Application of Fluorescence Spectroscopy to Quantify Shear-Induced Protein Conformation Change

Rapid and robust methods are required to quantify the effect of hydrodynamic shear on protein conformation change. We evaluated such strategies in this work and found that the binding of the fluorescent probe 4,4'-dianilino-1, 1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to hydrophobic pockets in the blood protein von Willebrand factor (VWF) is enhanced upon the application of fluid shear to the isolated protein. Significant structural changes were observed when the protein was sheared at shear rates >= 6000/s for similar to 3.5 min. The binding of bis-ANS to multimeric VWF, but not dimeric VWF or control protein bovine serum albumin, was enhanced upon fluid shear application. Thus, high-molecular-weight VWF is more susceptible to conformation change upon tensile loading. Although bis-ANS itself did not alter the conformation of VWF, it stabilized protein conformation once it bound the sheared molecule. Bis-ANS binding to VWF was reduced when the sheared protein was allowed to relax before dye addition. Taken together with functional data in the literature, our results suggest that shear-induced conformation changes in VWF reported by bis-ANS correlate well with the normal function of the protein under physiological/pathological fluid flow conditions. Further, this study introduces the fluorescent dye bis-ANS as a tool that may be useful in studies of shear-induced protein conformation change.

Physical and dynamical characterisation of low ΔV NEA (190491) 2000 FJ10

Aims: We investigated the physical properties and dynamical evolution of near-Earth asteroid (NEA) (190491) 2000 FJ10 in order to assess the suitability of this accessible NEA as a space mission target. Methods: Photometry and colour determination were carried out with the 1.54 m Kuiper Telescope (Mt Bigelow, USA) and the 10 m Southern African Large Telescope (SALT; Sutherland, South Africa) during the object's recent favourable apparition in 2011-12. During the earlier 2008 apparition, a spectrum of the object in the 6000-9000 Angstrom region was obtained with the 4.2 m William Herschel Telescope (WHT; Canary Islands, Spain). Interpretation of the observational results was aided by numerical simulations of 1000 dynamical clones of 2000 FJ10 up to 106 yr in the past and in the future. Results: The asteroid's spectrum and colours determined by our observations suggest a taxonomic classification within the S-complex although other classifications (V, D, E, M, P) cannot be ruled out. On this evidence, it is unlikely to be a primitive, relatively unaltered remnant from the early history of the solar system and thus a low priority target for robotic sample return. Our photometry placed a lower bound of 2 h to the asteroid's rotation period. Its absolute magnitude was estimated to be 21.54 ± 0.1 which, for a typical S-complex albedo, translates into a diameter of 130 ± 20 m. Our dynamical simulations show that it has likely been an Amor for the past 105 yr. Although currently not Earth-crossing, it will likely become so during the period 50-100 kyr in the future. It may have arrived from the inner or central main belt >1 Myr ago as a former member of a low-inclination S-class asteroid family. Its relatively slow rotation and large size make it a suitable destination for a human mission. We show that ballistic Earth-190491-Earth transfer trajectories with ΔV <2 km s-1 at the asteroid exist between 2052 and 2061. Based on observations made with the Southern African Large Telescope (SALT).

Tip Leakage Flow and Heat Transfer on Turbine Stage Tip and Casing: Effect of Unsteady Stator-Rotor Interactions

Unsteady simulations were performed to investigate time dependent behaviors of the leakage flow structures and heat transfer on the rotor blade tip and casing in a single stage gas turbine engine. This paper mainly illustrates the unsteady nature of the leakage flow and heat transfer, particularly, that caused by the stator–rotor interactions. In order to obtain time-accurate results, the effects of varying the number of time steps, sub iterations, and the number of vane passing periods was firstly examined. The effect of tip clearance height and rotor speeds was also examined. The results showed periodic patterns of the tip leakage flow and heat transfer rate distribution for each vane passing. The relative position of the vane and vane trailing edge shock with respect to time alters the flow conditions in the rotor domain, and results in significant variations in the tip leakage flow structures and heat transfer rate distributions. It is observed that the trailing edge shock phenomenon results in a critical heat transfer region on the blade tip and casing. Consequently, the turbine blade tip and casing are subjected to large fluctuations of Nusselt number (about Nu = 2000 to 6000 and about Nu = 1000 to 10000, respectively) at a high frequency (coinciding with the rotor speed).

