cultured cells and in transgenic mice, PED overexpression induces insulin-resistance and impairs glucose tolerance. Recent evidence indicates that increased interaction of PED with Phospholipase D1 (hPLD1) is a key event leading to these abnormalities in vivo and that this interaction is mediated by a C-terminal domain of PLD1 comprising residues 712–1070, D44. To further study this interaction, we prepared the recombinant proteins/sub-domains and after purification and preliminary characterization by CD, we measured the KDby SPR. Furthermore, following an approach of protein fragmentation and peptide fractionation we have been able to identify regions on PED that are involved in the...

The toxic properties of beta-amyloid protein, Abeta(1-42), the major component of senile plaques in Alzheimer's disease, depend on nucleation-dependent oligomerization and aggregation. In addition, Abeta(1-42) toxicity is favored by the presence of trace metals, which affect the secondary structure of the peptide. A peptide comprising 11 residues within Abeta(1-42) [Abeta(25-35)] aggregates and retains the neurotoxic activity of Abeta(1-42). We have used both Abeta(25-35) and its C-amidated or N-acetylated/C-amidated derivatives to investigate the role of copper(II) in modulating the conformation and aggregation state as well as the neurotoxic properties of amyloid peptides. Electrospray ionization mass spectrometry (ESI-MS) and electron paramagnetic resonance (EPR) measurements were performed to verify the formation of copper(II)/Abeta(25-35) complexes and to determine the coordination mode, respectively. Abeta(25-35) and its derivatives were analyzed by circular dichroism spectroscopy to assess their secondary structure, subjected to thioflavine-T (Th-T) binding assay to reveal beta-sheet structured aggregates formation, and imaged by scanning force microscopy. Toxicity was assessed on mature cultures of rat cortical neurons. We found that beta-sheet-structured species of Abeta(25-35) were neurotoxic, whereas the random-coil-structured derivatives were devoid of effect. Interestingly, copper promoted the random-coil/beta-sheet transition of Abeta(25-35), with ensuing peptide toxicity, but it induced the toxicity of the N-acetylated/C-amidated derivative without affecting peptide folding. Moreover, copper did not influence either the folding or the activity of the C-amidated Abeta(25-35), suggesting that blockade of the C-terminus of Abeta peptides might be sufficient to prevent Abeta toxicity.