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The brain is always confronted with the dilemma of the protection from noxious substances from the blood and the delivery of vital metabolites. Endothelial cells, forming together with other cells the blood-brain barrier (BBB), are known as the “gatekeepers” of this trafficking. Recent applications in nanomedicine focus on nanoparticles and liposomes as they are promising tools for site-specific delivery of drugs and diagnostic agents, through the possibility to functionalize their surface with target-specific ligands. Treatment options for Alzheimer’s disease (AD) are limited because of the inability of drugs to cross the BBB. We disclosed that intraperitoneal administration of liposomes functionalized with phosphatidic acid and an ApoE-derived peptide (mApoE-PA-LIP) reduces brain beta-amyloid (Aβ) burden and ameliorates impaired memory in AD mice. Among the different administration routes, pulmonary delivery is a field of increasing interest not only for the local treatment of airway diseases but also for the systemic administration. Here we prove that mApoE-PA-LIP are able to cross the pulmonary epithelium in vitro and reach the brain following in vivo intratracheal instillations. Lung administration of mApoE-PA-LIP to AD mice significantly decreases total brain Aβ (–60%; p < 0.05) compared to untreated mice. These results suggest that pulmonary administration could be exploited for brain delivery of nanoparticles designed for AD therapy.

Background: Traditional quantitative structure – property / activity relationships (QSPRs/QSARs) are based on representation of molecular structure by molecular graph or simplified molecular input-line entry system (SMILES). It is attractive idea to develop predictive models for large molecules in general and for peptides in particular. However, the representation of these molecules by molecular graph or SMILES is problematic owing to large size of these molecules. A possible alternative of SMILES is representation of peptides via sequence of abbreviations of amino acids. Method: Models for hemolysis and cytotoxicity of peptides are suggested. These models are based on representation of the peptides by sequences of amino acids. Correlation weights, which are calculated for each amino acid using the Monte Carlo method are basis for quantitative sequence - activity relationships (QSAR) for antimicrobial peptides. The correlation weights are basis for optimal descriptors, which are correlated with experimental data for hemolysis and cytotoxicity. The basic hypothesis is that if optimal descriptors correlated with endpoints of peptides for the training set, also they should correlate with the endpoints for validation set. Results: Checking up of correlations between the above-mentioned descriptors and antimicrobial activity of peptides (cytotoxicity or hemolysis) has shown that these models have good predictive potential. Conclusion: Suggested approach can be used as a tool to develop predictive models of biological activity of peptides as a mathematical function of sequences of amino acids.

In the search of new drug delivery carriers for the brain, self-assembled nanoparticles (NP) were prepared from poly(N,N-dimethylacrylamide)-block-polystyrene polymer. NP displayed biocompatibility on cultured endothelial cells, macrophages and differentiated SH-SY5Y neuronal-like cells. The surface-functionalization of NP with a modified fragment of human Apolipoprotein E (mApoE) enhanced the uptake of NP by cultured human brain capillary endothelial cells, as assessed by confocal microscopy, and their permeability through a Transwell Blood Brain Barrier model made with the same cells, as assessed by fluorescence. Finally, mApoE-NP embedding doxorubicin displayed an enhanced release of drug at low pH, suggesting the potential use of these NP for the treatment of brain tumors. In the search of novel drug carriers for brain drug delivery, poly(N,N-dimethylacrylamide)-b-polystyrene nanoparticles (NP) have been functionalized with a modified fragment of Apolipoprotein E (mApoE). These NP show to be uptaken and to overcome a Blood-Brain Barrier (BBB) in vitro cellular model. The cytotoxicity of NP is also investigated on different cell lines, showing high biocompatibility.

The reduction of amyloid-beta peptides (Ab) is considered a primary therapeutic target to minimize the neurodegeneration in Alzheimer's disease (AD), a pathology characterized by accumulation of Ab in the brain. A possible strategy to reduce Ab levels is to exploit the peripheral-sink effect that is based on the removal of plasma Ab, drawing out soluble Ab from the brain. Objectives. The aim of the present study was to investigate the potential effect of multi-functional liposomes binding Ab on the peptide exchange across an in vitro model of the blood-brain barrier (BBB). Methods. Liposomes were prepared by extrusion procedure and bi-functionalized with phosphatidic acid and with a modified ApoE-derived peptide (mApoE-PA-LIP) for Ab binding. An in vitro BBB model made of human brain endothelial cells (hCMEC/D3) was set up on a transwell system and soluble small Ab42 assemblies were added to the basolateral compartment (brain side). The Ab translocation across the BBB was evaluated both alone or with mApoE-PA-LIP present in the apical compartment (blood side). Also the permeability of liposomes from basolateral-to-apical compartment in the presence of Ab was analyzed. Results. The presence of mApoE-PA-LIP strongly enhanced (5-fold) the basolateral-to-apical transcytosis of Ab across the BBB model, compared to the peptide alone, in a lipid dosedependent manner. Moreover, the transcytosis of Ab was due to a peripheral-sink effect since mApoE-PA-LIP were not able to transport the peptide across the cellular monolayer. Conclusions. This study provide a rationale for the use of Ab-binding particles as a potential therapeutic strategy for AD.

Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by deposition of the β-amyloid (Ab) peptide in the brain. Although many Ab-centric therapies have been attempted, they all failed and no efficacious therapy is available yet. Objectives. Main objectives of this study were to investigate the efficacy of multifunctional liposomes (Lip), designed to target the brain, to 1. promote disaggregation of brain Ab assemblies in AD mouse brain, 2. reduce brain Ab burden and 3. restore mouse memory, at a post-symptomatic stage. 4. Arrest pathology progression at a presymptomatic stage. Methods. Functionalized Lip with a peptide derived from apolipoprotein-E receptorbinding domain (mApoE) for blood-brain barrier targeting, and with phosphatidic acid (PA) for Ab binding (mApoE-PA-Lip) were administered (I.P.) for 3 weeks in post- and 4 months in pre-symptomatic APP/PS1 mice subsequently tested in the novel object recognition memory test (NORT). At the end mouse brains were collected and analyzed through histology and biochemistry for Ab deposition. Results. Administration of mApoE-PA-Lip decreased total insoluble brain Ab1-42, total plaque area and Ab oligomers. Plaque reduction was confirmed by PET imaging with [11C]-PIB. The reduction of brain Ab was associated with its increase in liver and spleen. Treatment also restored mouse impaired memory to normal at post-symptomatic ages and prevented its loss when administered at pre-symptomatic ages. Conclusions. These data suggest that bi-functionalized Lip destabilize brain Ab aggregates and promote peptide removal from the brain and its peripheral clearance. This innovative therapeutic approach can be considered as a new candidate for AD treatment.

We report the near full length genome sequence, of an hepatitis C virus isolate from a man originating from Democratic Republic of Congo, whose genotype could not be determined by the routinely used sequence technique.The near-complete genome sequence of this variant BAK1 was obtained by the association of two next generation sequencing technologies.Evolutionary analysis indicates that this isolate BAK1 could be the first reported strain belonging to a new HCV-7b subtype. This new subtype has been incorrectly identified as genotype 2 by Versant HCV Genotype 2.0 assay (LiPA).The requirement of three independent isolates has been filled and a new subtype can be assigned. More examples of HCV-7 are required to better understand its origin, its pathogenicity and its relationship with genotype 2.

The role of first-stage β–amyloid aggregation in the development of the Alzheimer disease, is widely accepted but still unclear. Intimate interaction with the cell membrane is invoked. We designed Neutron Reflectometry experiments to reveal the existence and extent of the interaction between β–amyloid (Aβ) peptides and a lone customized biomimetic membrane, and their dependence on the aggregation state of the peptide. The membrane, asymmetrically containing phospholipids, GM1 and cholesterol in biosimilar proportion, is a model for a raft, a putative site for amyloid-cell membrane interaction. We found that the structured-oligomer of Aβ(1-42), its most acknowledged membrane-active state, is embedded as such into the external leaflet of the membrane. Conversely, the Aβ(1-42) unstructured early-oligomers deeply penetrate the membrane, likely mimicking the interaction at neuronal cell surfaces, when the Aβ(1-42) is cleaved from APP protein and the membrane constitutes a template for its further structural evolution. Moreover, the smaller Aβ(1-6) fragment, the N-terminal portion of Aβ, was also used. Aβ N-terminal is usually considered as involved in oligomer stabilization but not in the peptide-membrane interaction. Instead, it was seen to remove lipids from the bilayer, thus suggesting its role, once in the whole peptide, in membrane leakage, favouring peptide recruitment.

