One-step colloidal synthesis of biocompatible water-soluble ZnS quantum dot/chitosan nanoconjugates

Quantum dots (QDs) are luminescent semiconductor nanocrystals with great prospective for use in biomedical and environmental applications. Nonetheless, eliminating the potential cytotoxicity of the QDs made with heavy metals is still a challenge facing the research community. Thus, the aim of this work was to develop a novel facile route for synthesising biocompatible QDs employing carbohydrate ligands in aqueous colloidal chemistry with optical properties tuned by pH. The synthesis of ZnS QDs capped by chitosan was performed using a single-step aqueous colloidal process at room temperature. The nanobioconjugates were extensively characterised by several techniques, and the results demonstrated that the average size of ZnS nanocrystals and their fluorescent properties were influenced by the pH during the synthesis. Hence, novel 'cadmium-free’ biofunctionalised systems based on ZnS QDs capped by chitosan were successfully developed exhibiting luminescent activity that may be used in a large number of possible applications, such as probes in biology, medicine and pharmacy

Functionalized chitosan derivatives as nonviral vectors: Physicochemical properties of acylated N,N,N-trimethyl chitosan/oligonucleotide nanopolyplexes

Cationic polymers have recently attracted attention due to their proven potential for nonviral gene delivery. In this study, we report novel biocompatible nanocomplexes produced using chemically functionalized N,N,N-trimethyl chitosan (TMC) with different N-acyl chain lengths (C 5 -C 18 ) associated with single-stranded oligonucleotides. The TMC derivatives were synthesized by covalent coupling reactions of quaternized chitosan with n-pentanoic (C 5 ), n-decanoic (C 10 ), and n-octadecanoic (C 18 ) fatty acids, which were extensively characterized by Fourier transform-infrared spectroscopy (FT-IR) and proton nuclear magnetic resonance ( 1 H NMR). These N-acylated TMC derivatives (TMC n ) were used as cationic polymeric matrices for encapsulating anionic 18-base single-stranded thiophosphorylated oligonucleotides (ssONs), leading to the formation of polyplexes further characterized by zeta potential (ZP), dynamic light scattering (DLS), binding affinity, transfection efficiency and in vitro cytotoxicity assays. The results demonstrated that the length of the grafted hydrophobic N-acyl chain and the relative amino:phosphate groups ratio (N/P ratio) between the TMC derivatives and ssON played crucial roles in determining the physicochemical properties of the obtained nanocomplexes. While none of the tested derivatives showed appreciable cytotoxicity, the type of acyl chain had a remarkable influence on the cell transfection capacity of TMC-ssON nanocomplexes with the derivatives based on stearic acid showing the best performance based on the results of in vitro assays using a model cell line expressing luciferase (HeLa/Luc705).We acknowledge the financial support from the following Brazilian agencies: CAPES, FAPEMIG, CNPq, and FINEP. This work was co-financed by Fundação para a Ciência e a Tecnologia (FCT, Portugal) within the projects PTDC/CTM-NAN/NAN/115124/2009 and HMSP-ICT/0020/2010. Additionally, PMDM thanks the European Commission – Marie Curie Actions (PIEF-GA-2011-300485) for the postdoctoral fellowship. VL thanks the FCT fo the fellowship (SFRH/BPD/69110/2010). We are grateful to Dr Sandhra Carvalho (UFMG, Brazil) for the bioimaging analyses. The authors acknowledge the Centro de Materiais daUniversidade do Porto (CEMUP) for SEM and1H NMR analysis

Reflected Light from Sand Grains in the Terrestrial Zone of a Protoplanetary Disk

We show that grains have grown to ~mm size (sand sized) or larger in the terrestrial zone (within ~3 AU) of the protoplanetary disk surrounding the 3 Myr old binary star KH 15D. We also argue that the reflected light in the system reaches us by back scattering off the far side of the same ring whose near side causes the obscuration.Comment: 22 pages, 5 figures. To be published in Nature, March 13, 2008. Contains a Supplemen

Delivery of Splice Switching Oligonucleotides by Amphiphilic Chitosan-Based Nanoparticles

Splice switching oligonucleotides (SSOs) are a class of single-stranded antisense oligonucleotides (ssONs) being used as gene therapeutics and demonstrating great therapeutic potential. The availability of biodegradable and biocompatible delivery vectors that could improve delivery efficiencies, reduce dosage, and, in parallel, reduce toxicity concerns could be advantageous for clinical translation. In this work we explored the use of quaternized amphiphilic chitosan-based vectors in nanocomplex formation and delivery of splice switching oligonucleotides (SSO) into cells, while providing insights regarding cellular uptake of such complexes. Results show that the chitosan amphiphilic character is important when dealing with SSOs, greatly improving colloidal stability under serum conditions, as analyzed by dynamic light scattering, and enhancing cellular association. Nanocomplexes were found to follow an endolysosomal route with a long lysosome residence time. Conjugation of a hydrophobic moiety, stearic acid, to quaternized chitosan was a necessary condition to achieve transfection, as an unmodified quaternary chitosan was completely ineffective. We thus demonstrate that amphiphilic quaternized chitosan is a biomaterial that holds promise and warrants further development as a platform for SSO delivery strategies.This work was cofinanced by Fundacão para a Ciência e a Tecnologia (FCT, Portugal) within projects HMSP-ICT/0020/2010 and PTDC/CTM-NAN/NAN/115124/2009. Additionally, P.M.D.M.acknowledges the support from the Marie Curie Actions of the European Community’s 7th Framework Program (PIEF-GA-2011-300485); J.C.S. acknowledges the graduate fellowship from Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq, Ministry of Science and Technology, Brazil); C.P.G. and V.L. acknowledge FCT for their scholarships (SFRH/BD/79930/2011 and SFRH/BPD/69110/2010). We thank M. Lázaro from the Bioimaging Center for Biomaterials and Regenerative Therapies (b.IMAGE) for help with confocal microscopy. 1H NMR and Cryo-SEM were performed at the Centro de Materiais daUniversidade do Porto (CEMUP)

