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In this work, a theranostic nanoparticle was developed for multimodal imaging and siRNA delivery. The core of the nanoparticles (NP) was formed by encapsulation of superparamagnetic iron oxides and indocyanine green in a PLGA matrix to serve as a multimodal probe for near-infrared (NIFR) and magnetic resonance (MR) imaging. The surface of the particle was coated with polyethylenimine (PEI) for siRNA delivery. Macrophages efficiently took up the nanoparticles and emitted strong NIFR and MR contrast. When transfected with siRNA targeting the pro-inflammatory enzyme cyclooxygenase-2 (COX-2), significant down-regulation of COX-2 was achieved in activated macrophages. Furthermore, after injection into a unilateral ureteral obstruction (UUO)-induced kidney injury model, NIFR and MRI imaging revealed accumulation of nanoparticles in the injury kidney. In addition, in vivo silencing of COX-2 was achieved by NP/PEI/siCOX-2, which further attenuated kidney injury. Our theranostic platform represents a promising approach for simultaneous diagnosis and treatment of inflammatory diseases. [ABSTRACT FROM AUTHOR]

Combining imaging and drug delivery of “theranostic” nanoparticles has enabled concurrent diagnosis and therapy of diseases. Here, we describe a novel theranostic system that combines two imaging tracers, perfluorooctyl bromide (PFOB) for 19 F magnetic resonance imaging (MRI) and indocyanine green (ICG) for near infrared (NIR) imaging, with the chemotherapeutic agent doxorubicin (Dox) into poly (lactic-co-glycolic acid)- poly (ethylene-glycol)-folate (PLGA-PEG-folate) nanoparticles. Cell culture studies using flow cytometry, confocal laser scanning microscope imaging, and 19 F MRI showed enhanced uptake of nanoparticles via folate receptors expressed on human nasopharyngeal epidermal carcinoma (KB) cells. In vivo , higher MRI and fluorescence signals were obtained from tumors with 19 F MRI and NIR, respectively, using folate-receptor-targeted nanoparticles compared with non-targeted equivalents. An in vitro cytotoxicity assay showed that folate-targeted nanoparticles were able to kill cancer cells more efficiently than non-folate conjugated particles. Our results suggest a potential use of PLGA-PEG-folate PFOB/ICG/Dox nanoparticles as a targeted chemotherapy agent traceable by either 19 F MRI or NIR imaging. [ABSTRACT FROM AUTHOR]

Noninvasive therapeutic cell tracking methods in living animals are important for understanding cell function and fate in connection with cell therapy. Here we report a new particle system based on chitosan-coated poly(lactic-co-glycolic acid) perfluorooctyl bromide (PLGA PFOB) nanoparticles designed for 19 F magnetic resonance imaging (MRI) cell tracking. Chitosan was adsorbed onto the PLGA PFOB nanoparticles through electric interactions, which led to an increase in the hydrodynamic size and a surface charge proportional to the coating weight ratio. Confocal laser scanning microscopy, flow cytometry analysis and 19 F-MRI showed that to achieve the highest labeling efficiency in vitro , the optimal weight ratio of chitosan to the PLGA PFOB nanoparticles was 1:10 for human mesenchymal stem cells (hMSCs) and 1:100 for Raw 264.7 macrophages. In vivo 19 F-MRI showed that 19 F labeled hMSCs remained at the injected site 24 h after injection. Thus, this study validates that chitosan-coated PLGA PFOB nanoparticles have the potential to track cell migration in vivo . [ABSTRACT FROM AUTHOR]

Heteronuclear spin decoupling in solid-state magic-angle spinning NMR is investigated to present methods overcoming interferences between rf irradiation and sample spinning in the intermediate to fast spinning regime. We demonstrate that a recent phase-alternated variant of refocused CW irradiation ( r CW ApA ) provides efficient and robust decoupling in this regime. An extensive experimental and numerical comparison is presented for r CW ApA and PISSARRO (phase-inverted supercycled sequence for attenuation of rotary resonance), previously introduced to quench rotary-resonance recoupling effects, under conditions with spinning frequencies between 30 and 60 kHz. Simulations are used to identify the effect of decoupling for various nuclear spin interactions. [ABSTRACT FROM AUTHOR]

