Luciana Dente

• University of Pisa

Neuroprotective drug delivery to the posterior segment of the eye represents a major challenge to counteract vision loss. This work focuses on the development of a polymer-based nanocarrier, specifically designed for targeting the posterior eye. Polyacrylamide nanoparticles (ANPs) were synthesised and characterised, and their high binding efficienc...

The use of zebrafish embryos for personalized medicine has become increasingly popular. We present a co-clinical trial aiming to evaluate the use of zPDX (zebrafish Patient-Derived Xenografts) in predicting the response to chemotherapy regimens used for colorectal cancer patients. zPDXs are generated by xenografting tumor tissues in two days post-f...

It is increasingly evident the necessity of new predictive tools for the treatment of pancreatic ductal adenocarcinoma in a personalized manner. We present a co-clinical trial testing the predictiveness of zPDX (zebrafish patient-derived xenograft) for assessing if patients could benefit from a therapeutic strategy (ClinicalTrials.gov: XenoZ, NCT03...

Several nanomedicine-based platforms, including polymeric micelles, dendrimers, and liposomes, have been developed and explored for targeted delivery of therapeutics in cancer. These nanoparticles are capable of delivering selectively antineoplastic agents to the tumor, reduce the untoward toxicity, and improve the therapeutic effect. In the presen...

Stretch-growth has been defined as a process that extends axons via the application of mechanical forces. In the present paper, we used a protocol based on magnetic nanoparticles for labeling the entire axon tract of hippocampal neurons, and an external magnetic field gradient to generate a dragging force. We found that the application of forces be...

Animal “avatars” and co-clinical trials are being developed for possible use in personalized medicine in oncology. In a co-clinical trial, the cancer cells of the patient’s tumor are xenotransplanted into the animal avatar for drug efficacy studies, and the data collected in the animal trial are used to plan the best drug treatment in the patient t...

Animal “Avatars” and co-clinical trials represent an emerging concept for implementing schemes of personalized medicine in oncology. In a co-clinical trial, the cancer cells of the patient tumor are xenotransplanted in the animal Avatar for drug efficacy studies and data collected in the animal trial are used to plan the best drug treatment in the...

The 3-D spatial and mechanical features of nano-topography can create alternative environments, which influence cellular response. In this paper, murine fibroblast cells were grown on surfaces characterized by protruding nanotubes. Cells cultured on such nano-structured surface exhibit stronger cellular adhesion compared to control groups but, desp...

Glaucoma and other optic neuropathies are characterized by a loss of retinal ganglion cells (RGCs), a cell layer located in the posterior eye segment. Several preclinical studies demonstrate that neurotrophins (NTs) prevent RGC loss. However, NTs are rarely investigated in the clinic due to various issues, such as difficulties in reaching the retin...

Despite the higher rate of blindness due to population aging, minimally invasive and selective drug delivery to the eye still remains an open challenge, especially in the posterior segment. The retina, the retinal pigment epithelium (RPE) and the choroid are posterior segment cell layers, which may be affected by several diseases. In particular, da...

The only clinically approved alternative to autografts for treating large peripheral nerve injuries is the use of synthetic nerve guidance conduits (NGCs), which provide physical guidance to the regenerating stump and limit scar tissue infiltration at the injury site. Several lines of evidence suggest that a potential future strategy is to combine...

We validated the anticancer potential of a nanoformulation made by (+)-catechin, gelatin and carbon nanotubes in terms of inhibition of cancer cell proliferation, migration and associated neo-angiogenesis. Gelatin was selected to stabilize the catechin without compromising its anti-oxidant potential and the carbon nanotubes were used to increase it...

Pdzrn3, a multidomain protein with E3-ubiquitin ligase activity, has been reported to play a role in myoblast and osteoblast differentiation and, more recently, in neuronal and endo-thelial cell development. The expression of the pdzrn3 gene is developmentally regulated in various vertebrate tissues, including muscular, neural and vascular system....

Background We recently identified pfdn6a and tcp1α (also known as cct-α) as genes coregulated by the transcription factor Rx1. The proteins encoded by these genes belong to two interacting complexes (Prefoldin and "chaperonin containing t-complex polypeptide 1"), which promote the folding of actin and tubulin and have more recently been reported to...