Efficient Drug Delivery and Induction of Apoptosis in Colorectal Tumors Using a Death Receptor 5-Targeted Nanomedicine

Death Receptor 5 (DR5) is a pro-apoptotic cell-surface receptor that is a potential therapeutic target in cancer. Despite the potency of DR5-targeting agents in preclinical models, the translation of these effects into the clinic remains disappointing. Herein, we report an alternative approach to exploiting DR5 tumor expression using antibody-targeted, chemotherapy-loaded nanoparticles. We describe the development of an optimized polymer-based nanotherapeutic incorporating both a functionalized polyethylene glycol (PEG) layer and targeting antibodies to limit premature phagocytic clearance whilst enabling targeting of DR5-expressing tumor cells. Using the HCT116 colorectal cancer model, we show that following binding to DR5, the nanoparticles activate caspase 8, enhancing the anti-tumor activity of the camptothecin payload both in vitro and in vivo. Importantly, the combination of nanoparticle-induced DR5 clustering with camptothecin delivery overcomes resistance to DR5-induced apoptosis caused by loss of BAX or overexpression of anti-apoptotic FLIP. This novel approach may improve the clinical activity of DR5-targeted therapeutics while increasing tumor-specific delivery of systemically toxic chemotherapeutics.Molecular Therapy (2014); doi:10.1038/mt.2014.137.

Graphene Nanoribbons as a Drug Delivery Agent for Lucanthone Mediated Therapy of Glioblastoma Multiforme

We report use of PEG-DSPE coated oxidized graphene nanoribbons (O-GNR-PEG-DSPE) as agent for delivery of anti-tumor drug Lucanthone (Luc) into Glioblastoma Multiformae (GBM) cells targeting base excision repair enzyme APE-1 (Apurinic endonuclease-1). Lucanthone, an endonuclease inhibitor of APE-1, was loaded onto O-GNR-PEG-DSPEs using a simple non-covalent method. We found its uptake by GBM cell line U251 exceeding 67% and 60% in APE-1-overexpressing U251, post 24 h. However, their uptake was ~ 38% and 29% by MCF-7 and rat glial progenitor cells (CG-4), respectively. TEM analysis of U251 showed large aggregates of O-GNR-PEG-DSPE in vesicles. Luc-O-GNR-PEG-DSPE was significantly toxic to U251 but showed little/no toxicity when exposed to MCF-7/CG-4 cells. This differential uptake effect can be exploited to use O-GNR-PEG-DSPEs as a vehicle for Luc delivery to GBM, while reducing nonspecific cytotoxicity to the surrounding healthy tissue. Cell death in U251 was necrotic, probably due to oxidative degradation of APE-1.

Graphical abstract

We used O-GNR-PEG-DSPE as a reliable, non-toxic vehicle for delivery of APE-1 inhibiting Lucanthone into GBM tumor cell lines. LUC-O-GNR-PEG-DSPE particles showed 60% or more uptake by CMV/U251 and A1-5/CMV/U251 where as the uptake by MCF7 and normal CG4 glial cells was much lower (38% and 29% respectively). Different concentrations of Luc (5–80 μM) loaded onto O-GNR-PEG-DSPE showed lower toxicity in the exposed cells compared to the free drug, due to possible slow release of the drug from this particle, which ensures minimum non-specific release of the drug from the particle once it is injected in vivo.
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Water and Temperature Relations of Growth of the Entomogenous Fungi Beauveria bassiana, Metarhizium anisopliae, and Paecilomyces farinosus

