Determination of Nanoparticle Localisation Within Subcellular Organelles in Vitro Using Raman Spectroscopy

Ease of sample preparation, narrow spectral bandwidth and minimal influence from water are features of Raman spectroscopy which make it a powerful, label-free way to study a wide range of biological structures and phenomena. In this context, given the concerns over their toxicology arising from their increased production and use, evaluation of nanoparticle uptake and localisation in biological systems and determination of the mechanisms of subcellular interaction and trafficking can provide long-term solutions for nanotoxicology, and potential strategies for nanomedicine. In this study, Raman spectroscopy is explored to monitor the sequential trafficking of nanoparticles through subcellular organelles in-vitro and to establish the spectroscopic signatures of those organelles. A549 human lung carcinoma cells were exposed to 40 nm carboxylate-modified and fluorescently-labelled polystyrene nanoparticles for 4, 12 and 24hrs. Raman spectroscopy was applied to nanoparticle exposed cells to determine the localisation within cellular compartments. Confocal laser scanning fluorescence microscopy (CLSM) with different organelle staining kits confirmed the localisation of the nanoparticles in organelles at the chosen exposure periods and co-localization was quantified using ImageJ with the JACoP colocalisation plugin. To confirm nanoparticle localisation and elucidate the spectroscopic signatures of the different subcellular organelles, a combination of K-means clustering (KMCA) and Principal components analysis (PCA) was applied to the Raman spectroscopic maps. The study showed the applicability of the techniques for elucidation of the localisation of polystyrene nanoparticles within the cell as well as determination of their local environment, differentiating the spectral signatures of intracellular compartments such as endosomes, lysosomes and endoplasmic reticulum, in a completely label free manner

Quenching of the radio jet during the X-ray high state of GX 339-4

We have observed the black hole candidate X-ray binary GX 339-4 at radio wavelengths before, during and after the 1998 high/soft X-ray state transition. We find that the radio emission from the system is strongly correlated with the hard X-ray emission and is reduced by a factor > 25 during the high/soft state compared to the more usual low/hard state. At the points of state transition we note brief periods of unusually optically-thin radio emission which may correspond to discrete ejection events. We propose that in the low/hard state black hole X-ray binaries produce a quasi-continuous outflow, in the high/soft state this outflow is suppressed, and that state transitions often result in one or more discrete ejection events. Future models for low/hard states, such as ADAF/ADIOS solutions, need to take into account strong outflow of relativistic electrons from the system. We propose that the inferred Comptonising corona and the base of the jet-like outflow are the same thing, based upon the strong correlation between radio and hard X-ray emission in GX 339-4 and other X-ray binaries, and the similarity in inferred location and composition of these two components.Comment: Accepted for publication in ApJ Letter

Is telomere length a biomarker for aging: cross-sectional evidence from the west of Scotland?

Background <p> The search for biomarkers of aging (BoAs) has been largely unsuccessful to-date and there is widespread skepticism about the prospects of finding any that satisfy the criteria developed by the American Federation of Aging Research. This may be because the criteria are too strict or because a composite measure might be more appropriate. Telomere length has attracted a great deal of attention as a candidate BoA. We investigate whether it meets the criteria to be considered as a single biomarker of aging, and whether it makes a useful contribution to a composite measure. </p> Methodology/Principal Findings <p> Using data from a large population based study, we show that telomere length is associated with age, with several measures of physical and cognitive functioning that are related to normal aging, and with three measures of overall health. In the majority of cases, telomere length adds predictive power to that of age, although it was not nearly as good a predictor overall. We used principal components analysis to form two composites from the measures of functioning, one including telomere length and the other not including it. These composite BoAs were better predictors of the health outcomes than chronological age. There was little difference between the two composites. </p> Conclusions <p> Telomere length does not satisfy the strict criteria for a BoA, but does add predictive power to that of chronological age. Equivocal results from previous studies might be due to lack of power or the choice of measures examined together with a focus on single biomarkers. Composite biomarkers of aging have the potential to outperform age and should be considered for future research in this area.</p&gt

Evaluation of cytotoxicity profile and intracellular localisation of doxorubicin-loaded chitosan nanoparticles

