Limited proteolysis in the investigation of beta2-microglobulin amyloidogenic and fibrillar states.

Amyloid fibrils of patients treated with regular haemodialysis essentially consists of β2-microglobulin (β2-m) and its truncated species ΔN6β2-m lacking six residues at the amino terminus. The truncated fragment shows a higher propensity to self-aggregate and constitutes an excellent candidate for the analysis of a protein in the amyloidogenic conformation. The surface topology and the conformational analysis of native β2-m and the truncated ΔN6β2-m species both in the soluble and in the fibrillar forms were investigated by the limited proteolysis/mass spectrometry strategy. The conformation in solution of a further truncated mutant ΔN3β2-m lacking three residues at the N-terminus was also examined. This approach appeared particularly suited to investigate the regions that are solvent-exposed, or flexible enough to be accessible to protein-protein interactions and to describe the conformation of transient intermediates. Moreover, proteolysis experiments can also be tailored to investigate amyloid fibrils by discriminating the protein regions constituting the unaccessible core of the fibrils and those still flexible and exposed to the solvent. Although native β2-m and ΔN3β2-m shared essentially the same conformation, significative structural differences exist between the native and the ΔN6β2-m proteins in solution with major differences located at the end moiety of strand V and subsequent loop with strand VI and at both the N- and C-termini of the proteins. On the contrary, an identical distribution of preferential proteolytic sites was observed in both proteins in the fibrillar state, which was nearly superimposible to that observed for the soluble form of ΔN6β2-m. These data revealed that synthetic fibrils essentially consists of an unaccessible core comprising residues 20-87 of the β2-m protein with exposed and flexible N- and C-terminal ends. Moreover, proteolytic cleavages observed in vitro at Lys 6 and Lys 19 reproduce specific cleavages that have to take place in vivo to generate the truncated forms of β2-m occurring in natural fibrils. On the basis of these results, a molecular mechanism for fibril formation has been propose

Evaluation of epigenetic and radiomodifying effects during radiotherapy treatments in zebrafish

Radiotherapy is still a long way from personalizing cancer treatment plans, and its effective-ness depends on the radiosensitivity of tumor cells. Indeed, therapies that are efficient and successful for some patients may be relatively ineffective for others. Based on this, radiobiological research is focusing on the ability of some reagents to make cancer cells more responsive to ionizing radiation, as well as to protect the surrounding healthy tissues from possible side effects. In this scenario, zebrafish emerged as an effective model system to test for radiation modifiers that can potentially be used for radiotherapeutic purposes in humans. The adoption of this experimental organism is fully justified and supported by the high similarity between fish and humans in both their genome sequences and the effects provoked in them by ionizing radiation. This review aims to provide the literature state of the art of zebrafish in vivo model for radiobiological studies, particularly focusing on the epigenetic and radiomodifying effects produced during fish embryos’ and larvae’s exposure to radiotherapy treatments

Neuronavigation and intraoperative imaging system in orbital tumor surgery: a review of recent literature

Orbit is a small complex anatomic space that contains important structures, ocular globe, extrinsic muscles, cranial nerves, blood vessels, fat, lacrimal gland. In presence of orbital tumors it is mandatory to use a surgical approach that allows to achieve an adequate surgical field while preserving neurological function. Neuronavigation is the set of computer-assisted technologies used to guide or "navigate” the edges of the tumor to allow the surgeon during resection or biopsy. This technology started with use of CT data to get some landmarks of human anatomy defined “targets” that could be readily used in surgery. Finally, the evolution of modern neuroimaging technologies such intraoperative CT and MRI boosted the surgery accuracy. In order to identify advantages and practical use of these technologies we performed a nonsystematic review of the current literature using the keywords “orbital tumor or orbital neoplasia or orbital mass or orbital lesion” and “neuronavigation or navigation” published in last 10 years. We evaluated 29 papers and we can conclude that navigation in orbital surgery helps to reduce surgical damage while at the same time, allowing a more radical tumor resection. CT and MRI scans are complementary in diagnosing and in intraoperative navigation allow the surgeon to avoid and preserve vital structures, particularly in a complex surgical procedure without real anatomical landmarks for intraoperative orientation. Future is going towards rapid changes and the integration with intraoperative procedures is carrying on to new technologies further our contemporary bounds

CysMap and CysJoin: Database and tools for protein disulphide localization

We have developed a computer program able to make user-customised databases derived from the public PIR non-redundant reference protein database. When the database of interest has been created, the user will generate the map of all the possible linear peptides containing one and two cysteines for each protein and combine them to calculate the mass of all the possible clusters of linear peptides linked by a disulphide bridge with a cysteine pair. It is also possible to create selected maps corresponding to peptides formed by the action of specific proteases. In this way, mass spectrometric data obtained from the hydrolysis of proteins of unknown sequence can be related to that contained in the database for quick disulphide assignment and protein identification. To confirm signal attribution, the program will also furnish the expected mass of cluster peptides after performing a cycle of Edman degradation. The utility of the program is discussed and examples of application are given. © 2005 Federation of European Biochemical Societies

Langmuir-Schaefer films of a polyaniline-gold nanoparticle composite material for applications in organic memristive devices

Langmuir-Shaefer films of a polyaniline-gold nanoparticle composite were fabricated and characterized. The thickness of each deposited monolayer, determined with AFM, was found to be about 0.8 nm. The film morphology was studied by SEM, revealing the presence of embedded spherical-shaped gold nanoparticles of about 5-10 nm in diameter. The fabricated films were used as the active channel of the organic memristor. Its electric characterisation has revealed new phenomena, such as an increased working voltage range and sigmoidal voltage current characteristics that were connected to the charge trappin

Design of a Fractional Order PI (FOPI) for the speed control of a high-performance electrical drive with induction motor

This paper describes the application of the Fractional Order PIs (FOPI) in the speed loop of a high performance induction motor electrical drive. In particular the speed tracking and load rejection capability of FOPI controller has been investigated and compared with both an integer-order PI and an IP both in simulation and experimentally with constant settling time. Illustrative study proves the simplicity and efficiency of the presented design method over integer controllers

Hb Vila Real [beta36(C2)Pro®His] in Italy: characterization of the amino acid substitution and the DNA mutation

A rare high oxygen affinity hemoglobin variant was identified in a 22-year-old male patient from Napoli (Naples, Italy) affected by erythrocytosis. A detailed structural characterization of the variant hemoglobin was carried out, both at the protein and DNA levels essentially by mass spectrometric procedures and allele-specific amplification techniques. The amino acid substitution was determined by liquid chromatography tandem mass spectrometric analysis of the tryptic digest as β36(C2)Pro → His; the corresponding DNA mutation was identified as C → A at the second position of codon 36 of the β chain (CCT → CAT). These variations identified the presence of Hb Vila Real, described only once before in a Portuguese woman. Haplotype analysis of DNA polymorphisms showed that the β-globin gene of Hb Vila Real was associated with haplotype I