The effects of temperature (5-50°C), water availability (0.998-0.88 water activity, aw), and aw × temperature interactions (15-45°C) on growth of three entomogenous fungi, Beauveria bassiana, Metarhizium anisopliae, and Paecilomyces farinosus, were evaluated on a Sabouraud dextrose-based medium modified with the ionic solute KCl, the non-ionic solute glycerol, and an inert solute, polyethylene glycol (PEG) 600. The temperature ranges for growth of B. bassiana, M. anisopliae, and P. farinosus were 5-30, 5-40, and 5-30°C, and optimum growth temperatures were 25, 30, and 20°C, respectively. All three species grew over a similar aw range (0.90-0.998) at optimum temperatures for growth. However, there were significant interspecies variations in growth rates on media modified with each of the three aw-modifying solutes. Growth aw optima ranged between 0.99 and 0.97 on KCl-, glycerol-, and PEG 600-modified media for M. anisopliae and P. farinosus. B. bassiana grew optimally at 0.998 aw, regardless of aw. Comprehensive two-dimensional profiles of aw × temperature relations for growth of these three species were constructed for the first time. The results are discussed in relation to the environmental limits that determine efficacy of entomogenous fungi as biocontrol agents in nature. © 1999 Academic Press.

Hydrogel-Forming Microneedles Prepared from ‘‘Super Swelling’’ Polymers Combined with Lyophilised Wafers for Transdermal Drug Delivery

We describe, for the first time, hydrogel-forming microneedle arrays prepared from "super swelling" polymeric compositions. We produced a microneedle formulation with enhanced swelling capabilities from aqueous blends containing 20% w/w Gantrez S-97, 7.5% w/w PEG 10,000 and 3% w/w Na2CO3 and utilised a drug reservoir of a lyophilised wafer-like design. These microneedle-lyophilised wafer compositions were robust and effectively penetrated skin, swelling extensively, but being removed intact. In in vitro delivery experiments across excised neonatal porcine skin, approximately 44 mg of the model high dose small molecule drug ibuprofen sodium was delivered in 24 h, equating to 37% of the loading in the lyophilised reservoir. The super swelling microneedles delivered approximately 1.24 mg of the model protein ovalbumin over 24 h, equivalent to a delivery efficiency of approximately 49%. The integrated microneedle-lyophilised wafer delivery system produced a progressive increase in plasma concentrations of ibuprofen sodium in rats over 6 h, with a maximal concentration of approximately 179 µg/ml achieved in this time. The plasma concentration had fallen to 71±6.7 µg/ml by 24 h. Ovalbumin levels peaked in rat plasma after only 1 hour at 42.36±17.01 ng/ml. Ovalbumin plasma levels then remained almost constant up to 6 h, dropping somewhat at 24 h, when 23.61±4.84 ng/ml was detected. This work represents a significant advancement on conventional microneedle systems, which are presently only suitable for bolus delivery of very potent drugs and vaccines. Once fully developed, such technology may greatly expand the range of drugs that can be delivered transdermally, to the benefit of patients and industry. Accordingly, we are currently progressing towards clinical evaluations with a range of candidate molecules.

Thermo-Mechanical Properties of Poly ε-Caprolactone/Poly L-Lactic Acid Blends: Addition of Nalidixic Acid and Polyethylene Glycol Additives Journal of the Mechanical Behavior of Biomedical Materials

The search for ideal biomaterials is still on-going for tissue regeneration. In this study, blends of Poly ε-caprolactone (PCL) with Poly l-lactic acid (PLLA), Nalidixic Acid (NA) and Polyethylene glycol (PEG) were prepared. Mechanical and thermal properties of the blends were investigated by tensile and flexural analysis, DSC, TGA, WXRD, MFI, BET, SEM and hot stage optical microscopy. Results showed that the loading of PLLA caused a significant decrease in tensile strength and almost total eradication of the elongation at break of PCL matrix, especially after PEG and NA addition. Increased stiffness was also noted with additional NA, PEG and PLLA, resulting in an increase in the flexural modulus of the blends.
Isothermal degradation indicated that bulk PCL, PLLA and the blends were thermally stable at 200°C for the duration of 2h making extrusion of the blends at this temperature viable. Morphological study showed that increasing the PLLA content and addition of the very low viscosity PEG and powder NA decreased the Melt Flow Indexer and increased the viscosity.
At the higher temperature the PLLA begins to soften and eventually melts allowing for increased flow and, coupling this with, the natural increase in MFI caused by temperature is enhanced further. The PEG and NA addition increased dramatically the pore volume which is important for cell growth and flow transport of nutrients and metabolic waste.