This thesis is an account of a curriculum reform initiative that took place in 2005 at the Pathways College of Australia (PCA) [a pseudonym]. It is an investigation of an innovative collaborative educational development project in an Australian higher education pathway institution. The research highlights the neglect of the professional development of casually employed teachers and makes contributions to the literatures of educational development, curriculum and collaboration. It suggests ways to improve quality in the current higher education context through a process of action research enquiry and organisational change In recent times the higher education landscape in Australia has transformed with growing numbers of casual and part-time teachers, many more international students and an increasing focus on quality assurance. This changing context has led to the emergence of a number of private institutions providing an alternative entry pathway to tertiary study for students who do not meet standard university entrance requirements. The story of PCA and its growth during this time comes out of an increasing focus on quality and accountability underpinning the funding changes to, and the internationalisation of, higher education. This study presents a curriculum development framework which engages casually employed teachers and supports curriculum reform. It addresses a need to ensure quality in the teaching and learning at PCA by developing an integrated curriculum. The framework allows for the professional development of casualised teaching staff in a pathways higher education institution and encourages a critical reflection on the process through action research. An exploration of the usefulness of communities of practice theory for examining the workings of this group-based educational development process frames the data analysis. The research contributes to the literature by analysing how the participants engaged in the project cycles and illuminates the different ways in which they were working. Insights into curriculum reform are given through building collaboration under adverse conditions. The discussion adds a new dimension to communities of practice theory as it does not account for the important set of tensions found in the data. It furthers our understanding of its application in an environment with mostly casually employed teachers. The story about this research reveals the complexities in the relationships between the researcher, the participants and PCA and shows a successful collaboration can be achieved under challenging employment conditions.

J.BIOL.CHEM.

Veri toplama teknolojisinin hızlı gelişimi ve süreçlerin bilgisayarlar aracılığıyla izlenmesi, birden fazla kalite bileşeninin veya süreç değişkeninin eş zamanlı kontrolüne olan ilgiyi arttırmıştır. Çok değişkenli istatistiksel süreç kontrol yöntemleri olarak adlandırılan bu tekniklerin en önemli özellikleri değişkenler arasındaki ilişkileri de göz önünde bulundurarak süreci bir bütün olarak incelemeleridir. Hotelling T2, çok değişkenli süreçlerin incelenmesinde en yaygın olarak kullanılan kontrol diyagramı istatistiklerinden biridir. Çalışmamızda cam takviyeli plastik borular ve bağlantı manşonları üreten bir endüstri işletmesinde sürekli elyaf sarma sürecine çok değişkenli istatistiksel kalite kontrolü uygulanması hedeflenmiştir. Özellikle Hotelling T2 istatistiği üzerine kurduğumuz çalışma QualStat yazılım programı aracılığıyla gerçekleştirilmiştir. İstanbul Ticaret Üniversitesi

We report the cloning of the AOH1 and AOH2 genes, which encode two novel mammalian molybdo-flavoproteins. We have purified the AOH1 protein to homogeneity in its catalytically active form from mouse liver. Twenty tryptic peptides, identified or directly sequenced by mass spectrometry, confirm the primary structure of the polypeptide deduced from the AOH1 gene. The enzyme contains one molecule of FAD, one atom of molybdenum, and four atoms of iron per subunit and shows spectroscopic features similar to those of the prototypic molybdo-flavoprotein xanthine oxidoreductase. The AOH1 and AOH2 genes are 98 and 60 kilobases long, respectively, and consist of 35 coding exons. The AOH1 gene has the potential to transcribe an extra leader non-coding exon, which is located downstream of exon 26, and is transcribed in the opposite orientation relative to all the other exons. AOH1 and AOH2 map to chromosome 1 in close proximity to each other and to the aldehyde oxidase gene, forming a molybdo-flavoenzyme gene cluster. Conservation in the position of exon/intron junctions among the mouse AOH1, AOH2, aldehyde oxidase, and xanthine oxidoreductase loci indicates that these genes are derived from the duplication of an ancestral precursor.

The addition of acute-phase apolipoprotein serum amyloid A (SAA) to cultured aortic smooth-muscle cells caused a decrease in the incorporation of [(14)C]acetate into lipids. Optimal inhibition of lipid biosynthesis was achieved with 2 microM SAA, and the effect was maintained for up to 1 week when SAA was included in the culture medium. Lipid extracts were subjected to TLC and it was determined that the SAA-induced decrease in [(14)C]acetate incorporation into lipids was attributable to decreases in cholesterol, phospholipid and triglyceride levels. The accumulated mass of cholesterol and phospholipid in SAA-treated cultures was significantly less than that of controls, with no change in the accumulated protein. Moreover, SAA had no effect on either protein synthesis or DNA synthesis, suggesting that SAA specifically alters lipid synthesis. By using a peptide corresponding to the cholesterol-binding domain of acute-phase SAA (amino acids 1-18), it was shown that this region of the molecule was as effective as the full-length protein in decreasing lipid synthesis and the accumulation of cholesterol and phospholipid. The implications of these findings for atherosclerosis and Alzheimer's disease are discussed.