Pdzrn3, a multidomain protein with E3-ubiquitin ligase activity, has been reported to play a role in myoblast and osteoblast differentiation and, more recently, in neuronal and endothelial cell development. The expression of the pdzrn3 gene is developmentally regulated in various vertebrate tissues, including muscular, neural and vascular system. L...

All biomaterials initiate a tissue response when implanted in living tissues. Ultimately this reaction causes fibrous encapsulation and hence isolation of the material, leading to failure of the intended therapeutic effect of the implant. There has been extensive bioengineering research aimed at overcoming or delaying the onset of encapsulation. Na...

There is a growing body of evidence indicating the importance of physical stimuli for neuronal growth and development. Specifically, results from published experimental studies indicate that forces, when carefully controlled, can modulate neuronal regeneration. Here, we validate a non-invasive approach for physical guidance of nerve regeneration ba...

Unlabelled: Various in vivo biological models have been proposed for studying the interactions of nano-materials in biological systems. Unfortunately, the widely used small mammalian animal models (rodents) are costly and labor intensive and generate ethical issues and antagonism from the anti-vivisectionist movement. Recently, there has been incr...

Kidins220 (Kinase D interacting substrate of 220 kDa)/ARMS (Ankyrin Repeat-rich Membrane Spanning) is a conserved scaffold protein that acts as a downstream substrate for protein kinase D and mediates multiple receptor signalling pathways. Despite the dissecting of the function of this protein in mammals, using both in vitro and in vivo studies, a...

Various in-vivo biological models have been proposed for studying the interactions of nanomaterials with biological systems. Recently, there has been a significant increase in interest in the use of non-mammalian embryos, such as the frog Xenopus laevis as valid models for research in nanomedicine. In the present work, we demonstrate that X. laevis...

One of the most challenging efforts in drug delivery is the targeting of the eye. The eye structure and barriers render this organ poorly permeable to drugs. Quite recently the entrance of nanoscience in ocular drug delivery has improved the penetration and half-life of drugs, especially in the anterior eye chamber, while targeting the posterior ch...

We report the identification of a novel partner of Kidins220/ARMS (Kinase D-interacting substrate of 220 kDa/Ankyrin Repeat-rich Membrane Spanning) an adaptor of neurotrophin receptors playing crucial roles during neurogenesis. Screening a phage display library of brain cDNA products we found that D. rerio Pdzrn3, a protein containing RING-finger a...

Pdzrn3, a member of the PDZRN/SEMCAP/LNX protein family containing a RING finger and two PDZ domains, has been implicated in myoblast and osteoblast differentiation. However, its spatio-temporal expression pattern during embryonic development has not been defined. Here, we describe the cloning and expression pattern of pdzrn3 during zebrafish devel...

Here we report the dissection of a tripartite complex formed by CIPP (channel-interacting PDZ protein), IRSp53 (insulin receptor tyrosine kinase substrate protein) and Cypin (cytosolic PSD-95 interactor) in cultured cells. The three proteins are expressed in similar neuronal districts, where CIPP binds to different membrane channels and receptors,...

Neuronal CIPP (channel-interacting PDZ protein) is a multivalent PDZ protein that interacts with specific channels and receptors highly expressed in the brain. It is composed of four PDZ domains that behave as a scaffold to clusterize functionally connected proteins. In the present study, we selected a set of potential CIPP interactors that are inv...

PDZ (acronym of the synapse-associated protein PSD-95/SAP90, the septate junction protein Discs-large, and the tight junction protein ZO-1) domains are abundant small globular protein interaction domains that mainly recognize the carboxyl termini of their target proteins. Detailed knowledge on PDZ domain binding specificity is a prerequisite for un...

Dystroglycan (DG) is a transmembrane receptor linking the extracellular matrix to the internal cytoskeleton. Its structural function has been mainly characterized in muscle fibers, but DG plays signaling and developmental roles also in different tissues and cell types. We have investigated the effects of dystroglycan depletion during eye developmen...

PDZ domains are protein-protein interaction modules that are crucial for the assembly of structural and signalling complexes. They specifically bind to short C-terminal peptides and occasionally to internal sequences that structurally resemble such peptide termini. The binding of PDZ domains is dominated by the residues at the P(0) and P(-2) positi...

Dystroglycan is a transmembrane receptor protein that provides a structural linkage between extracellular matrix components and cytoskeletal proteins. It was originally characterized as a member of dystrophin associated protein complex in muscle but, unlike other proteins of this complex, mutations in the dystroglycan gene have not been implicated...

