Annalisa Lamberti

The translation elongation factor 1A in tumorigenesis, signal transduction and apoptosis: Review article

Summary.An increasing number of evidences suggest the involvement of the eukaryotic elongation factor 1A, a core component of the protein synthesis machinery, at the onset of cell transformation. In fact, eEF1A is shown to be up-regulated in cell death; moreover, it seems to be involved in the regulation of ubiquitin-mediated protein degradation. In addition, eEF1A undergoes several post-translational modifications, mainly phosphorylation and methylation, that generally influence the activity of the protein. This article summarizes the present knowledges on the several extra-translational roles of eEF1A also in order to understand as the protein synthesis regulatory mechanisms could offer tools for cancer intervention.

Marine Diatoms as Optical Biosensors

We have chemically modified the frustules of the marine diatom Coscinodiscus concinnus Wm. Smith to properly bind a highly selective bioprobe such as an antibody. By measuring the changes in the photoluminescence emission of diatoms frustules, we have monitored the molecular recognition event between the antibody and its ligand: the dissociation constant estimated is of the same order of that measured by standard Biacore. The nanostructured silica frustules, a low-cost and natural available material, have shown high sensitivity, equal to 1.2+/-0.2 nm microM(-1), and a detection limit of 100 nM, and thus are quite ideal candidates for lab-on-particle applications.

Optical sensors for vapors, liquids, and biological molecules based on porous silicon technology

The sensing of chemicals and biochemical molecules using several porous silicon optical microsensors, based both on single-layer interferometers and resonant-cavity-enhanced microstructures, is reported. The operation of both families of sensors is based on the variation of the average refractive index of the porous silicon region, due to the interaction with chemical substances either in vapor or liquid state, which results in marked shifts of the device reflectivity spectra. The well established single-layer configuration has been used to test a new chemical approach based on Si-C bonds for covalent immobilization of biological molecules, as probe, in a stable way on the porous silicon surface. Preliminary results on complementary oligonucleotide recognition, based on this technique, are also presented and discussed. Porous silicon optical microcavities, based on multilayered resonating structures, have been used to detect chemical substances and, in particular, flammable and toxic organic solvents, and some hydrocarbons. The results put in evidence the high sensitivity, the reusability, and the low response time of the resonant-cavity-enhanced sensing technique. The possibility of operating at room temperature, of remote interrogation, and the absence of electrical contacts are further advantages characterizing the sensing technique.

Fabrication and characterization of a porous silicon based microarray for label-free optical monitoring of biomolecular interactions

We have fabricated a microarray of porous silicon Bragg reflectors on a crystalline silicon substrate using a technological process based on standard photolithography and electrochemical anodization of the silicon. The array density is of 170 elements/cm2 and each element has a diameter of 200 μm. The porous silicon structures have been used as platform to immobilize an amino terminated DNA single strand probe. All fabrication steps have been monitored by spectroscopic reflectometry, optical and electron microscopy, and Fourier transform infrared spectroscopy. A label-free detection method has been employed to investigate the hybridization between micromolar DNA probe and its complementary target. Due to fast and low cost production, good reproducibility, and high quality optical features, this platform could be adopted also for other different microarray applications such as proteomics and medical diagnostics.

Porous Silicon Based Resonant Mirrors for Biochemical Sensing

We report on our preliminary results in the realization and characterization of a porous silicon (PSi) resonant mirror (RM) for optical biosensing. We have numerically and experimentally studied the coupling between the electromagnetic field, totally reflected at the base of a high refractive index prism, and the optical modes of a PSi waveguide. This configuration is very sensitive to changes in the refractive index and/or in thickness of the sensor surface. Due to the high specific area of the PSi waveguide, very low DNA concentrations can be detected confirming that the RM could be a very sensitive and label-free optical biosensor.

Porous silicon-based optical biochips

In this paper, we present our work on an optical biosensor for the detection of the interaction between a DNA single strand and its complementary oligonucleotide, based on the porous silicon (PSi) microtechnology. The crucial point in this sensing device is how to make a stable and repeatable link between the DNA probe and the PSi surface. We have experimentally compared some functionalization processes which modify the PSi surface in order to covalently fix the DNA probe on it: a pure chemical passivation procedure, a photochemical functionalization process, and a chemical modification during the electrochemical etching of the PSi. We have quantitatively measured the efficiency of the chemical bond between the DNA and the porous silicon surface using Fourier transform infrared spectroscopy (FT-IR) and light induced photoluminescence emission. From the results and for its intrinsic simplicity, photochemical passivation seems to be the most promising method. The interaction between a label-free 50 µM DNA probe with complementary and non-complementary oligonucleotides sequences has been also successfully monitored by means of optical reflectivity measurements.

