Inhibition of Secretion of Interleukin (IL)-12/IL-23 Family Cytokines by 4-Trifluoromethyl-celecoxib Is Coupled to Degradation via the Endoplasmic Reticulum Stress Protein HERP

Interleukin-12 (IL-12), p80, and IL-23 are structurally related cytokines sharing a p40 subunit. We have recently demonstrated that celecoxib and its COX-2-independent analogue 4-trifluoromethyl-celecoxib (TFM-C) inhibit secretion but not transcription of IL-12 (p35/p40) and p80 (p40/p40). This is associated with a mechanism involving altered cytokine-chaperone interaction in the endoplasmic reticulum (ER). In the present study, we found that celecoxib and TFM-C also block secretion of IL-23 (p40/p19 heterodimers). Given the putative ER-centric mode of these compounds, we performed a comprehensive RTPCR analysis of 23 ER-resident chaperones/foldases and associated co-factors. This revealed that TFM-C induced 1.5-3-fold transcriptional up-regulation of calreticulin, GRP78, GRP94, GRP170, ERp72, ERp57, ERdj4, and ERp29. However, more significantly, a 7-fold up-regulation of homocysteine-inducible ER protein (HERP) was observed. HERP is part of a high molecular mass protein complex involved in ER-associated protein degradation (ERAD). Using co-immunoprecipitation assays, we show that TFM-C induces protein interaction of p80 and IL-23 with HERP. Both HERP siRNA knockdown and HERP overexpression coupled to cycloheximide chase assays revealed that HERP is necessary for degradation of intracellularly retained p80 by TFM-C. Thus, our data suggest that targeting cytokine folding in the ER by small molecule drugs could be therapeutically exploited to alleviate in appropriate inflammation in autoimmune conditions.

Investigation of swelling and network parameters of poly (ethylene glycol)-crosslinked poly (methyl vinyl ether-co-maleic acid) hydrogels

he influence of poly(ethylene glycol) (PEG) plasticiser content and molecular weight on the physicochemical properties of films cast from aqueous blends of poly(methyl vinyl ether-co-maleic acid) was investigated using thermal analysis, swelling studies, scanning electron microscopy (SEM) and attenuated total reflectance (ATR)-Fourier transform infrared (FTIR) spectroscopy. FTIR spectroscopy revealed a shift of the CO peak from 1708 to 1731 cm-1, indicating that an esterification reaction had occurred upon heating, thus producing crosslinked films. Higher molecular weight PEGs (10,000 and 1000 Da, respectively), having greater chain length, producing hydrogel networks with lower crosslink densities and higher average molecular weight between two consecutive crosslinks. Accordingly, such materials exhibited higher swelling rates. Hydrogels crosslinked with a low molecular weight PEG (PEG 200) showed rigid networks with high crosslink densities and, therefore, lower swelling rates. Polymer:plasticizer ratio alteration did not yield any discernable patterns, regardless of the method of analysis. The polymer–water interaction parameter (?) increased with increases in the crosslink density. SEM studies showed that porosity of the crosslinked films increased with increasing PEG MW, confirming what had been observed with swelling studies and thermal analysis, that the crosslink density must be decreased as the Mw of the crosslinker is increased. Hydrogels containing PMVE/MA/PEG 10,000 could be used for rapid delivery of drug, due to their low crosslink density. Moderately crosslinked PMVE/MA/PEG 1000 hydrogels or highly crosslinked PMVE/MA/PEG 200 systems could then be used in controlling the drug delivery rates. We are currently evaluating these systems, both alone and in combination, for use in sustained release drug delivery devices.