Protein-phosphorylation is a post-translation modification that alters the binding or the enzymatic properties of the modified proteins. In particular, phosphorylation of tyrosine residues is a crucial event in protein-protein interactions occurring during intracellular signal transduction. Several approaches have been used to study these interacti...

We have designed a repertoire of 107 different SH3 domains by grafting the residues that are represented in the binding surfaces of natural SH3 domains onto the scaffold of the human Abl-SH3 domain. This phage-displayed library was screened by affinity selection for SH3 domains that bind to the synthetic peptides, APTYPPPLPP and LSSRPLPTLPSP, which...

PDZ domains are protein-protein interaction modules that typically bind to short peptide sequences at the carboxyl terminus of target proteins. Proteins containing multiple PDZ domains often bind to different trans-membrane and intracellular proteins, playing a central role as organizers of multimeric complexes. To characterize the rules underlying...

One of the goals of functional genomics is the description of reliable and complete protein interaction networks. To facilitate ligand discovery from complex protein mixtures, we have developed an improved approach that is affected by a negligible fraction of false positives. We have combined a novel technique based on the display of cDNA libraries...

Filamentous phage has been extensively used to implement various aspects of phage display technology. The success of these organisms as vectors to present foreign peptides and to link them to their coding sequences is a consequence of their structural and biological characteristics. Some of these properties, however, represent a limitation when one...

Several approaches, some of which are described in this issue, have been proposed to assemble a complete protein interaction map. These are often based on high throughput methods that explore the ability of each gene product to bind any other element of the proteome of the organism. Here we propose that a large number of interactions can be inferre...

The carboxy-terminal region of utrophin, like the homologous proteins dystrophin, Drp2 and dystrobrevins, contains structural domains frequently involved in protein-protein interaction. These domains (WW, EF hands, ZZ and H1-H2) mediate recognition and binding to a multicomponent complex of proteins, also known as dystrophin-associated proteins (DA...

The proline-rich domain of synaptojanin 1, a synaptic protein with phosphatidylinositol phosphatase activity, binds to amphiphysin and to a family of recently discovered proteins known as the SH3p4/8/13, the SH3-GL, or the endophilin family. These interactions are mediated by SH3 domains and are believed to play a regulatory role in synaptic vesicl...

The synapsins are a family of major neuron-specific synaptic vesicle-associated phosphoproteins which play important roles in synaptic function. In an effort to identify molecular tools which can be used to perturb the activity of the synapsins in in vitro as well as in vivo experiments, we have localized the epitopes of a panel of monoclonal antib...

Tyrosine phosphorylation and protein recognition, mediated by phosphotyrosine containing peptides, play an important role in determining the specific response of a cell, when stimulated by external signals. We have used peptide repertoires displayed by filamentous phage as a tool to study the substrate specificity of the protein tyrosine kinase (PT...

Shc proteins are targets of activated tyrosine kinases and have been implicated in the transmission of activation signals to Ras. Upon phosphorylation, Shc proteins form stable complexes with cellular tyrosine-phosphorylated proteins and with the Grb2 adaptor protein. Two Shc isoforms of 52 and 46 kDa have been characterized. They share a C-termina...

We generated six hybridoma cell lines that secrete monoclonal antibodies (mAb) which specifically bind filamentous phage coat proteins. Two of these mAb recognise epitopes that include the N terminus of the coat protein III (pIII), while two others are specific for the N terminus of the major coat protein VIII (pVIII). These mAb are valuable tools...

The occurrence and the glycosylation of human alpha 1-acid glycoprotein (AGP) was studied in two classes of transgenic mice expressing either the A, B and B' genes (ABB'-mice) or only the A gene of human AGP (A-mice). The glycosylation of the human AGP molecules in the transgenic mouse sera was compared with the glycosylation of mouse AGP in the sa...

The relative occurrence of genetic variants of human alpha 1-acid glycoprotein (AGP) in relation to changes in glycosylation was studied in sera of patients with burn injury, media of cytokine-treated primary cultures of human hepatocytes and Hep 3B cells, and sera of transgenic mice expressing the human AGP-A gene. It is concluded (i) that the gly...

Sera from transgenic mice (TM) carrying human genes of alpha 1-acid glycoprotein (orosomucoid or ORM) have been analyzed by isoelectrofocusing and subsequent immunoblotting with antihuman ORM antibodies. With this technique it is possible to reveal selectively the human protein secreted in the TM sera. Orosomucoid bands present in TM sera have been...

