De-novo design of complementary (antisense) peptide mini-receptor inhibitor of interleukin 18 (IL-18).

Complementary (antisense) peptide mini-receptor inhibitors are complementary peptides designed to be receptor-surrogates that act by binding to selected surface features of biologically important proteins thereby inhibiting protein-cognate receptor interactions and subsequent biological effects. Previously, we described a complementary peptide mini-receptor inhibitor of interleukin-1beta (IL-1beta) that was designed to bind to an external surface loop (beta-bulge) of IL-1beta (Boraschi loop) clearly identified in the X-ray crystal structure of this cytokine. Here, we report the de-novo design and rational development of a complementary peptide mini-receptor inhibitor of cytokine interleukin-18 (IL-18), a protein for which there is no known X-ray crystal structure. Using sequence homology comparisons with IL-1beta, putative IL-18 surface loops are identified and used as a starting point for design, including a loop region 1 thought to be equivalent with the Boraschi loop of IL-1beta. Only loop region 1 complementary peptides are found to be promising leads as mini-receptor inhibitors of IL-18 but these are prevented from being properly successful owing to solubility problems. The application of "M-I pair mutagenesis" and inclusion of a C-terminal arginine residue are then sufficient to solve this problem and convert one lead peptide into a functional complementary peptide mini-receptor inhibitor of IL-18. This suggests that the biophysical and biological properties of complementary peptides can be improved in a rational and logical manner where appropriate, further strengthening the potential importance of complementary peptides as inhibitors of protein-protein interactions, even when X-ray crystal structural information is not readily available

Biological properties of adriamycin bound to biodegradable polymeric carriers

Three different conjugates having adriamycin (ADR) bound to the side chain carboxyl groups of high-molecular weight poly (¿--glutamic acid) (PGA) either directly or by interpolation of GlyGly and GlyGlyGlyLeu spacers, respectively, were compared with respect to immunogenicity and cytotoxicity in mice as well as release of drug by lysosomal enzymes. The cytotoxic efficacy of a single i.p. dose of each conjugate (5 mg ADR-equiv./kg) against L1210 leukemia cells implanted i.p. in DBA2 mice was studied by monitoring the survival time, the body weight and the number of long-term survivors (LTS). PGA-GlyGlyGlyLeu-ADR and PGA-GlyGly-ADR significantly enhanced the mean survival time (MST) of treated animals compared with the untreated control group (T/C 148¿149%) as did free ADR (T/C 147%). The tetrapeptide-spacer containing conjugate effected the presence of LTS at day 50 (2/5) as did free ADR (1/5).\ud \ud The secondary antibody response of the drug conjugates elicited in A/J mice after repeated dosage (125 ¿g/mouse) at day 0, 14 and 28 was evaluated at day 35 using the ELISA technique. IgG titers varied from a very low value (PGA-GlyGlyGlyLeu-ADR) to moderately high levels (PGA-ADR, PGA-GlyGly-ADR) which are 2¿3 orders of magnitude below that obtained for the strong immunogen bovine IgG. The data suggest that certain parts on the surface of the conjugates are immunogenic.\ud \ud The release of extractable low-molecular weight products from the conjugates mediated by lysosomal enzymes was analyzed using reversed-phase HPLC. The release profile of ADR as well as Gly-ADR, Leu-ADR or GlyLeu-ADR was determined. The total amount of ADR released after 77 h was 3.6% for PGA-GlyGlyGly-Leu-ADR, 1.0% for PGA-GlyGly-ADR and 0.5% for PGA-ADR. With all conjugates unidentified products were produced.\ud \ud It is proposed that the mechanism of action of the polymeric conjugates under in vivo conditions may be due to pinocytic capture followed by lysosomal degradation with release of ADR

Private governance of human and labor rights in seafood supply chains:The case of the modern slavery crisis in Thailand

A growing recognition of human rights in business has shed light on labor violations and abusive practices that prevail in many global supply chains. The recent 'modern slavery' crisis in the Thai fishing industry not only brings the question of government's responsibility to the fore but also increasingly highlights the role of private governance in global supply chains. This paper provides an updated analysis on the state of labor rights protection in the Thai fishing industry by analyzing responses from private business and civil society to the modern slavery scandal. We focus on three responses in particular: ethical recruitment policies, worker grievance mechanisms and worker associations. We analyse the effectiveness of these responses and delineate the potential of private governance as well as the limits that need to be overcome to ensure the protection of human and labor rights in global seafood supply chains

