Cytoprotective mechanisms of erythropoietin and erythropoietin derivatives in peripheral arterial disease

A third of patients with critical limb ischaemia (CLI) eventually require amputation. Inconsistencies between successful revascularisation and functional outcomes exist, and underlying musculopathy in CLI patients has been identified. Erythropoietin (EPO) has tissue-protective effects in response to ischaemic injury, but its clinical use is often precluded by thromboembolic side effects. Non-haematopoietic EPO-derivatives have been designed to retain only tissueprotective functions of EPO. We hypothesised that ARA-290 (EPO-derivative) may have tissue-protective potential that could represent a novel therapeutic adjunct in patients with CLI. The effect of EPO and ARA-290 in mediating cytoprotection in an in vitro simulated ischaemia model of skeletal muscle was assessed firstly in the immortalised murine C2C12 myoblast cell line and subsequently in skeletal myoblasts isolated from CLI and control donors. In human and murine cells, simulated ischaemia alone demonstrated a detrimental effect on cell function and survival. Addition of EPO or ARA-290 demonstrated significant improvements in function and survival and utilised JAK2/STAT3, PI3k/Akt and NF!B signalling molecules. Isolation of human skeletal myoblasts from CLI patients has not previously been described. Comparison of CLI and control myoblasts elucidated significant differences in their function and survival under both normoxic and simulated ischaemic conditions. CLI myoblasts and myotubes exhibited increased proliferative capacity but reduced migratory and contractile function and importantly a reduced susceptibility to a second ischaemic-insult compared with control myoblasts and myotubes. Evaluation of several variations in the hindlimb ischaemia model allowed the creation of a model which closely recapitulated the muscular pathology observed in human CLI patients. ARA-290 demonstrated improved functional, histological and perfusion outcomes compared to EPO or vehicle-control treated animals. These studies demonstrate the potential of ARA-290 to protect tissues and cells from ischaemic-injury and encourages the development of novel pharmacological therapies for use in patients with “no option” CLI or severe functional deficit

Reconstruction of plasma density profiles by measuring spectra of radiation emitted from oscillating plasma dipoles

We suggest a new method for characterising non-uniform density distributions of plasma by measuring the spectra of radiation emitted from a localised plasma dipole oscillator excited by colliding electromagnetic pulses. The density distribution can be determined by scanning the collision point in space. Two-dimensional particle-in-cell simulations demonstrate the reconstruction of linear and nonlinear density profiles corresponding to laser-produced plasma. The method can be applied to a wide range of plasma, including fusion and low temperature plasmas. It overcomes many of the disadvantages of existing methods that only yield average densities along the path of probe pulses, such as interferometry and spectroscopy

Evaluation of Phage Display Discovered Peptides as Ligands for Prostate-Specific Membrane Antigen (PSMA)

The aim of this study was to identify potential ligands of PSMA suitable for further development as novel PSMA-targeted peptides using phage display technology. The human PSMA protein was immobilized as a target followed by incubation with a 15-mer phage display random peptide library. After one round of prescreening and two rounds of screening, high-stringency screening at the third round of panning was performed to identify the highest affinity binders. Phages which had a specific binding activity to PSMA in human prostate cancer cells were isolated and the DNA corresponding to the 15-mers were sequenced to provide three consensus sequences: GDHSPFT, SHFSVGS and EVPRLSLLAVFL as well as other sequences that did not display consensus. Two of the peptide sequences deduced from DNA sequencing of binding phages, SHSFSVGSGDHSPFT and GRFLTGGTGRLLRIS were labeled with 5-carboxyfluorescein and shown to bind and co-internalize with PSMA on human prostate cancer cells by fluorescence microscopy. The high stringency requirements yielded peptides with affinities KD∼1 μM or greater which are suitable starting points for affinity maturation. While these values were less than anticipated, the high stringency did yield peptide sequences that apparently bound to different surfaces on PSMA. These peptide sequences could be the basis for further development of peptides for prostate cancer tumor imaging and therapy. © 2013 Shen et al

Antitumor lectin Sclerotium rolfsii (SRL) induces apoptosis in human colon cancer cells by activation of multiple signaling pathways; A microarray analysis