International audience; This paper reports the occurrence of a spongiform encephalopathy (SE) in young adult monkeys housed in a zoological park. Three rhesus monkeys (Macaca mulatta) acquired from the same zoo and maintained on feed containing animal protein, developed a progressive neurological disorder with behavioural abnormalities and physical deterioration and died at the age of 10-year-old. Neuropathological examination of one of these animals revealed a spongiform encephalopathy similar to that observed in monkeys following experimental transmission of Creutzfeldt-Jakob disease (CJD). In particular, several brain regions exhibited vacuolation of nerve cell bodies and processes accompanied by astrogliosis. Immunohistochemical analysis showed prion protein (PrP) immunoreactivity at the periphery of vacuolated neurons. The spontaneous occurrence of a SE in these young monkeys might be related to consumption of protein of animal origin.

Following the report of an increased mortality among patients with amyotrophic lateral sclerosis given high daily doses of branched-chain aminoacids, we assessed the plasma concentrations of large neutral aminoacids and glutamic acid and the large neutral aminoacid brain influx in 24 amyotrophic lateral sclerosis patients receiving placebo or branched-chain aminoacids (L-leucine 12 g, L-isoleucine 6 g, L-valine 6 g daily), in 15 untreated amyotrophic lateral sclerosis patients and in 15 healthy volunteers. The branched-chain aminoacid plasma concentrations increased three- to six-fold in the treated group compared to the patients receiving placebo or no treatment and to the healthy controls. Plasma glutamic acid concentrations in healthy volunteers were 51.59 +/- 7.53 nmol/ml while in the amyotrophic lateral sclerosis patients receiving no treatment, placebo or branched-chain aminoacids were 92.33 +/- 12.15 nmol/ml, 91.21 +/- 15.86 nmol/ml and 95.08 +/- 17.96 nmol/ml respectively. The glutamic acid concentration was significantly higher (P0.01) in amyotrophic lateral sclerosis patients than in healthy individuals. Plasma phenylalanine and tyrosine were lower in the amyotrophic lateral sclerosis patients than in healthy controls, regardless of treatment, whereas tryptophan levels were not significantly different. The branched-chain aminoacid brain influx of the treated group was 110-140% of that measured in the patients receiving placebo and in the healthy controls. The aromatic aminoacid brain influx was lower in the treated group than in the placebo group or healthy controls. An impairment of brain large neutral aminoacid availability might possible contribute to enhancing the progression of symptoms in patients with amyotrophic lateral sclerosis.

Macrophages (M phi diameter) from three mouse strains with genetically distinct M phi diameter deficits (C3H/HeJ, A/J, and P/J) were unable to develop high cytolytic and cytotoxic activity against tumor cells in vitro when exposed to agents (MAF and IFN-beta) that strongly increased the tumoricidal capacity of M phi diameter from nondefective C3H/HeN mice. Nevertheless, the tumoricidal deficits of M phi diameter from the defective strains did not affect their suppressive capacity on Con A-induced lymphoproliferation, nor their ability to react to IFN-beta by decreasing suppressive activity. In fact, natural suppressive activity and IFN-beta-induced changes in the suppression of M phi diameter from C3H/HeJ, A/J, and P/J mice were highly comparable to those of C3H/HeN M phi diameter, thus stressing the dissociation between the mechanisms governing M phi diameter suppression and M phi diameter tumoricidal activity. Analysis of the modulation by MAF and IFN-beta of M phi diameter ability to release the oxygen metabolites O2- and H2O2, molecules possibly involved in the effector mechanism of both M phi diameter cytotoxicity and suppression, revealed a close correlation with the patterns of suppressive activity in both nondefective and defective strains. In contrast, no correlation between the production of oxygen-reactive species and M phi diameter tumoricidal activity was observed. The ability of MAF- and IFN-beta-treated M phi diameter to produce PGE, a molecule of major importance in M phi diameter-mediated suppression and possibly involved also in the regulation of M phi diameter tumoricidal activity, again paralleled M phi diameter suppressive capacity. Thus, the mechanisms controlling M phi diameter antitumor activity appeared to be clearly distinct from those involved in M phi diameter suppression.