A negative screen for mutations in calstabin 1 and 2 genes in patients with dilated cardiomyopathy

<p>Abstract</p> <p>Background</p> <p>Calstabins 1 and 2 bind to Ryanodine receptors regulating muscle excitation-contraction coupling. Mutations in Ryanodine receptors affecting their interaction with calstabins lead to different cardiac pathologies. Animal studies suggest the involvement of calstabins with dilated cardiomyopathy.</p> <p>Results</p> <p>We tested the hypothesis that calstabins mutations may cause dilated cardiomyopathy in humans screening 186 patients with idiopathic dilated cardiomyopathy for genetic alterations in calstabins 1 and 2 genes (<it>FKBP12 </it>and <it>FKBP12.6)</it>. No missense variant was found. Five no-coding variations were found but not related to the disease.</p> <p>Conclusions</p> <p>These data corroborate other studies suggesting that mutations in <it>FKBP12 </it>and <it>FKBP12.6 </it>genes are not commonly related to cardiac diseases.</p

Impact of complex NOTCH1 mutations on survival in paediatric T-cell leukaemia

<p>Abstract</p> <p>Background</p> <p>Molecular alterations occur frequently in T-ALL and the potential impact of those abnormalities on outcome is still controversial. The current study aimed to test whether <it>NOTCH1 </it>mutations and additional molecular abnormalities would impact T-ALL outcome in a series of 138 T-ALL paediatric cases.</p> <p>Methods</p> <p>T-ALL subtypes, status of <it>SIL-TAL1 </it>fusion, ectopic expression of <it>TLX3</it>, and mutations in <it>FBXW7</it>, <it>KRAS</it>, <it>PTEN </it>and <it>NOTCH1 </it>were assessed as overall survival (OS) and event-free survival (EFS) prognostic factors. OS and EFS were determined using the Kaplan-Meier method and compared using the log-rank test.</p> <p>Results</p> <p>The frequencies of mutations were 43.5% for <it>NOTCH1</it>, while <it>FBXW7</it>, <it>KRAS </it>and <it>PTEN </it>exhibited frequencies of 19.1%, 9.5% and 9.4%, respectively. In 78.3% of cases, the coexistence of <it>NOTCH1 </it>mutations and other molecular alterations was observed. In multivariate analysis no statistical association was revealed between <it>NOTCH1 </it>mutations and any other variable analyzed. The mean length of the follow-up was 68.4 months and the OS was 50.7%. <it>SIL-TAL1 </it>was identified as an adverse prognostic factor. <it>NOTCH1 </it>mutation status was not associated with outcome, while the presence of <it>NOTCH1 </it>complex mutations (indels) were associated with a longer overall survival (<it>p </it>= 0.031) than point mutations.</p> <p>Conclusion</p> <p><it>NOTCH1 </it>mutations alone or in combination with <it>FBXW7 </it>did not impact T-ALL prognosis. Nevertheless, complex <it>NOTCH1 </it>mutations appear to have a positive impact on OS and the <it>SIL-TAL1 </it>fusion was validated as a negative prognostic marker in our series of T-ALL.</p

Survival Analysis of Patients with Heart Failure: Implications of Time-Varying Regression Effects in Modeling Mortality

Background: Several models have been designed to predict survival of patients with heart failure. These, while available and widely used for both stratifying and deciding upon different treatment options on the individual level, have several limitations. Specifically, some clinical variables that may influence prognosis may have an influence that change over time. Statistical models that include such characteristic may help in evaluating prognosis. The aim of the present study was to analyze and quantify the impact of modeling heart failure survival allowing for covariates with time-varying effects known to be independent predictors of overall mortality in this clinical setting. Methodology: Survival data from an inception cohort of five hundred patients diagnosed with heart failure functional class III and IV between 2002 and 2004 and followed-up to 2006 were analyzed by using the proportional hazards Cox model and variations of the Cox's model and also of the Aalen's additive model. Principal Findings: One-hundred and eighty eight (188) patients died during follow-up. For patients under study, age, serum sodium, hemoglobin, serum creatinine, and left ventricular ejection fraction were significantly associated with mortality. Evidence of time-varying effect was suggested for the last three. Both high hemoglobin and high LV ejection fraction were associated with a reduced risk of dying with a stronger initial effect. High creatinine, associated with an increased risk of dying, also presented an initial stronger effect. The impact of age and sodium were constant over time. Conclusions: The current study points to the importance of evaluating covariates with time-varying effects in heart failure models. The analysis performed suggests that variations of Cox and Aalen models constitute a valuable tool for identifying these variables. The implementation of covariates with time-varying effects into heart failure prognostication models may reduce bias and increase the specificity of such models.CNPq Brazilian Foundation for Scientific and Technological DevelopmentCNPq - Brazilian Foundation for Scientific and Technological Development [150653/2008-5