In the emerging field of nanomedicine, targeted delivery of nanoparticle encapsulated active pharmaceutical ingredients (API) is seen as a potential significant development, promising improved pharmacokinetics and reduced side effects. In this context, understanding the cellular uptake of the nanoparticles and subsequent subcellular distribution of the API is of critical importance. Doxorubicin (DOX) was encapsulated within chitosan nanoparticles to investigate its intracellular delivery in A549 cells in vitro. Unloaded (CS-TPP) and doxorubicin-loaded (DOX-CS-TPP) chitosan nanoparticles were characterised for size (473±41 nm), polydispersity index (0.3±0.2), zeta potential (34±4 mV), drug content (76±7 µM) and encapsulation efficiency (95±1%). The cytotoxic response to DOX-CS-TPP was substantially stronger than to CS-TPP, although weaker than that of the equivalent free DOX. Fluorescence microscopy showed a dissimilar pattern of distribution of DOX within the cell, being predominantly localised in the nucleus for free form and in cytoplasm for DOX-CS-TPP. Confocal microscopy demonstrated endosomal localisation of DOX-CS-TPP. Numerical simulations, based on a rate equation model to describe the uptake and distribution of the free DOX, nanoparticles and DOX loaded nanoparticles within the cells, and the subsequent dose and time dependent cytotoxic responses, were used to further elucidate the API distribution processes. The study demonstrates that encapsulation of the API in nanoparticles results in a delayed release of the drug to the cell, resulting in a delayed cellular response. This work further demonstrates the potential of mathematical modelling in combination with intracellular imaging techniques to visualise and further understand the intracellular mechanisms of action of external agents, both APIs and nanoparticles in cells

Advancing Raman Microspectroscopy for Cellular and Subcellular Analysis: Towards in Vitro High Content Spectralomic Analysis

In the confocal mode, Raman microspectroscopy can profile the biochemical content of biological cells at a subcellular level, and any changes to it by exogenous agents, such as therapeutic drugs or toxicants. As an exploration of the potential of the technique as a high content, label free analysis technique, this report reviews work to monitor the spectroscopic signatures associated with the uptake and response pathways of commercial chemotherapeutic agents and polymeric nanoparticles by human lung cells. It is demonstrated that the signatures are reproducible and characteristic of the cellular event, and can be used, for example, to identify the mode of action of the agent as well as the subsequent cell death pathway, and even mechanisms of cellular resistance. Data mining approaches are discussed and a spectralomics approach is proposed

MicroRNA regulation of glycolytic metabolism in glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most aggressive and common malignant brain and central nervous system tumour. A well-known hallmark of GMB, and many other tumours, is aerobic glycolysis. microRNAs (miRNAs) are a class of short non-protein coding sequences that exert post-transcriptional controls on gene expression and represent critical regulators of aerobic glycolysis in GBM. In GBM, miRNAs regulate the expression of glycolytic genes directly and via the regulation of metabolism-associated oncogenic pathways, such as the PI3K/Akt signalling pathway. The aim of this review is to establish links between miRNA expression levels, disease grade and prognosis, and the glycolytic phenotype of GBM. In this review, the involvement of 25 miRNAs in the regulation of glycolytic metabolism of GBM is discussed. Seven of these miRNAs have been shown to regulate glycolytic metabolism in other tumour types. Further eight miRNAs, which have been shown to be differentially expressed in GBM, were also reported to play a regulatory role in glycolysis in other cancer types. Such miRNAs could serve as potential glycolytic regulators in GBM but require functional validation. This review concludes with presenting a number of glycolytic regulatory miRNAs that have demonstrated their therapeutic potential either alone or as adjuvants in GBM, despite the major challenges that have to be solved before miRNA-based therapies can widely be used for the treatment of GBM patients

Synthetic, structural and magnetic implications of introducing 2,2'-dipyridylamide to sodium-ferrate complexes

Using a transamination approach to access novel Fe(II) complexes, this study presents the synthesis, X-ray crystallographic and magnetic characterisation of a series of new iron complexes containing the multifunctional 2,2-dipyridylamide (DPA) ligand using iron bis(amide) [{Fe(HMDS)2}2] and sodium ferrate [{NaFe (HMDS)3}∞] (1) as precursors (HMDS = 1,1,1,3,3,3-hexamethyldisilazide). Reactions of DPA(H) with 1 show exceptionally good stoichiometric control, allowing access to heteroleptic [(THF)2·NaFe(DPA)(HMDS)2] (3) and homoleptic [{THF·NaFe(DPA)3}∞] (4) by using 1 and 3 equivalents of DPA(H) respectively. Linking this methodology and co-complexation, which is a more widely used approach to prepare heterobimetallic complexes, 3 can also be prepared by combining NaHMDS with heteroleptic [{Fe(DPA)(HMDS)}2] (2). In turn, 2 has been also synthesised and structurally defined by reacting [{Fe(HMDS)2}2] with two equivalents of DPA(H). Structural studies demonstrate the coordination flexibility of the N-bridged bis(heterocycle) ligand DPA, with 2 and 3 exhibiting discrete monomeric motifs, whereas 4 displays a much more intricate supramolecular structure, with one of its DPA ligands coordinating in an anti/anti fashion (as opposed to 2 and 3 where DPA shows a syn/syn conformation), which facilitates propagation of the structure via its central amido N. Magnetic studies confirmed the high-spin electron configuration of the iron(II) centres in all three compounds and revealed the existence of weak ferromagnetic interactions in dinuclear compound 2 ( J = 1.01 cm−1)

Un peu moins de vingt mille incipit inédits de Georges Perec

Un peu moins de vingt mille incipit inédits de Georges Pere