The successful application of solid-state NMR spectroscopy for structural study of biological macromolecules requires high spectral resolution. In presence of abundant H 1 spins, the resolution of the prevailing C 13 or N 15 chemical shift encoding experiments critically depends on the availability of efficient and robust heteronuclear decoupling methods in addition to the use of high-field instrumentation and fast sample spinning. Robustness of the decoupling method towards alterations in amplitude/offset of radio frequency fields due to varying sample states is important to ensure recording of spectra with high resolution over long sampling periods for insensitive samples. Here, we present a phase-alternated refocused continuous-wave decoupling method offering better resolution, easier setup, and higher robustness than previous methods. Improved decoupling is in part ascribed to more efficient cancellation of the residual heteronuclear, H 1 – C 13 , dipolar coupling interactions which are induced by homonuclear, H 1 – H 1 , dipolar coupling interactions. [ABSTRACT FROM AUTHOR]

Mutations in the transforming growth factor β-induced protein (TGFBIp) are linked to the development of corneal dystrophies in which abnormal protein deposition in the cornea leads to a loss of corneal transparency and ultimately blindness. Different mutations give rise to phenotypically distinct corneal dystrophies. Most mutations are located in the fourth fasciclin-1 domain (FAS1-4). The amino acid substitution A546T in the FAS1-4 domain is linked to the development of lattice corneal dystrophy with amyloid deposits in the superficial and deep stroma, classifying it as an amyloid disease. Here we provide a detailed description of the fibrillation of the isolated FAS1-4 domain carrying the A546T substitution. The A546T substitution leads to a significant destabilization of FAS1-4 and induces a partially folded structure with increased surface exposure of hydrophobic patches. The mutation also leads to two distinct fibril morphologies. Long straight fibrils composed of pure β-sheet structure are formed at lower concentrations, whereas short and curly fibrils containing a mixture of α-helical and β-sheet structures are formed at higher concentrations. The formation of short and curly fibrils is preceded by the formation of a small number of oligomeric species with high membrane permeabilization potential and rapid fibril formation. The long straight fibrils are formed more slowly and through progressively bigger oligomers that lose their membrane permeabilization potential as fibrillation proceeds beyond the lag phase. These different fibril classes and associated biochemical differences may lead to different clinical symptoms associated with the mutation. [ABSTRACT FROM AUTHOR]

Abstract: Membrane proteins are vital for biological function, and their action is governed by structural properties critically depending on their interactions with the membranes. This has motivated considerable interest in studies of membrane protein folding and unfolding. Here the structural changes induced by unfolding of an integral membrane protein, namely TFE-induced unfolding of KcsA solubilized by the n-dodecyl β-d-maltoside (DDM) surfactant is investigated by the recently introduced GPS-NMR (Global Protein folding State mapping by multivariate NMR) (Malmendal et al., PlosONE 5, e10262 (2010)) along with dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). GPS-NMR is used as a tool for fast analysis of the protein unfolding processes upon external perturbation, and DLS and SAXS are used for further structural characterization of the unfolding states. The combination allows addressing detergent properties and protein conformations at the same time. The mapping of the states reveals that KcsA undergoes a series of rearrangements which include expansion of the tetramer in several steps followed by dissociation into monomers at 29% TFE. Supplementary studies of DDM and TFE in the absence of KcsA suggest that the disintegration of the tetramer at 29% TFE is caused by TFE dissolving the surrounding DDM rim. Above 34% TFE, KcsA collapses to a new structure that is fully formed at 44% TFE. [Copyright &y& Elsevier]

Abstract: Cold tolerance and metabolic responses to freezing of three slug species common in Scandinavia (Arion ater, Arion rufus and Arion lusitanicus) are reported. Autumn collected slugs were cold acclimated in the laboratory and subjected to freezing conditions simulating likely winter temperatures in their habitat. Slugs spontaneously froze at about −4°C when cooled under dry conditions, but freezing of body fluids was readily induced at −1°C when in contact with external ice crystals. All three species survived freezing for 2days at −1°C, and some A. rufus and A. lusitanicus also survived freezing at −2°C. 1H NMR spectroscopy revealed that freezing of body fluids resulted in accumulation of lactate, succinate and glucose. Accumulation of lactate and succinate indicates that ATP production occurred via fermentative pathways, which is likely a result of oxygen depletion in frozen tissues. Glucose increased from about 6 to 22μg/mg dry tissue upon freezing in A. rufus, but less so in A. ater and A. lusitanicus. Glucose may thus act as a cryoprotectant in these slugs, although the concentrations are not as high as reported for other freeze tolerant invertebrates. [Copyright &y& Elsevier]