Melt-compounded nanocomposites of titanium dioxide atomic-layer-deposition-coated polyamide and polystyrene powders

Polyamide and polystyrene particles were coated with titanium dioxide films by atomic layer deposition (ALD) and then melt-compounded to form polymer nanocomposites. The rheological properties of the ALD-created nanocomposite materials were characterized with a melt flow indexer, a melt flow spiral mould, and a rotational rheometer. The results suggest that the melt flow properties of polyamide nanocomposites were markedly better than those of pure polyamide and polystyrene nanocomposites. Such behavior was shown to originate in an uncontrollable decrease in the polyamide molecular weight, likely affected by a high thin-film impurity content, as shown in gel permeation chromatography (GPC) and scanning electron microscope (SEM) equipped with an energy-dispersive spectrometer. Transmission electron microscope image showed that a thin film grew on both studied polymer particles, and that subsequent melt-compounding was successful, producing well dispersed ribbon-like titanium dioxide with the titanium dioxide filler content ranging from 0.06 to 1.12wt%. Even though we used nanofillers with a high aspect ratio, they had only a minor effect on the tensile and flexural properties of the polystyrene nanocomposites. The mechanical behavior of polyamide nanocomposites was more complex because of the molecular weight degradation. Our approach here to form polymeric nanocomposites is one way to tailor ceramic nanofillers and form homogenous polymer nanocomposites with minimal work-related risks in handling powder form nanofillers. However, further research is needed to gauge the commercial potential of ALD-created nanocomposite materials. Copyright (C) 2011 John Wiley & Sons, Ltd.

Biaxial Deformation and Experimental Study of PET at Conditions Applicable to Stretch Blow Molding

This article is concerned with understanding the behavior of polyethylene terephthalate (PET) in the injection stretch blow molding (ISBM) process where it is typically biaxially stretched to form bottles for the packaging industry. A comprehensive experimental study was undertaken, analyzing the behavior of three different grades of PET under constant width (CW), simultaneous (EB), and sequential (SQ) equal biaxial deformation. Experiments were carried out at temperature and strain rate ranges of 80–110C and 1 /s to 32 /s, respectively, to different stretch ratios. Results show that the biaxial deformation behavior of PET exhibits a strong dependency on forming temperature, strain rate, stretch ratio,deformation mode, and molecular weight. The tests were also monitored via a high speed thermal image camera which showed an increase in temperature between 5C and 15C observed depending on the stretch conditions.

The identification of potential alternative biomarkers of nitrofurazone abuse in animal derived food products

Semicarbazide (SEM) was considered to be a characteristic protein-bound side-chain metabolite of the banned veterinary drug nitrofurazone and used as a marker of nitrofurazone abuse. It was recently discovered that SEM can arise in food from sources other than nitrofurazone. This uncertainty over the source of SEM may be overcome if alternative markers specific to tissue-bound nitrofurazone residues can be determined. The structure of nitrofurazone metabolites in vivo and particular proteins to which they are bound are not known. These proteins with altered structure due to the presence of the drug metabolites can be considered as potential alternative biomarkers of nitrofurazone abuse. The proteins implicated in the in vivo binding of nitrofurazone were separated and identified. A crude mixture of proteins extracted from the liver of a rat treated with the drug was separated using a series of different techniques such as preparative isoelectric focusing and size exclusion HPLC. Multiple fractions were assayed by LC-MS/MS to detect the presence of SEM. The proteins containing SEM residues were identified by peptide mass mapping using trypsin digestion and MALDI-TOF. The first protein identified as containing high concentration of SEM was albumin. It was also shown that low molecular weight species within a protein mixture whose main constituent was glutathione S-transferase contained a high concentration of SEM. The chemical composition of these components is under investigation. Preliminary data suggest the SEM forms part of a nitrofurazone metabolite conjugated to glutathione. (C) 2008 Elsevier Ltd. All rights reserved.