Homing of stem cells to sites of inflammatory brain injury after intracerebral and intravenous administration: a longitudinal imaging study

Introduction This study aimed to determine the homing potential and fate of epidermal neural crest stem cells (eNCSCs) derived from hair follicles, and bone marrow-derived stem cells (BMSCs) of mesenchymal origin, in a lipopolysaccharide (LPS)-induced inflammatory lesion model in the rat brain. Both eNCSCs and BMSCs are easily accessible from adult tissues by using minimally invasive procedures and can differentiate into a variety of neuroglial lineages. Thus, these cells have the potential to be used in autologous cell-replacement therapies, minimizing immune rejection, and engineered to secrete a variety of molecules. Methods Both eNCSCs and BMSCs were prelabeled with iron-oxide nanoparticles (IO-TAT-FITC) and implanted either onto the corpus callosum in healthy or LPS-lesioned animals or intravenously into lesioned animals. Both cell types were tracked longitudinally in vivo by using magnetic resonance imaging (MRI) for up to 30 days and confirmed by postmortem immunohistochemistry. Results Transplanted cells in nonlesioned animals remained localized along the corpus callosum. Cells implanted distally from an LPS lesion (either intracerebrally or intravenously) migrated only toward the lesion, as seen by the localized MRI signal void. Fluorescence microscopy of the FITC tag on the nanoparticles confirmed the in vivo MRI data, Conclusions This study demonstrated that both cell types can be tracked in vivo by using noninvasive MRI and have pathotropic properties toward an inflammatory lesion in the brain. As these cells differentiate into the glial phenotype and are derived from adult tissues, they offer a viable alternative autologous stem cell source and gene-targeting potential for neurodegenerative and demyelinating pathologies. </br

Pretreatment Effects on the Uptake/Retention Kinetics of L-Dopa in Harding-Passey Melanoma

Malignant melanoma cells possess a unique biochemical pathway that converts L-3,4-dihydroxyphenylalanine (L-dopa) to the biopigment melanin. Selective cytotoxic incorporation of exogenous L-dopa into melanoma cells in vivo may provide a means of designing specific chemotherapeutic agents useful in the treatment of this disease. Using the Harding-Passey murine melanotic tumor model, a preferential uptake of [3H]L-dopa by the tumor was characterized. Following pretreatment of the tumor-bearing mice with nonradioactive L-dopa, a significant enhancement (p < 0.01) of [3H]L-dopa incorporation and retention into melanoma for a period of 24h was observed, when compared with the concomitant tissue distribution and clearance of radioactivity in the control animals. This finding suggests that by initial pretreatment of melanoma with nonradioactive L-dopa, the subsequent selective accumulation of [3H]L-dopa in tumor may provide a useful tool in testing new modalities of therapy in malignant melanoma

Bacterial adhesion and biofilms on surfaces

Bacterial adhesion has become a significant problem in industry and in the domicile, and much research has been done for deeper understanding of the processes involved. A generic biological model of bacterial adhesion and population growth called the bacterial biofilm growth cycle, has been described and modified many times. The biofilm growth cycle encompasses bacterial adhesion at all levels, starting with the initial physical attraction of bacteria to a substrate, and ending with the eventual liberation of cell clusters from the biofilm matrix. When describing bacterial adhesion one is simply describing one or more stages of biofilm development, neglecting the fact that the population may not reach maturity. This article provides an overview of bacterial adhesion, cites examples of how bac-terial adhesion affects industry and summarises methods and instrumentation used to improve our understanding of the adhesive prop-erties of bacteria

Biocompatible Peptide-Coated Ultrasmall Superparamagnetic Iron Oxide Nanoparticles for In Vivo Contrast-Enhanced Magnetic Resonance Imaging

