NEUROPEPTIDE-F (MONIEZIA-EXPANSA) - LOCALIZATION AND CHARACTERIZATION USING SPECIFIC ANTISERA

Immunocytochemical techniques used in conjunction with confocal scanning laser microscopy (CSLM) and electron microscopy have been used to demonstrate, for the first time, the distribution of the parasitic platyhelminth neuropeptide, neuropeptide F (NPF) in the cestode, Moniezia expansa. Antisera were raised to intact NPF(1-39) and to the C-terminal decapeptide of NPF(30-39). These antisera were characterized and validated for use in both immunocytochemistry and radioimmunoassay (RIA). NPF immunoreactivity (IR) was detected using both antisera throughout all of the major components of the central and peripheral nervous systems of the worm. The pattern of NPF-IR was found to mirror the IR obtained using a C-terminally directed pancreatic polypeptide (PP) antiserum and FMRFamide antisera; blocking studies using these antisera revealed that FMRFamide and PP antisera cross-react with NPF(M. expansa). RIA of acid-alcohol extracts of the worm measured 114 ng/g using the C-terminal NPF antiserum and 56 ng/g using the whole-molecule-directed antiserum. While the C-terminally-directed NPF antiserum cross-reacts with NPF-related peptides from other invertebrates, the whole-molecule-directed NPF antiserum is specific for NPF(M. expansa). The C-terminal NPF antiserum has potential for use in the identification and purification of NPF analogues from other platyhelminth parasites.

Identification of an acetylation-dependant Ku70/FLIP complex that regulates FLIP expression and HDAC inhibitor-induced apoptosis

FLIP is a potential anti-cancer therapeutic target that inhibits apoptosis by blocking caspase 8 activation by death receptors. We report a novel interaction between FLIP and the DNA repair protein Ku70 that regulates FLIP protein stability by inhibiting its polyubiquitination. Furthermore, we found that the histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) enhances the acetylation of Ku70, thereby disrupting the FLIP/Ku70 complex and triggering FLIP polyubiquitination and degradation by the proteasome. Using in vitro and in vivo colorectal cancer models, we further demonstrated that SAHA-induced apoptosis is dependant on FLIP downregulation and caspase 8 activation. In addition, an HDAC6-speci?c inhibitor Tubacin recapitulated the effects of SAHA, suggesting that HDAC6 is a key regulator of Ku70 acetylation and FLIP protein stability. Thus, HDAC inhibitors with anti-HDAC6 activity act as ef?cient post-transcriptional suppressors of FLIP expression and may, therefore, effectively act as ‘FLIP inhibitors’ © 2012 Macmillan Publishers Limited.

Fetch Gating Control through Speculative Instruction Window Weighting

In a dynamic reordering superscalar processor, the front-end fetches instructions and places them in the issue queue. Instructions are then issued by the back-end execution core. Till recently, the front-end was designed to maximize performance without considering energy consumption. The front-end fetches instructions as fast as it can until it is stalled by a filled issue queue or some other blocking structure. This approach wastes energy: (i) speculative execution causes many wrong-path instructions to be fetched and executed, and (ii) back-end execution rate is usually less than its peak rate, but front-end structures are dimensioned to sustained peak performance. Dynamically reducing the front-end instruction rate and the active size of front-end structure (e.g. issue queue) is a required performance-energy trade-off. Techniques proposed in the literature attack only one of these effects.
In previous work, we have proposed Speculative Instruction Window Weighting (SIWW) [21], a fetch gating technique that allows to address both fetch gating and instruction issue queue dynamic sizing. SIWW computes a global weight on the set of inflight instructions. This weight depends on the number and types of inflight instructions (non-branches, high confidence or low confidence branches, ...). The front-end instruction rate can be continuously adapted based on this weight. This paper extends the analysis of SIWW performed in previous work. It shows that SIWW performs better than previously proposed fetch gating techniques and that SIWW allows to dynamically adapt the size of the active instruction queue.

Platinum-catalysed intravaginal rings

The present invention provides improved intravaginal drug delivery devices, i.e., intravaginal rings, useful for the prophylactic administration of an antimicrobial compound, e.g., Dapivirine, to a human. The intravaginal rings of the invention address previous stability issues by utilizing a platinum catalyst (e.g., in the form of a platinum-siloxane complex) for the cross-linking reaction. The vaginal rings surprisingly achieve relatively high and steady release rates in vivo with a matrix ring containing a relatively small loading dose. While the matrix rings of the present invention have in vivo the steady release rates associated with reservoir rings, they are easier and less expensive to manufacture. The present invention also provides methods of blocking DNA polymerization by an HIV reverse transcriptase enzyme, methods of preventing HIV infection in a female human, methods of treating HIV infection in a female human, and methods of preparing platinum-catalyzed intravaginal rings.