Effects of storage on thermomechanical properties of poly(epsilon-caprolactone) blends containing poly(vinyl pyrrolidone/iodine)

This study reports the effects of: the molecular weight ratio of poly(epsilon -caprolactone) (PCL) in blends containing polymer of high (50 000 g mol(-1)) and low (4000 g mol(-1)) molecular weight; the concentration (0, 1, and 5 wt-%) of poly(vinyl pyrrolidone/iodine) (PVP/I); and storage at 30 degreesC and 75% relative humidity; on the thermomechanical properties of films prepared by solvent evaporation from solutions containing both PCL and PVP/I. The tensile properties were found to be statistically dependent on the molecular weight ratio of PCL but not on the concentration of PVP/I. The reductions in tensile strength and elongation at break associated with increasing amounts of low molecular weight PCL were attributed to a reduction in the concentration of chain entanglements. No changes were observed in viscoelastic properties or the glass transition temperature. Following storage there were no changes in the tensile strength, glass transition temperature, or viscoelastic properties of the films; however, significant reductions in elongation at break were observed. It is suggested that this is due to hydrolytic chain scission of amorphous PCL. Inclusion of 5 wt-% PVP/I increased this process in films containing 100:0 and 80:20 high/low molecular weight PCL (but not 60.40), but the extent of this was small. This study highlighted significant aging properties of PCL in a moist atmosphere. Consequently, it is recommended that suitable packaging materials should be employed to control the exposure of PCL films to water during storage.

DNA double-strand break distributions in X-ray and alpha-particle irradiated V79 cells: Evidence for non-random breakage

Many studies have shown that with increasing LET of ionizing radiation the RBE (relative biological effectiveness) for dsb (double strand breaks) induction remains around 1.0 despite the increase in the RBE for cell killing. This has been attributed to an increase in the complexity of lesions, classified as dsb with current techniques, at multiply damaged sites. This study determines the molecular weight distributions of DNA from Chinese hamster V79 cells irradiated with X-rays or 110 keV/mu m alpha-particles. Two running conditions for pulsed-field gel-electrophoresis were chosen to give optimal separation of fragments either in the 225 kbp-5.7 Mbp range or the 0.3 kbp to 225 kbp range. Taking the total fraction of DNA migrating into the gel as a measure of fragmentation, the RBE for dsb induction was less than 1.0 for both molecular weight regions studied. The total yields of dsb were 8.2 x 10(-9) dsb/Gy/bp for X-rays and 7.8 x 10(-9) dsb/Gy/bp for a-particles, measured using a random breakage model. Analysis of the RBE of alpha-particles versus molecular weight gave a different response. In the 0.4 Mbp-57 Mbp region the RBE was less than 1.0; however, below 0.4 Mbp the RBE increased above 1.0. The frequency distributions of fragment sizes were found to differ from those predicted by a model assuming random breakage along the length of the DNA and the differences were greater for alpha-particles than for X-rays. An excess of fragments induced by a single-hit mechanism was found in the 8-300 kbp region and for X-rays and alpha-particles these corresponded to an extra 0.8 x 10(-9) and 3.4 x 10(-9) dsb/bp/Gy, respectively. Thus for every alpha-particle track that induces a dsb there is a 44% probability of inducing a second break within 300 kbp and for electron tracks the probability is 10%. This study shows that the distribution of damage from a high LET alpha-particle track is significantly different from that observed with low LET X-rays. In particular, it suggests that the fragmentation patterns of irradiated DNA may be related to the higher-order chromatin repealing structures found in intact cells.

Secretion and processing of a novel multi-domain cystatin-like protein by intracellular stages of Trichinella spiralis