Abstract: In the present study the joint transcriptomic and metabolomic responses in Folsomia candida (Collembola) to temperature changes on a short-term scale were studied. Change in heat tolerance was examined as survival after a 35°C heat shock (2h) in the course of either a fluctuating temperature regime (8 to 32°C; pre-treated) or a constant temperature (20°C; control) over a period of 24h. Exposure to a temperature increase from 20 to 32°C (2.4°Cmin− 1) induced a significantly increased heat tolerance which continued throughout the experiment. Expression of the gene encoding heat shock protein Hsp70 was assessed at the mRNA level using real time quantitative polymerase chain reaction (QPCR). Hsp70 was rapidly induced and significantly increased by the temperature increase. The relative concentrations of low molecular weight metabolites were analysed in F. candida using nuclear magnetic resonance spectroscopy (1H NMR). A significant metabolomic divergence between pre-treated and control collembolans was evident; partly due to a significantly reduced relative concentration of five free amino acids (arginine, leucine, lysine, phenylalanine and tyrosine) in pre-treated collembolans. These results, obtained from ecological transcriptomics and metabolomics jointly generated insight on various levels into the combined responses to a changing environment. [Copyright &y& Elsevier]

Abstract: In this study, we show that human extracellular superoxide dismutase (EC-SOD) binds to low-density lipoprotein receptor-related protein (LRP). This interaction is most likely responsible for the removal of EC-SOD from the blood circulation via LRP expressed in liver tissue. The receptor recognition site was located within the extracellular matrix-binding region of EC-SOD. This region encompasses the naturally occurring Arg213Gly amino acid substitution, which affects the affinity of EC-SOD for ligands in the extracellular space. Interestingly, the binding between LRP and Arg213Gly EC-SOD was significantly reduced, thus clarifying the observation that hetero- or homozygous carriers present with a significant increase in EC-SOD in their blood. On the basis of our results, we speculate that EC-SOD synthesized locally in tissues diffuses slowly into the circulation, from where it is removed by binding to LRP present in the liver. The interaction between LRP and EC-SOD is thus likely to be important for maintaining redox balance in the circulation. [Copyright &y& Elsevier]

Abstract: Heteronuclear dipolar recoupling experiments without 1H decoupling based on simple cross polarization are introduced for applications in biological solid-state NMR. It is shown that standard or adiabatic variants of the cross-polarization experiment with irradiation on the low-γ (e.g., 13C,15N) spins even at modest spinning frequencies enable efficient band-selective or broadband dipolar recoupling without the need for intense 1H decoupling. This facilitates experiments on expensive isotope-labelled protein samples for which sample heating by intense 1H decoupling may lead to sample detoriation. The principle is demonstrated numerically and experimentally on uniformly 13C,15N-labelled samples of GB1 and fibrils of hIAPP from the human islet amyloid labelled on the FGAIL part. [Copyright &y& Elsevier]

Abstract: A short exposure to a mild cold stress is sufficient to increase cold tolerance in many insects. This phenomenon, termed rapid cold hardening (RCH) expands the thermal interval that can be exploited by the insect. To investigate the possible role of altered metabolite levels during RCH, the present study used untargeted 1H NMR metabolomic profiling to examine the metabolomic response in Drosophila melanogaster during the 72h following RCH and cold shock treatment. These findings are discussed in relation to the costs and benefits of RCH that are measured in terms of survival and reproductive output. Cold shock caused a persistent disturbance of the metabolite profile that correlated well with a delayed onset of cold shock mortality. The disruption of metabolite homeostasis was smaller following RCH, where control levels were fully recovered after 72h. RCH improved both survival and reproductive output after a subsequent cold shock but the RCH treatment alone was associated with costs in terms of reduced survival and reproductive output. The most pronounced changes following the RCH treatment were elevated levels of glucose and trehalose. Although, it is difficult to discern if a change in a specific metabolite is linked to physiological processes of adaptive, neutral or detrimental nature we observed that the onset and magnitude of the increased sugar levels correlated tightly with the improved chill tolerance following RCH. These findings suggest a putative role of cryoprotectants during RCH which are discussed in the light of the existing literature on the mechanistic background of RCH. [Copyright &y& Elsevier]