Late Holocene sea-level fall and turn-off of reef flat carbonate production: Rethinking bucket fill and coral reef growth models

Relative sea-level rise has been a major factor driving the evolution of reef systems during the Holocene. Most models of reef evolution suggest that reefs preferentially grow vertically during rising sea level then laterally from windward to leeward, once the reef flat reaches sea level. Continuous lagoonal sedimentation ("bucket fill") and sand apron progradation eventually lead to reef systems with totally filled lagoons. Lagoonal infilling of One Tree Reef (southern Great Barrier Reef) through sand apron accretion was examined in the context of late Holocene relative sea-level change. This analysis was conducted using sedimentological and digital terrain data supported by 50 radiocarbon ages from fossil microatolls, buried patch reefs, foraminifera and shells in sediment cores, and recalibrated previously published radiocarbon ages. This data set challenges the conceptual model of geologically continuous sediment infill during the Holocene through sand apron accretion. Rapid sand apron accretion occurred between 6000 and 3000 calibrated yr before present B.P. (cal. yr B.P.); followed by only small amounts of sedimentation between 3000 cal. yr B.P. and present, with no significant sand apron accretion in the past 2 k.y. This hiatus in sediment infill coincides with a sea-level fall of similar to 1-1.3 m during the late Holocene (ca. 2000 cal. yr B.P.), which would have caused the turn-off of highly productive live coral growth on the reef flats currently dominated by less productive rubble and algal flats, resulting in a reduced sediment input to back-reef environments and the cessation in sand apron accretion. Given that relative sea-level variations of similar to 1 m were common throughout the Holocene, we suggest that this mode of sand apron development and carbonate production is applicable to most reef systems.

City Profile: Erbil

Erbil (Hawler in Kurdish), is the capital and the largest city of Iraqi Kurdistan. Having been continuously inhabited for about 6000 years, the city has recently been regarded by UNESCO World Heritage as one of the world’s oldest urban settlements. The city is witnessing remarkable urban growth and rapid spatial expansion compounded by a dramatic increase in population due to emigration from the countryside and rural areas over the last three decades. Following the changing geopolitical landscape of post-war Iraq, urban changes and socio-political transformation are largely driven by Erbil’s growing autonomous status as the capital of northern region of Kurdistan since 2003. This paper explores the layers of historical, spatial and social developments of the contemporary urban context of Kurdistan in general and of Erbil in particular as a reflection of the changing status of the city, as well as the polarization of Iraq and the emergence of neoliberal urbanism. The tension between the global and modern from one side and traditional and authentic from another is ever present and evident in everyday challenges in the planning of the city. In large part, Erbil’s built fabric embodies the dichotomy of identity and contests between its past and future, in which the present remains a transition between two disconnected realities.

Active site labeling of cysteine cathepsins by a straightforward diazomethylketone probe derived from the N-terminus of human cystatin C

We designed a straightforward biotinylated probe using the N-terminal substrate-like region of the inhibitory site of human cystatin C as a scaffold, linked to the thiol-specific reagent diazomethylketone group as a covalent warhead (i.e. Biot-(PEG)2-Ahx-LeuValGly-DMK). The irreversible activity-based probe bound readily to cysteine cathepsins B, L, S and K. Moreover affinity labeling is sensitive since active cathepsins were detected in the nM range using an ExtrAvidin®-peroxidase conjugate for disclosure. Biot-(PEG)2-Ahx-LeuValGly-DMK allowed a slightly more pronounced labeling for cathepsin S with a compelling second-order rate constant for association (kass = 2,320,000 M−1 s−1). Labeling of the active site is dose-dependent as observed using 6-cyclohexylamine-4-piperazinyl-1,3,5-triazine-2-carbonitrile, as competitive inhibitor of cathepsins. Finally we showed that Biot-(PEG)2-Ahx-LeuValGly-DMK may be a simple and convenient tool to label secreted and intracellular active cathepsins using a myelomonocytic cell line (THP-1 cells) as model.