The excretory-secretory (ES) proteins of nematode parasites are of major interest as they function at the host-parasite interface and are likely to have roles crucial for successful parasitism. Furthermore, the ES proteins of intracellular nematodes such as Trichinella spiralis may also function to regulate gene expression in the host cell. In a recent proteomic analysis we identified a novel secreted cystatin-like protein from T. spiralis L1 muscle larva. Here we show that the protein, MCD-1 (multi-cystatin-like domain protein 1), contains three repeating cystatin-like domains and analysis of the mcd-1 gene structure suggests that the repeated domains arose from duplication of an ancestral cystatin gene. Cystatins are a diverse group of cysteine protease inhibitors and those secreted by parasitic nematodes are important immuno-modulatory factors. The cystatin superfamily also includes cystatin-like proteins that have no cysteine protease inhibitory activity. A recombinant MCD-1 protein expressed as a GST-fusion protein in Escherichia coli failed to inhibit papain in vitro suggesting that the T. spiralis protein is a new member of the non-inhibitory cystatin-related proteins. MCD-1 secreted from T. spiralis exists as high- and low-molecular weight isoforms and we show that a recombinant MCD-1 protein secreted by HeLa cells undergoes pH-dependent processing that may result in the release of individual cystatin-like domains. Furthermore, we found that mcd-1 gene expression is largely restricted to intracellular stages with the highest levels of expression in the adult worms. It is likely that the major role of the protein is during the intestinal stage of T. spiralis infections.

Investigation of swelling and network parameters of poly(ethylene glycol)-crosslinked poly(methyl vinyl ether-co-maleic acid) hydrogels

The influence of poly(ethylene glycol) (PEG) plasticiser content and molecular weight on the physicochemical properties of films cast from aqueous blends of poly(methyl vinyl ether-co-maleic acid) was investigated using thermal analysis, swelling studies, scanning electron microscopy (SEM) and attenuated total reflectance (ATR)-Fourier transform infrared (FTIR) spectroscopy. FTIR spectroscopy revealed a shift of the C{double bond, long}O peak from 1708 to 1731 cm, indicating that an esterification reaction had occurred upon heating, thus producing crosslinked films. Higher molecular weight PEGs (10,000 and 1000 Da, respectively), having greater chain length, producing hydrogel networks with lower crosslink densities and higher average molecular weight between two consecutive crosslinks. Accordingly, such materials exhibited higher swelling rates. Hydrogels crosslinked with a low molecular weight PEG (PEG 200) showed rigid networks with high crosslink densities and, therefore, lower swelling rates. Polymer:plasticizer ratio alteration did not yield any discernable patterns, regardless of the method of analysis. The polymer-water interaction parameter (?) increased with increases in the crosslink density. SEM studies showed that porosity of the crosslinked films increased with increasing PEG MW, confirming what had been observed with swelling studies and thermal analysis, that the crosslink density must be decreased as the M of the crosslinker is increased. Hydrogels containing PMVE/MA/PEG 10,000 could be used for rapid delivery of drug, due to their low crosslink density. Moderately crosslinked PMVE/MA/PEG 1000 hydrogels or highly crosslinked PMVE/MA/PEG 200 systems could then be used in controlling the drug delivery rates. We are currently evaluating these systems, both alone and in combination, for use in sustained release drug delivery devices. © 2008 Elsevier Ltd. All rights reserved.

Cloning and characterisation of three novel disintegrin precursors from the venoms of three Atheris species: Atheris chlorechis, Atheris nitschei and Atheris squamigera

Snake venom constitutes one of the most complex mixtures of naturally-occurring toxic proteins/polypeptides and a large number of these possess very profound biological activities. Disintegrins, that are commonly found in viper venoms, are low molecular weight proteins that usually contain an -Arg-Gly-Asp- (-RGD-) motif that is known to be involved in cell adhesion ligand recognition, binding specifically to cell surface integrin receptors and also exhibiting platelet anti-aggregation activity.

Here, we report for the first time, the successful cloning of three cDNAs encoding disintegrin precursors from lyophilised venom-derived libraries of Atheris chlorechis, Atheris nitschei and Atheris squamigera, respectively. All of these disintegrins belong to the short-coding class and all exhibit high degrees of structural identity, both in their amino acid sequences and in the arrangement of their functional domains. Mass spectrometric analyses of the HPLC-separated/in-gel digested venom proteins was performed to characterise the mature disintegrins as expressed in the venom proteome. Studies on both the structures and conserved sites within these disintegrins are of considerable theoretical interest in the field of biological evolution and in the development of new research tools or novel templates for drug design.