Improved procedure for protein binder analysis in mural painting by LC-ESI/Q-q-TOF mass spectrometry: detection of different milk species by casein proteotypic peptides

Diagnostic techniques applied to the field of cultural heritage represent a very important aspect of scientific investigation. Recently, proteomic approaches based on mass spectrometry coupled with traditional spectroscopic methods have been used for painting analysis, generating promising results for binder’s protein identification. In the present work, an improved procedure based on LC-ESI/Q-q-TOF tandem mass spectrometry for the identification of protein binders has been developed for the molecular characterization of samples from an early-twentieth-century mural painting from the St. Dimitar Cathedral in Vidin, Bulgaria. The proteomic investigation has led to the identification of both egg white and egg yolk proteins, according to traditional old recipes for tempera paintings. In addition, beyond the egg components, the presence of caseins was also revealed, thus suggesting the use of milk as binding medium, fixative or stabilising agent. Furthermore, for the first time, the capability to discriminate the milk origin on the basis of alpha casein proteotypic peptides is reported, that are diagnostic for a given species, thus opening interesting perspectives in art and archaeological fields.

A microfluidics assisted porous silicon array for optical label-free biochemical sensing.

A porous silicon (PSi) based microarray has been integrated with a microfluidic system, as a proof of concept device for the optical monitoring of selective label-free DNA-DNA interaction. A 4 × 4 square matrix of PSi one dimensional photonic crystals, each one of 200 μm diameter and spaced by 600 μm, has been sealed by a polydimethylsiloxane (PDMS) channels circuit. The PSi optical microarray elements have been functionalized by DNA single strands after sealing: the microfluidic circuit allows to reduce significantly the biologicals and chemicals consumption, and also the incubation time with respect to a not integrated device. Theoretical calculations, based on finite element method, taking into account molecular interactions, are in good agreement with the experimental results, and the developed numerical model can be used for device optimization. The functionalization process and the interaction between DNA probe and target has been monitored by spectroscopic reflectometry for each PSi element in the microchannels.

Analysis of interaction partners for eukaryotic translation elongation factor 1A M-domain by functional proteomics

The eukaryotic translation elongation factor 1A (eEF1A), besides to its canonical role in protein synthesis, is also involved in several other cellular processes, depending on changes in cellular location, cell type, concentration of ligands, substrates or cofactors. Therefore eEF1A is a moonlighting protein that participates to a network of molecular interactions involving its structural domains. Since the identification of novel protein-protein interactions represents important tasks in post-genomic era, the interactome of eEF1A1 M-domain was investigated by using a proteomic approach. To this purpose, the eEF1A1 M-domain was fused with glutathione-S-transferase (GST) and Strep-tag (ST) at its N- and C-terminal, respectively. The recombinant protein (GST-M-ST) was purified and incubated with a mouse embryo lysate by applying an affinity chromatography strategy. The interacting proteins were separated by SDS-PAGE and identified by peptide mass fingerprinting using MALDI-TOF mass spectrometry. Besides the known partners, the pool of interacting proteins contained sorbin, a polypeptide of 153 amino acids present in SH3 domain-containing adaptor proteins, such as SORBS2. This interaction was also assessed by Western blot on immunoprecipitate from mouse embryo or H1355 cell lysates with anti-eEF1A or anti-SORBS2 antibodies and on eEF1A1-His pull-down from H1355 cell lysate with antibody anti-SORBS2. Furthermore, the interaction between eEF1A and SORBS2 was also confirmed by confocal microscopy and FRET analysis. Interestingly, a co-localization of SORBS2 and eEF1A was evidenced at level of plasma membrane, thus suggesting the involvement of eEF1A1 in novel key signal transduction complexes.

Bioengineered Silicon Diatoms: Adding Photonic Features to a Nanostructured Semiconductive Material for Biomolecular Sensing

Native diatoms made of amorphous silica are first converted into silicon structures via magnesiothermic process, preserving the original shape: electron force microscopy analysis performed on silicon-converted diatoms demonstrates their semiconductor behavior. Wet surface chemical treatments are then performed in order to enhance the photoluminescence emission from the resulting silicon diatoms and, at the same time, to allow the immobilization of biological probes, namely proteins and antibodies, via silanization. We demonstrate that light emission from semiconductive silicon diatoms can be used for antibody-antigen recognition, endorsing this material as optoelectronic transducer.