The biocompatibility and performance of reagents for in vivo contrast-enhanced magnetic resonance imag-ing are essential for their translation to the clinic. The quality of the surface coating of nanoparticle-based MRI contrast agents, such as ultra-small superparamagnetic iron oxide nanoparticles (USPIONs), is criti-cal to ensure high colloidal stability in biological environments, improved magnetic performance and dis-persion in circulatory fluids and tissues. Herein, we report the design of a library of 21 peptides and lig-ands and identify highly stable self-assembled monolayers on the USPIONs surface. A total of 86 differ-ent peptide coated USPIONs are prepared and selected using several stringent criteria, e.g., stability against electrolyte-induced aggregation in physiological conditions, prevention of non-specific binding to cells, absence of cellular toxicity and contrast-enhanced in vivo MRI. The bis-phosphorylated peptide 2PG-S∗VVVT-PEG4-ol provides highest biocompatibility and performance for USPIONs, with no de-tectable toxicity or adhesion to live cells. The 2PG-S∗VVVT-PEG4-ol coated USPIONs show enhanced magnetic resonance properties, r1 (2.4 mM-1.s-1) and r2 (217.8 mM-1.s-1) relaxivities, and greater r2/r1 relaxivity ratios (>90), when compared to commercially available MRI contrast agents. Furthermore, we demonstrate the utility of 2PG-S∗VVVT-PEG4-ol coated USPIONs as a T2 contrast agent for in vivo MRI applica-tions. High contrast enhancement of the liver is achieved as well as detection of liver tumors, with signifi-cant improvement of the contrast-to-noise ratio of tumor-to-liver contrast. It is envisaged that the reported peptide coated USPIONs have the potential to allow for the specific targeting of tumors, and hence early detection of cancer by MRI

Characterisation of secondary metabolites associated with neutrophil apoptosis

We studied changes in secondary metabolites in human neutrophils undergoing constitutive or tumour necrosis factor (TNFalpha) stimulated apoptosis by a combination of high-performance liquid chromatography (HPLC) and NMR spectroscopy. Our results show that in contrast to freshly isolated neutrophils, neutrophil cells aged for 20 h in vitro had marked differences in the levels of a number of endogenous metabolites including lactate, amino acids and phosphocholine (PCho). There was no change in the concentration of taurine or glutamate and the ATP/ADP ratio was not affected. Levels of glutamine and lactate actually decreased. Identical changes were also observed in neutrophils stimulated to undergo apoptosis over a shorter time period (6 h) in the presence of TNFalpha and the phosphatidylinositol-3-kinase inhibitor wortmannin (WM). The changes in the concentration of PCho suggest possible activation of phospholipase associated with apoptosis or a selective failure of phosphatidycholine synthesis. The increased levels of apoptosis obtained with WM+TNFalpha, compared to TNFalpha by itself, suggest a synergistic effect by these compounds. The acceleration in rate of apoptosis probably arises from suppression by WM of pathway(s) that normally delay the onset of apoptosis. Changes in PCho and other endogenous metabolites, if proven to be characteristic of apoptosis in other cell systems, may permit non-invasive quantification of apoptosis.

Apoptosis is associated with triacylglycerol accumulation in Jurkat T-cells

Magnetic resonance spectroscopy is increasingly used as a non-invasive method to investigate apoptosis. Apoptosis was induced in Jurkat T-cells by Fas mAb. 1H magnetic resonance spectra of live cells showed an increase in methylene signal as well as methylene/methyl ratio of fatty acid side chains at 5 and 24 h following induction of apoptosis. To explain this observation, 1H magnetic resonance spectra of cell extracts were investigated. These demonstrated a 70.0±7.0%, 114.0±8.0% and 90.0±5.0% increase in the concentration of triacylglycerols following 3, 5 and 7 h of Fas mAb treatment (P<0.05). Confocal microscopy images of cells stained with the lipophilic dye Nile Red demonstrated the presence of lipid droplets in the cell cytoplasm. Quantification of the stained lipids by flow cytometry showed a good correlation with the magnetic resonance results (P⩾0.05 at 3, 5 and 7 h). 31P magnetic resonance spectra showed a drop in phosphatidylcholine content of apoptosing cells, indicating that alteration in phosphatidylcholine metabolism could be the source of triacylglycerol accumulation during apoptosis. In summary, apoptosis is associated with an early accumulation of mobile triacylglycerols mostly in the form of cytoplasmic lipid droplets. This is reflected in an increase in the methylene/methyl ratio which could be detected by magnetic resonance spectroscopy