Identification of the F1-ATPase at the cell surface of colonic epithelial cells:role in mediating cell proliferation

F1F0-ATPase was initially believed to be strictly expressed in the mitochondrial membrane. Interestingly, recent reports have shown that the F1 complex can serve as a cell surface receptor for apparently unrelated ligands. Here, we show for the first time the presence of the F1-ATPase at the cell surface of normal or cancerous colonic epithelial cells. Using Surface Plasmon Resonance technology and mass spectrometry, we identified a peptide hormone product of the gastrin gene (glycine-extended gastrin, G-gly), as a new ligand for the F1-ATPase. By molecular modeling, we identified the motif in the peptide sequence (EE/DxY), which directly interacts with the F1-ATPase and the amino-acids in the F1-ATPase which bind this motif. Replacement of the E9 residue by an alanine in the EE/DxY motif resulted in a strong decrease of G-gly binding to the F1-ATPase and the loss of its biological activity. In addition we demonstrated that F1-ATPase mediates the growth effects of the peptide. Indeed, blocking ATPase activity decreases G-gly-induced cell growth. The mechanism likely involves ADP production by the membrane F1-ATPase which is induced by G-gly. These results suggest an important contribution of cell surface ATPase in the pro-proliferative action of this gastrointestinal peptide.

Low MAD2 expression levels associate with reduced progression-free survival in patients with high-grade serous epithelial ovarian cancer

Epithelial ovarian cancer (EOC) has an innate susceptibility to become chemoresistant. Up to 30% of patients do not respond to conventional chemotherapy [paclitaxel (Taxol®) in combination with carboplatin] and, of those who have an initial response, many patients relapse. Therefore, an understanding of the molecular mechanisms that regulate cellular chemotherapeutic responses in EOC cells has the potential to impact significantly on patient outcome. The mitotic arrest deficiency protein 2 (MAD2), is a centrally important mediator of the cellular response to paclitaxel. MAD2 immunohistochemical analysis was performed on 82 high-grade serous EOC samples. A multivariate Cox regression analysis of nuclear MAD2 IHC intensity adjusting for stage, tumour grade and optimum surgical debulking revealed that low MAD2 IHC staining intensity was significantly associated with reduced progression-free survival (PFS) (p = 0.0003), with a hazard ratio of 4.689. The in vitro analyses of five ovarian cancer cell lines demonstrated that cells with low MAD2 expression were less sensitive to paclitaxel. Furthermore, paclitaxel-induced activation of the spindle assembly checkpoint (SAC) and apoptotic cell death was abrogated in cells transfected with MAD2 siRNA. In silico analysis identified a miR-433 binding domain in the MAD2 3' UTR, which was verified in a series of experiments. Firstly, MAD2 protein expression levels were down-regulated in pre-miR-433 transfected A2780 cells. Secondly, pre-miR-433 suppressed the activity of a reporter construct containing the 3'-UTR of MAD2. Thirdly, blocking miR-433 binding to the MAD2 3' UTR protected MAD2 from miR-433 induced protein down-regulation. Importantly, reduced MAD2 protein expression in pre-miR-433-transfected A2780 cells rendered these cells less sensitive to paclitaxel. In conclusion, loss of MAD2 protein expression results in increased resistance to paclitaxel in EOC cells. Measuring MAD2 IHC staining intensity may predict paclitaxel responses in women presenting with high-grade serous EOC.

Managing diabetic nephropathy

The earliest sign of DN is the development of microalbuminuria which is associated with a significant risk of both progressive renal failure and premature death from cardiovascular disease. Optimal glycaemic and BP control, including the use of RAAS blocking drugs, can prevent, slow and even reverse the processes causing DN.

Up-regulation of the granulocyte adhesion molecule Mac-1 by autoantibodies in autoimmune vasculitis

The characteristic finding of autoantibodies in patients with vasculitis has raised the possibility that these antibodies play a role in the pathogenesis of the disease. The expression of adhesion molecules (AM) on leucocytes and endothelial cells is believed to be integral to the development of vasculitis. We therefore investigated the effect of sera, positive for anti-neutrophil cytoplasmic antibodies (ANCA) or anti-nuclear antibodies (ANA) from patients with vasculitis, on granulocyte expression of the adhesion molecule Mac-1 (CD11b). Autoantibody-positive sera from 15 out of 35 patients with vasculitis stimulated an up-regulation of Mac-1 on granulocytes. In most cases this effect was reproduced by the autoantibody-positive purified IgG fraction. Autoantibody-negative samples did not stimulate AM up-regulation. Of interest, preincubation of sera with purified antigens did not inhibit AM up-regulation by the autoantibody samples. Blocking the Fc receptors on granulocytes did result in a decrease of Mac-1 up-regulation, but this trend was not statistically significant. These results suggest that both ANCA and ANA have the capacity to up-regulate granulocyte AM expression, and that while Fc interaction with granulocyte Fc receptors is important, it is not the only mechanism whereby such autoantibodies activate cells.