Next generation sequencing-based molecular diagnosis of retinitis pigmentosa: identification of a novel genotype-phenotype correlation and clinical refinements

Retinitis pigmentosa (RP) is a devastating form of retinal degeneration, with significant social and professional consequences. Molecular genetic information is invaluable for an accurate clinical diagnosis of RP due to its high genetic and clinical heterogeneity. Using a gene capture panel that covers 163 of the currently known retinal disease genes, including 48 RP genes, we performed a comprehensive molecular screening in a collection of 123 RP unsettled probands from a wide variety of ethnic backgrounds, including 113 unrelated simplex and 10 autosomal recessive RP (arRP) cases. As a result, 61 mutations were identified in 45 probands, including 38 novel pathogenic alleles. Interestingly, we observed that phenotype and genotype were not in full agreement in 21 probands. Among them, eight probands were clinically reassessed, resulting in refinement of clinical diagnoses for six of these patients. Finally, recessive mutations in CLN3 were identified in five retinal degeneration patients, including four RP probands and one cone-rod dystrophy patient, suggesting that CLN3 is a novel non-syndromic retinal disease gene. Collectively, our results underscore that, due to the high molecular and clinical heterogeneity of RP, comprehensive screening of all retinal disease genes is effective in identifying novel pathogenic mutations and provides an opportunity to discover new genotype-phenotype correlations. Information gained from this genetic screening will directly aid in patient diagnosis, prognosis, and treatment, as well as allowing appropriate family planning and counseling.

Morphology and compression behaviour of biodegradable scaffolds produced by the sintering process

Porous poly(L-lactic acid) (PLA) scaffolds of 85 per cent and 90 per cent porosity are prepared using polymer sintering and porogen leaching method. Different weight fractions of 10 per cent, 30 per cent, and 50 per cent of hydroxyapatite (HA) are added to the PLA to control the acidity and degradation rate. The three-dimensional (3D) morphology and surface porosity are tested using micro-computer tomography (micro-CT), optical microscopy, and scanning electron microscopy (SEM). Results indicate that the surface porosity does not change on the addition of HA. The micro-CT examinations show a slight decrease in the pore size and increase in the wall thickness accompanied by reduced anisotropy for the scaffolds containing HA. Scanning electron micrographs show detectable interconnected pores for the scaffold with pure PLA. Addition of the HA results in agglomeration of the HA particles and reduced leaching of the porogen. Compression tests of the scaffold identify three stages in the stress-strain curve. The addition of HA results in a reduction in the modulus of the scaffold at the first stage of elastic bending of the wall, but this is reversed for the second and third stages of collapse of the wall and densification in the compression tests. In the scaffolds with 85 per cent porosity, the addition of a high percentage of HA could result in 70 per cent decrease in stiffness in the first stage, 200 per cent increase in stiffness in the second stage, and 20 per cent increase in stiffness in the third stage. The results of these tests are compared with the Gibson cellular material model that is proposed for prediction of the behaviour of cellular material under compression. The pH and molecular weight changes are tracked for the scaffolds within a period of 35 days. The addition of HA keeps the pH in the alkaline region, which results in higher rate of degradation at an early period of observation, followed by a reduced rate of degradation later in the process. The final molecular weight is higher for the scaffolds with HA than for scaffolds of pure PLA. The manufactured scaffolds offer acceptable properties in terms of the pore size range and interconnectivity of the pores and porosity for non-load-bearing bone graft substitute; however, improvement to the mixing of the phases of PLA and HA is required to achieve better integrity of the composite scaffolds. © 2008 IMechE.

