Ship in a bottle: confinement-promoted self-assembly

Understanding self-assembly in confined spaces is essential to fully understand molecular processes in confined cell compartments and will offer clues on the behaviour of simple confined systems, such as protocells and lipid-vesicle based devices. Using a model system composed of lipid vesicles, a membrane impermeable receptor and a membrane-permeable ligand, we have studied in detail how compartmentalization modulates the interaction between the confined receptor and its ligand. We demonstrate that confinement of one of the building blocks stabilizes complex self-assembled structures to the extent that dilution leads, counterintuitively, to the formation of long range assemblies. The behaviour of the system can be explained by considering a confinement factor that is analogous, although not identical, to the effective molarity for intramolecular binding events. The confinement effect renders complex self-assembled species robust and persistent under conditions where they do not form in bulk solution. Moreover, we show that the formation of stable complex assemblies in systems compartmentalized by semi-permeable membranes does not require the prior confinement of all components, but only that of key membrane impermeable building blocks. To use a macroscopic analogy, lipid vesicles are like ship-in-a bottle constructs that are capable of directing the assembly of the confined ship following the confinement of a few key wooden planks. Therefore, we believe that the confinement effect described here would have played an important role in shaping the increase of chemical complexity within protocells during the first stages of abiogenesis. Additionally, we argue that this effect can be exploited to design increasingly efficient functional devices based on comparatively simple vesicles for applications in biosensing, nanoreactors and drug delivery vehicles

Chronic kidney disease in children: the global perspective

In contrast to the increasing availability of information pertaining to the care of children with chronic kidney disease (CKD) from large-scale observational and interventional studies, epidemiological information on the incidence and prevalence of pediatric CKD is currently limited, imprecise, and flawed by methodological differences between the various data sources. There are distinct geographic differences in the reported causes of CKD in children, in part due to environmental, racial, genetic, and cultural (consanguinity) differences. However, a substantial percentage of children develop CKD early in life, with congenital renal disorders such as obstructive uropathy and aplasia/hypoplasia/dysplasia being responsible for almost one half of all cases. The most favored end-stage renal disease (ESRD) treatment modality in children is renal transplantation, but a lack of health care resources and high patient mortality in the developing world limits the global provision of renal replacement therapy (RRT) and influences patient prevalence. Additional efforts to define the epidemiology of pediatric CKD worldwide are necessary if a better understanding of the full extent of the problem, areas for study, and the potential impact of intervention is desired

Advanced imaging of tau pathology in Alzheimer Disease: New perspectives from super resolution microscopy and label-free nanoscopy.

Alzheimer's disease (AD) is the main cause of dementia in the elderly population. Over 30 million people worldwide are living with dementia and AD prevalence is projected to increase dramatically in the next two decades. In terms of neuropathology, AD is characterized by two major cerebral hallmarks: extracellular β-amyloid (Aβ) plaques and intracellular Tau inclusions, which start accumulating in the brain 15-20 years before the onset of symptoms. Within this context, the scientific community worldwide is undertaking a wide research effort to detect AD pathology at its earliest, before symptoms appear. Neuroimaging of Aβ by positron emission tomography (PET) is clinically available and is a promising modality for early detection of Aβ pathology and AD diagnosis. Substantive efforts are ongoing to develop advanced imaging techniques for early detection of Tau pathology. Here, we will briefly describe the key features of Tau pathology and its heterogeneity across various neurodegenerative diseases bearing cerebral Tau inclusions (i.e., tauopathies). We will outline the current status of research on Tau-specific PET tracers and their clinical development. Finally, we will discuss the potential application of novel super-resolution and label-free techniques for investigating Tau pathology at the experimental level and their potential application for AD diagnosis. Microsc. Res. Tech. 79:677-683, 2016. © 2016 Wiley Periodicals, Inc.LG acknowledges funding from the European Community's Seventh Framework Program (FP7/2012-2015) under grant agreement n°280804. GSSK acknowledges funding from the U.K. Medical Research Council (MR/K015850/1 and MR/K02292X/1), Alzheimer's Research UK (ARUK-EG2012A-1), U.K. Engineering and Physical Sciences Research Council (EPSRC) (EP/H018301/1) and the Wellcome Trust (089703/Z/09/Z).This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1002/jemt.2269

Interactions between a luminescent conjugated polyelectrolyte and amyloid fibrils investigated with flow linear dichroism spectroscopy