Modeling colloid deposition on a protein layer adsorbed to iron-oxide-coated sand

Our recent study reported that conformation change of granule-associated Bovine Serum Albumin (BSA) may influence the role of the protein controlling colloid deposition in porous media (Flynn et al., 2012). The present study conceptualized the observed phenomena with an ellipsoid morphology model, describing BSA as an ellipsoid taking a side-on or end-on conformation on granular surface, and identified the following processes: (1) at low adsorbed concentrations, BSA exhibited a side-on conformation blocking colloid deposition; (2) at high adsorbed concentrations, BSA adapted to an end-on conformation promoted colloid deposition; and (3) colloid deposition on the BSA layer may progressively generate end-on molecules (sites) by conformation change of side-on BSA, resulting in sustained increasing deposition rates. Generally, the protein layer lowered colloid attenuation by the porous medium, suggesting the overall effect of BSA was inhibitory at the experimental time scale. A mathematical model was developed to interpret the ripening curves. Modeling analysis identified the site generation efficiency of colloid as a control on the ripening rate (declining rate in colloid concentrations), and this efficiency was higher for BSA adsorbed from a more dilute BSA solution. © 2012 Elsevier B.V.

The Anti-Migratory Effects of FKBPL and Its Peptide Derivative, AD-01: Regulation of CD44 and the Cytoskeletal Pathway

FK506 binding protein-like (FKBPL) and its peptide derivatives exert potent anti-angiogenic activity and and control tumour growth in xenograft models, when administered exogenously. However, the role of endogenous FKBPL in angiogenesis is not well characterised. Here we investigated the molecular effects of the endogenous protein and its peptide derivative, AD-01, leading to their anti-migratory activity. Inhibition of secreted FKBPL using a blocking antibody or siRNA-mediated knockdown of FKBPL accelerated the migration of human microvascular endothelial cells (HMEC-1). Furthermore, MDA-MB-231 tumour cells stably overexpressing FKBPL inhibited tumour vascular development suggesting that FKBPL secreted from tumour cells could inhibit angiogenesis. Whilst FKBPL and AD-01 target CD44, the nature of this interaction is not known and here we have further interrogated this aspect. We have demonstrated that FKBPL and AD-01 bind to the CD44 receptor and inhibit tumour cell migration in a CD44 dependant manner; CD44 knockdown abrogated AD-01 binding as well as its anti-migratory activity. Interestingly, FKBPL overexpression and knockdown or treatment with AD-01, regulated CD44 expression, suggesting a co-regulatory pathway for these two proteins. Downstream of CD44, alterations in the actin cytoskeleton, indicated by intense cortical actin staining and a lack of cell spreading and communication were observed following treatment with AD-01, explaining the anti-migratory phenotype. Concomitantly, AD-01 inhibited Rac-1 activity, up-regulated RhoA and the actin binding proteins, profilin and vinculin. Thus the anti-angiogenic protein, FKBPL, and AD-01, offer a promising and alternative approach for targeting both CD44 positive tumours and vasculature networks.

Multiplex Detection of Plant Pathogens Using a Microsphere Immunoassay Technology

Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), chilli vein-banding mottle virus (CVbMV, potyvirus), watermelon silver mottle virus (WSMoV, tospovirus serogroup IV) and melon yellow spot virus (MYSV, tospovirus). An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE)-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA) when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour) was much shorter than that of ELISA (4 hours). This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection. © 2013 Charlermroj et al.