The phenomenon of acute phase (AP) response can be reproduced in vitro using cultured cells of hepatic origin by stimulation with the crude supernatant of activated monocytes (MoCM). Several monocyte-derived factors have been identified which might be responsible, alone or in combination, for the induction of AP response, but recently the attention...

The phenomenon of acute phase (AP) response can be reproduced in vitro using cultured cells of hepatic origin by stimulation with the crude supernatant of activated monocytes (MoCM). Several monocyte-derived factors have been identified which might be responsible, alone or in combination, for the induction of AP response, but recently the attention...

The human genome contains three alpha 1-glycoprotein genes (AGP-A, AGP-B, and AGP-B') encoding for slightly different forms of the protein. The major component of human alpha 1-acid glycoprotein found in plasma is coded by AGP-A, which is expressed in liver and in hepatoma cell lines and is induced by inflammatory stimuli. We have studied the regul...

alpha 1-acid glycoprotein (alpha AGP) is a well-characterized human plasma protein. Its structural properties have been studied for many years but little is known about its function. Amino acid sequence analysis of purified human alpha AGP from plasma pooled from several individuals showed considerable heterogeneity. We have cloned the genomic DNA...

The G domain of elongation factor Tu (EF-Tu), representing the N-terminal half of the factor according to its three-dimensional model traced at high resolution, has been isolated by genetic manipulation of tufA and purified to homogeneity. The G domain, whose primary structure shares homology with the eukaryotic protein p21, is capable of supportin...

In this chapter we describe a series of plasmids, the 'pEMBL family', that have the property of being encapsidated as single-stranded DNA upon superinfection with phage F1. They can be used for DNA sequencing and for all the other uses that require single-stranded DNA, but, in the absence of F1 helper phage, they replicate as any other double-stran...

The previously unassigned gene coding for the anti-coagulatory protein C has been mapped on chromosome 2 using a cDNA probe and genomic blots from a human-hamster somatic cell hybrid panel. The assignments of the genes coding for the coagulation factor X to chromosome 13, and for alpha 1-acid glycoprotein to chromosome 9 have been confirmed using a...

We have cloned the human alpha 1-antitrypsin (alpha 1-AT) gene and identified the promoter and the transcription initiation point. The cloned gene, following transfection, is expressed in a cell-specific manner, being transcribed in a human hepatoma cell line (Hep3B) but not in HeLa cells. We show that the 5' flanking region of the alpha 1-AT gene...

We have determined the sequence coding for human α1-acid glycoprotein from two independently isolated cDNA clones and a genomic clone. The aminoacid sequences deduced from the three clones, deriving from three different individuals, are identical. Southern blot analysis on human DNA indicates that there are at least two genes coding for α1AGP. We p...

We have constructed a series of plasmids, the pEMBL family, characterized by the presence of 1) the bla gene as selectable marker, 2) a short segment coding for the β–peptide of β-galactosidase and containing a multiple cloning sites polylinker, 3) the intragenic region of phage Fl. pEMBL plasmids have the property of being encapsidated as single...

We have shown that the 34 bp internal control region of the somatic 5S RNA gene from Xenopus borealis can be split into two separable components. A maxigene carrying an insertion between nucleotide 71 and nucleotide 74 of the coding region is actively transcribed in the nucleus of X. laevis oocytes, giving rise to a maxitranscript with initiation a...

A human cDNA library was constructed using M13 derivative vectors. The simple and rapid procedures for sequencing single-stranded DNA by the dideoxy chain termination method allowed a screening of individual clones directly by DNA sequence analysis. Some of these clones were identified as coding for: serum albumin, alpha1-antitrypsin, retinol-bindi...

Pyruvate kinase (PK) has been purified from the red blood cells of two sisters who had suffered severe chronic non-spherocytic haemolytic anaemia since infancy, and of one patient who had haemolytic anaemia during pregnancy. The two sisters showed remarkable clinical improvement following splenectomy. The enzyme from their red cells was found to ex...

Eukaryotic tDNA promoters are composed of two essential regions contained within the coding sequence (Box A and Box B). Due to the highly conserved structure of prokaryotic and eukaryotic tRNA, most prokaryotic tRNA genes are expected to be active templates in eukaryotic transcriptional systems. In this paper we show that Escherichia coli tDNATyr i...