Luminescent conjugated polyelectrolytes (LCPs) have emerged as novel stains to detect and distinguish between various amyloidogenic species, including prefibrillar aggregates and mature fibril deposits, both in vitro and in histological tissue samples, offering advantages over traditional amyloid stains. We here use linear dichroism (LD) spectroscopy under shear alignment to characterize interactions between the LCP poly(3-thiophene acetic acid) (PTAA) and amyloid fibrils. The positive signature in the LD spectrum of amyloid-bound PTAA suggests that it binds in the grooves between adjacent protein side-chains in the amyloid fibril core, parallel to the fibril axis, similar to thioflavin-T and congo red. Moreover, using LD we record the absorption spectrum of amylokl-bound PTAA in isolation from free dye showing a red-shift by ca 30 nm compared to in solution. This has important implications for the use of PTAA as an amyloid probe in situ and in vitro and we demonstrate how to obtain optimal amyloid-specific fluorescence read-outs using PTAA. We use the shift in maximum absorption to estimate the fraction of bound PTAA at a given concentration. PTAA binding reaches saturation when added in 36 times excess and at this concentration the PTAA density is 4-5 monomer units per insulin monomer in the fibril. Finally, we demonstrate that changes in LD intensity can be related to alterations in persistence length of amyloid fibrils resulting from changes in solution conditions, showing that this technique is useful to assess macroscopic properties of these biopolymers. (C) 2011 Elsevier Inc. All rights reserved

Tryptophan Orientations in Membrane-Bound Gramicidin and Melittin - a Comparative Linear Dichroism Study on Transmembrane and Surface-Bound Peptides

In the search for methods to study structure and function of membrane-associated proteins and peptides flow linear dichroism, LD, spectroscopy has emerged as a promising technique. Using shear-aligned lipid vesicles, conformations and binding geometries of membrane-bound bio-macromolecules can be assessed. Here we investigate anchoring properties and specific orientations of tryptophan relative to the peptide backbone and to the membrane normal for the model peptides gramicidin and melittin. We have monitored the conformational change associated with the refolding of non-channel gramicidin into its channel form, and quantitatively determined the average orientations of its tryptophan transition moments, suggesting that these residues adopt a well-defined orientation at the membrane interface. An important conclusion regards the structural variation of gramicidin between these two distinct transmembrane forms. Whilst circular dichroism (CD) spectra, as has been reported before, vary strongly between the two forms suggesting their structures might be quite different, the LD results clearly evidence both the peptide backbone orientation and tryptophan side-chain positioning to be very similar. The latter are oriented in accord with what is expected from their role to anchor peptide termini to the membrane surface. The variations in CD could be due to, the in LD observed, minor shifts in mutual orientation and distance between neighbouring tryptophans sensitively determining their exciton interactions. Our data dispute that the non-channel form of membrane-bound gramicidin would be any of the intertwined forms often observed in crystal as the positioning of tryptophans along the peptide axis would not be compatible with the strong interfacial positioning observed here. The general role of tryptophans as interfacial anchors is further assessed for melittin whose conformation shows considerable angular spread, consistent with a carpet model of its mechanism for induced membrane leakage, and a predominantly surface-aligned membrane orientation governed by amphipathic interactions

Using Tetracysteine-Tagged TDP-43 with a Biarsenical Dye To Monitor Real-Time Trafficking in a Cell Model of Amyotrophic Lateral Sclerosis

TAR DNA-binding protein 43 (TDP-43) has been identified as the major constituent of the proteinaceous inclusions that are characteristic of most forms of amyotrophic lateral sclerosis (ALS) and ubiquitin positive frontotemporal lobar degeneration (FTLD). Wild type TDP-43 inclusions are a pathological hallmark of \u3e95% of patients with sporadic ALS and of the majority of familial ALS cases, and they are also found in a significant proportion of FTLD cases. ALS is the most common form of motor neuron disease, characterized by progressive weakness and muscular wasting, and typically leads to death within a few years of diagnosis. To determine how the translocation and misfolding of TDP-43 contribute to ALS pathogenicity, it is crucial to define the dynamic behavior of this protein within the cellular environment. It is therefore necessary to develop cell models that allow the location of the protein to be defined. We report the use of TDP-43 with a tetracysteine tag for visualization using fluorogenic biarsenical compounds and show that this model displays features of ALS observed in other cell models. We also demonstrate that this labeling procedure enables live-cell imaging of the translocation of the protein from the nucleus into the cytosol

The Liver and Kidneys mediate clearance of cardiac troponin in the rat

Cardiac-specific troponins (cTn), troponin T (cTnT) and troponin I (cTnI) are diagnostic biomarkers when myocardial infarction is suspected. Despite its clinical importance it is still not known how cTn is cleared once it is released from damaged cardiac cells. The aim of this study was to examine the clearance of cTn in the rat. A cTn preparation from pig heart was labeled with fluorescent dye or fluorine 18 (F-18). The accumulation of the fluorescence signal using organ extracts, or the 18F signal using positron emission tomography (PET) was examined after a tail vein injection. The endocytosis of fluorescently labeled cTn was studied using a mouse hepatoma cell line. Close to 99% of the cTnT and cTnI measured with clinical immunoassays were cleared from the circulation two hours after a tail vein injection. The fluorescence signal from the fluorescently labeled cTn preparation and the radioactivity from the 18F-labeled cTn preparation mainly accumulated in the liver and kidneys. The fluorescently labeled cTn preparation was efficiently endocytosed by mouse hepatoma cells. In conclusion, we find that the liver and the kidneys are responsible for the clearance of cTn from plasma in the rat