Micro-abrasion mechanisms of cast CoCrMo in simulated body fluids

The abrasion seen on some of the retrieved CoCrMo hip joints has been reported to be caused by entrained hard particles in vivo. However, little work has been reported on the abrasion mechanisms of CoCrMo alloy in simulated body environments. Therefore. this study covers the mapping of micro-abrasion wear mechanisms of cast CoCrMo induced by third body hard particles under a wide range of abrasive test conditions. This study has a specific focus on covering the possible in vivo wear modes seen on metal-on-metal (MoM) surfaces. Nano-indentation and nano-scratch tests were also employed to further investigate the secondary wear mechanisms-nano-scale material deformation that involved in micro-abrasion processes. This work addresses the potential detrimental effects of third body hard particles in vivo such as increased wear rates (debris generation) and corrosion (metal-ion release). The abrasive wear mechanisms of cast CoCrMo have been investigated under various wear-corrosion conditions employing two abrasives, SiC (similar to 4 mu m) and Al(2)O(3) (similar to 1 mu m), in two test solutions, 0.9% NaCl and 25% bovine serum. The specific wear rates, wear mechanisms and transitions between mechanisms are discussed in terms of the abrasive size, volume fraction and the test solutions deployed. The work shows that at high abrasive volume fractions, the presence of protein enhanced the wear loss due to the enhanced particle entrainment, whereas at much lower abrasive volume fractions, protein reduced the wear loss by acting as a boundary lubricant or rolling elements which reduced the abrasivity (load per particle) of the abrasive particles. The abrasive wear rate and wear mechanisms of the CoCrMo are dependent on the nature of the third body abrasives, their entrainment into the contact and the presence of the proteins. (C) 2009 Elsevier B.V. All rights reserved.

A rapid HPLC protocol for detection of polyols and trehalose

A procedure was developed to extract polyols and trehalose (protectants against stress) from fungal conidia. Conidia were sonicated (120 s) and immersed in a boiling water bath (5.5 min) to optimize extraction of polyols and trehalose, respectively. A rapid method was developed to separate and detect low-molecular-weight polyols and trehalose using high-performance liquid chromatography (HPLC). An ion exchange column designed for standard carbohydrate analysis was used in preference to one designed for sugar alcohol separation. This resulted in rapid elution (less than 5 min), without sacrificing peak resolution. The use of a pulsed electrochemical detector (gold electrode) resulted in limits of reliable quantification as low as 1.6 μg ml-1 for polyols and 2.8 μg ml-1 for trehalose. This is very sensitive and rapid method by which these protectants can be analysed. It avoids polyol derivatization that characterizes analysis by gas chromatography and the long run times (up to 45 min) that typify HPLC analysis using sugar alcohol columns.

Culture Age, temperature, and pH affect the polyol and trehalose contents of fungal propagules

The growth and conidial physiology of the entomopathogenic fungi Beauveria bassiana, Metarhizium anisopliae, and Paecilomyces farinosus were studied under different conditions. The effects of culture age (up to 120 days), temperature (5 to 35°C), and pH (2.9 to 11.1) were determined. Growth was optimal at pH 5 to 8 for each isolate and between 20 and 35°C, depending on the isolate. The predominant polyol in conidia was mannitol, with up to 39, 134, and 61 mg g of conidia-1 for B. bassiana, M. anisopliae, and P. farinosus, respectively. Conidia of M. anisopliae contained relatively small amounts of lower-molecular-weight polyols and trehalose (less than 25 mg g-1 in total) in all treatments. Conidia of B. bassiana and P. farinosus contained up to 30, 32, and 25 mg of glycerol, erythritol, and trehalose, respectively, g-1, depending on the treatment. Conidia of P. farinosus contained unusually high amounts of glycerol and erythritol at pH 2.9. The apparent effect of pH on gene expression is discussed in relation to the induction of a water stress response. To our knowledge, this is the first report of polyols and trehalose in fungal propagules produced over a range of temperature or pH. Some conditions and harvesting times were associated with an apparent inhibition of synthesis or accumulation of polyols and trehalose. This shows that culture age and environmental conditions affect the physiological quality of inoculum and can thereby determine its potential for biocontrol.