Influence of the type of coarse lightweight aggregate on properties of Semi-Lightweight Self-Consolidating Concrete

This paper presents studies on the properties of fresh and hardened semilightweight self-consolidating concrete (SLWSCC) mixtures, produced with two types of manufactured coarse lightweight aggregates (LWA) and normal weight sand. The first type, a sintered pulverized fuel ash, was made from an industrial by-product, fly ash, whereas the second one, an expanded clay, was produced from a naturally sourced clay. For all mixtures, normal weight sand was used as a fine fraction of aggregates, and the portland cement was partially replaced with a limestone powder. The SLWSCC was produced with different water presaturation regimes of the LWAs. The desired initial slump-flow spread was set between 700 and 800 mm. The effect of three superplasticizers was evaluated by testing properties of SLWSCC, normal weight SCC, and paste mixtures. Three SCC fresh properties were measured: the slump-flow, the V-funnel flow time, and the J-ring blocking step. Moreover, the slump-flow loss was evaluated. The degree of segregation was assessed in both fresh and hardened states. Additionally, the hardened density and the compressive strengths were tested. All SLWSCC mixtures were produced with a desired range of slump-flow spread and with satisfactory passing ability assessed with the J-ring test. SLWSCCs prepared with the expanded clay LWA were less sensitive to the variation of water presaturation levels and showed lower viscosity than those made with the sintered pulverized fuel ash LWA. Only mixtures containing SP-3 superplasticizer showed acceptable workability loss resistance. The saturated surface-dry density of all of the mixtures varied in a range of 2,025–2,125??kg/m 3 . Mixtures containing 29% of coarse LWAs and 71% of sand (by mass) had 24-h and 28-day compressive strengths above 20 and 40 MPa, respectively, but the mixtures made with the expanded clay were slightly weaker.

Serological correlate of protection against norovirus-induced gastroenteritis

Norovirus infection is the leading cause of acute nonbacterial gastroenteritis. Histoblood group antigens (HBGAs) are host susceptibility determinants for Norwalk virus (NV) infection. We hypothesized that antibodies that block NV-HBGA binding are associated with protection from clinical illness following NV exposure.

VEIDase is a principal caspase-like activity involved in plant programmed cell death and essential for embryonic pattern formation

Plant embryogenesis is intimately associated with programmed cell death. The mechanisms of initiation and control of programmed cell death during plant embryo development are not known. Proteolytic activity associated with caspase-like proteins is paramount for control of programmed cell death in animals and yeasts. Caspase family of proteases has unique strong preference for cleavage of the target proteins next to asparagine residue. In this work, we have used synthetic peptide substrates containing caspase recognition sites and corresponding specific inhibitors to analyse the role of caspase-like activity in the regulation of programmed cell death during plant embryogenesis. We demonstrate that VEIDase is a principal caspase-like activity implicated in plant embryogenesis. This activity increases at the early stages of embryo development that coincide with massive cell death during shape remodeling. The VEIDase activity exhibits high sensitivity to pH, ionic strength and Zn2+ concentration. Altogether, biochemical assays show that VEIDase plant caspase-like activity resembles that of both mammalian caspase-6 and yeast metacaspase, YCA1. In vivo, VEIDase activity is localised specifically in the embryonic cells during both the commitment and in the beginning of the execution phase of programmed cell death. Inhibition of VEIDase prevents normal embryo development via blocking the embryo-suspensor differentiation. Our data indicate that the VEIDase activity is an integral part in the control of plant developmental cell death programme, and that this activity is essential for the embryo pattern formation.

Gelsolin induces colorectal tumor cell invasion via modulation of the urokinase-type plasminogen activator cascade

Gelsolin is a cytoskeletal protein which participates in actin filament dynamics and promotes cell motility and plasticity. Although initially regarded as a tumor suppressor, gelsolin expression in certain tumors correlates with poor prognosis and therapy-resistance. In vitro, gelsolin has anti-apoptotic and pro-migratory functions and is critical for invasion of some types of tumor cells. We found that gelsolin was highly expressed at tumor borders infiltrating into adjacent liver tissues, as examined by immunohistochemistry. Although gelsolin contributes to lamellipodia formation in migrating cells, the mechanisms by which it induces tumor invasion are unclear. Gelsolin's influence on the invasive activity of colorectal cancer cells was investigated using overexpression and small interfering RNA knockdown. We show that gelsolin is required for invasion of colorectal cancer cells through matrigel. Microarray analysis and quantitative PCR indicate that gelsolin overexpression induces the upregulation of invasion-promoting genes in colorectal cancer cells, including the matrix-degrading urokinase-type plasminogen activator (uPA). Conversely, gelsolin knockdown reduces uPA levels, as well as uPA secretion. The enhanced invasiveness of gelsolin-overexpressing cells was attenuated by treatment with function-blocking antibodies to either uPA or its receptor uPAR, indicating that uPA/uPAR activity is crucial for gelsolin-dependent invasion. In summary, our data reveals novel functions of gelsolin in colorectal tumor cell invasion through its modulation of the uPA/uPAR cascade, with potentially important roles in colorectal tumor dissemination to metastatic sites.