Background: TF antigen specific Sclerotium rolfsii lectin (SRL) inhibits human colon epithelial cancer HT29 cell growth by induction of apoptosis through cell surface binding and has tumor suppressing effect in vivo as reported earlier. Here we report the purification, identification and characterization of SRL binding membrane proteins from HT29 cells. Methods and Findings: Membrane proteins from HT29 cells were isolated by phase separation and purified by affinity chromatography using SRL-Sepharose4B matrix. Affinity purified proteins were subjected to in-gel and in-solution trypsin digestion, analysed by ESI-Q-TOF LC-MS and spectrum mill software. Considering the specificity of SRL towards O-glycans, the presence of O-GalNAc sites in SRL interacting proteins were tested using NetOGlyc software. Western blotting was performed to validate the MS identified proteins. A major protein band around 25kDa following in-gel trypsin digestion was identified as Keratin 1 by MS. In-solution trypsin digestion followed by MS identified 25 SRL interacting proteins namely, keratins, heat shock proteins, tubulins, pyruvate kinase M1/M2, peroxiredoxin-1, ATP synthase subunit alpha, mitochondrial, retinal dehydrogenase 1, actin, annexin-A2, prohibitin, ADP/ATP translocase-2 and alpha enolase. NetOGlyc software analysis revealed 21 proteins positive for O-glycosylation sites including keratins alone containing 27 to 50 O-GalNAc sites. Keratin 1 identified and validated by western blotting as major SRL interacting protein showed 49 O-GalNAc sites. Conclusion: SRL binding membrane proteins from human colon epithelial cancer HT29 cells have been identified and characterized. Identified proteins contain O-GalNAc sites and are known to be involved in cell survival, apoptosis and tumorigenesis. The present study provides insights in studying the mechanism of SRL induced apoptosis and to explore lectin for its clinical implications. Key words: Sclerotium rolfsii lectin; HT29 cell membrane proteins; NetOGlyc version 4.0; Q-TOF-LC/MS; Spectrum Mill. Abbreviations: SRL: Sclerotium rolfsii lectin; LC/MS: Liquid chromatography/Mass spectrometry; ESI: Electro Spray Ionization; Q-TOF: Quadrupole- Time of Flight; PTM: Post Translational Modification; ACN: Acetonitrile; CBB: Coomassie Brilliant Blue; BSA: Bovine Serum Albumin

Universal time-dependent deformations of Schrodinger geometry

We investigate universal time-dependent exact deformations of Schrodinger geometry. We present 1) scale invariant but non-conformal deformation, 2) non-conformal but scale invariant deformation, and 3) both scale and conformal invariant deformation. All these solutions are universal in the sense that we could embed them in any supergravity constructions of the Schrodinger invariant geometry. We give a field theory interpretation of our time-dependent solutions. In particular, we argue that any time-dependent chemical potential can be treated exactly in our gravity dual approach.Comment: 24 pages, v2: references adde

Germline Genetic Variants Disturbing the Let-7/LIN28 Double-Negative Feedback Loop Alter Breast Cancer Susceptibility

Previous studies have shown that let-7 can repress the post-transcriptional translation of LIN28, and LIN28 in turn could block the maturation of let-7, forming a double-negative feedback loop. In this study, we investigated the effect of germline genetic variants on regulation of the homeostasis of the let-7/LIN28 loop and breast cancer risk. We initially demonstrated that the T/C variants of rs3811463, a single nucleotide polymorphism (SNP) located near the let-7 binding site in LIN28, could lead to differential regulation of LIN28 by let-7. Specifically, the C allele of rs3811463 weakened let-7–induced repression of LIN28 mRNA, resulting in increased production of LIN28 protein, which could in turn down-regulate the level of mature let-7. This effect was then validated at the tissue level in that the normal breast tissue of individuals with the rs3811463-TC genotype expressed significantly lower levels of let-7 and higher levels of LIN28 protein than those individuals with the rs3811463-TT genotype. Because previous in vitro and ex vivo experiments have consistently suggested that LIN28 could promote cellular transformation, we then systematically evaluated the relationship between rs3811463 as well as other common LIN28 SNPs and the risk of breast cancer in a stepwise manner. The first hospital-based association study (n = 2,300) demonstrated that two SNPs were significantly associated with breast cancer risk, one of which was rs3811463, while the other was rs6697410. The C allele of the rs3811463 SNP corresponded to an increased risk of breast cancer with an odds ratio (OR) of 1.25 (P = 0.0091), which was successfully replicated in a second independent study (n = 1,156) with community-based controls. The combined P-value of the two studies was 8.0×10−5. Taken together, our study demonstrates that host genetic variants could disturb the regulation of the let-7/LIN28 double-negative feedback loop and alter breast cancer risk

Genotoxicity evaluation of the insecticide ethion in root of Allium cepa L.

In this study, the genotoxic effects of ethion were investigated in the mitotic cell division of Allium cepa. Primary roots of A. cepa were treated with various concentrations (25, 50, 75, and 100%) of ethion solutions for different duration of time. The result revealed that increase in the concentration and duration of treatment decreases the mitotic indices. 24 h treatment at 100% concentration of ethion induced lowest mitotic index (20.08%) than that of the control (36.37%). The percentage of chromosomal abnormalities in different mitotic stages was significantly generally higher than that of the control in all the treatment period and concentrations. These abnormalities appeared in various degrees depending on the treatment duration and concentrations of ethion. The abnormalities in dividing cell reached a maximum value of 11.30% after 12 h of treatment at 75% concentration. The type of abnormalitiesproduced were scattered prophase, non-synchronized condensation of chromosome, disturbed prophase, equatorial plate shifting, sticky chromosomes, C-metaphase and sticky metaphase. Overall, it can be concluded that ethion has a potential genotoxic effects on mitotic divisions in A. cepa root tip cells. So, it will be necessary to test the mutagenic potential of ethion on a more intensive and extensive basis especially on non-target systems before it is recommended for wider use in agriculturalfield