Development of polymeric–cationic peptide composite nanoparticles, a nanoparticle-in-nanoparticle system for controlled gene delivery

We report the formulation of novel composite nanoparticles that combine the high transfection efficiency of cationic peptide-DNA nanoparticles with the biocompatibility and prolonged delivery of polylactic acid–polyethylene glycol (PLA-PEG). The cationic cell-penetrating peptide RALA was used to condense DNA into nanoparticles that were encapsulated within a range of PLA-PEG copolymers. The composite nanoparticles produced exhibited excellent physicochemical properties including size <200 nm and encapsulation efficiency >80%. Images of the composite nanoparticles obtained with a new transmission electron microscopy staining method revealed the peptide-DNA nanoparticles within the PLA-PEG matrix. Varying the copolymers modulated the DNA release rate >6 weeks in vitro. The best formulation was selected and was able to transfect cells while maintaining viability. The effect of transferrin-appended composite nanoparticles was also studied. Thus, we have demonstrated the manufacture of composite nanoparticles for the controlled delivery of DNA.

51 Pegasi - a planet-bearing Maunder minimum candidate

We observed 51 Peg, the first detected planet-bearing star, in a 55 ks XMM-Newton pointing and in 5 ks pointings each with Chandra HRC-I and ACIS-S. The star has a very low count rate in the XMM observation, but is clearly visible in the Chandra images due to the detectors' different sensitivity at low X-ray energies. This allows a temperature estimate for 51 Peg's corona of T⪉ 1 MK; the detected ACIS-S photons can be plausibly explained by emission lines of a very cool plasma near 200 eV. The constantly low X-ray surface flux and the flat-activity profile seen in optical Ca II data suggest that 51 Peg is a Maunder minimum star; an activity enhancement due to a Hot Jupiter, as proposed by recent studies, seems to be absent. The star's X-ray fluxes in different instruments are consistent with the exception of the HRC Imager, which might have a larger effective area below 200 eV than given in the calibration.

Chronic PEGylated obestatin treatment prevents the onset of high fat diet-induced hypertension and endothelial dysfunction in the absence of metabolic actions

Background: Obestatin is a gastrointestinal peptide with established metabolic actions and emerging vascular effects which involve activation of NO signalling. The aim of this study was to investigate effects of a recently-characterised stable analogue, PEGylated obestatin (PEG-OB), in the setting of diet-induced obesity which is associated with both metabolic and vascular dysfunction. Methods: Male Sprague Dawley rats (6 weeks; n=8) were maintained on standard (SD) or high fat (HF) diet (60% fat) for 8 weeks with once-daily injection of either PEG-OB (50nmol/kg/day) or saline from 2 weeks. Results: HF feeding for 8 weeks resulted in marked body weight gain which was not affected by chronic PEG-OB treatment (HF saline, 175.0±12.2; HF PEG-OB, 190.4±6.4g; P=NS). Similarly, blood glucose, as indicated by HbA1c (HF saline, 6.30±0.15; HF PEG-OB, 6.13±0.36%; P=NS) and insulin tolerance (HF saline, 105.2±52.5; HF PEG-OB, 90.3±45.4mmol/L.min; P=NS), were unaltered by PEG-OB. Despite the apparent lack of metabolic effects, chronic PEG-OB treatment markedly attenuated development of HF-induced hypertension (HF saline, 146.5±4.9mmHg; HF PEG-OB, 123.0±9.7mmHg; P<0.01), assessed by tail-cuff plethysmography. Furthermore, organ bath pharmacology in isolated aortic rings, indicated that HF diet-induced endothelial dysfunction was completely prevented by PEG-OB (acetylcholine, EC50: SD saline, 335±113; HF saline, 758±164; HF PEG-OB, 277±85nmol/L; P<0.05). However, contraction to phenylephrine and relaxation to the NO donor, sodium nitroprusside, were unaltered between groups. Conclusions: PEG-OB exerts beneficial effects on hypertension and endothelial function in diabetes independently of metabolic actions suggesting that obestatin signalling may represent a novel therapeutic target to reduce the risk of associated cardiovascular complications.