The use of thermographic imaging to evaluate therapeutic response in human tumour xenograft models

YesNon-invasive methods to monitor tumour growth are an important goal in cancer drug development. Thermographic imaging systems offer potential in this area, since a change in temperature is known to be induced due to changes within the tumour microenvironment. This study demonstrates that this imaging modality can be applied to a broad range of tumour xenografts and also, for the first time, the methodology’s suitability to assess anti-cancer agent efficacy. Mice bearing subcutaneously implanted H460 lung cancer xenografts were treated with a novel vascular disrupting agent, ICT-2552, and the cytotoxin doxorubicin. The effects on tumour temperature were assessed using thermographic imaging over the first 6 hours post-administration and subsequently a further 7 days. For ICT-2552 a significant initial temperature drop was observed, whilst for both agents a significant temperature drop was seen compared to controls over the longer time period. Thus thermographic imaging can detect functional differences (manifesting as temperature reductions) in the tumour response to these anti-cancer agents compared to controls. Importantly, these effects can be detected in the first few hours following treatment and therefore the tumour is observable non-invasively. As discussed, this technique will have considerable 3Rs benefits in terms of reduction and refinement of animal use.University of Bradfor

Increased Aβ pathology in aged Tg2576 mice born to mothers fed a high fat diet

Maternal obesity is associated with increased risk of developing diabetes, obesity and premature death in adult offspring. Mid-life diabetes, hypertension and hypercholesterolaemia are risk factors for the development of sporadic Alzheimer's disease (AD). A key pathogenic feature of AD is the accumulation of β-amyloid (Aβ) in the brain. The purpose of this study was to investigate the effect of high fat diet feeding during early life on Aβ pathology in the Tg2576 mouse model of AD. Female mice were fed a standard (C) or high fat (HF) diet before mating and during gestation and lactation. At weaning, male offspring were fed a C diet. Significantly higher levels of guanidine-soluble Aβ and plaque loads were observed in the hippocampi of 11-month old Tg2576 mice born to mothers fed a HF diet. Changes in the extracellular matrix led to increased retention of Aβ within the parenchyma. These data support a role for maternal and gestational health on the health of the aged brain and pathologies associated with AD and may provide a novel target for both the prevention and treatment of AD

TLR 9 Activation in Dendritic Cells Enhances Salmonella Killing and Antigen Presentation via Involvement of the Reactive Oxygen Species

Synthetic CpG containing oligodeoxynucleotide Toll like receptor-9 agonist (CpG DNA) activates innate immunity and can stimulate antigen presentation against numerous intracellular pathogens. It was observed that Salmonella Typhimurium growth can be inhibited by the CpG DNA treatment in the murine dendritic cells. This inhibitory effect was mediated by an increased reactive oxygen species production. In addition, it was noted that CpG DNA treatment of dendritic cells during Salmonella infection leads to an increased antigen presentation. Further this increased antigen presentation was dependent on the enhanced reactive oxygen species production elicited by Toll like receptor-9 activation. With the help of an exogenous antigen it was shown that Salmonella antigen could also be cross-presented in a better way by CpG induction. These data collectively indicate that CpG DNA enhance the ability of murine dendritic cells to contain the growth of virulent Salmonella through reactive oxygen species dependent killing

New approaches to measuring anthelminthic drug efficacy: parasitological responses of childhood schistosome infections to treatment with praziquantel

By 2020, the global health community aims to control and eliminate human helminthiases, including schistosomiasis in selected African countries, principally by preventive chemotherapy (PCT) through mass drug administration (MDA) of anthelminthics. Quantitative monitoring of anthelminthic responses is crucial for promptly detecting changes in efficacy, potentially indicative of emerging drug resistance. Statistical models offer a powerful means to delineate and compare efficacy among individuals, among groups of individuals and among populations.; We illustrate a variety of statistical frameworks that offer different levels of inference by analysing data from nine previous studies on egg counts collected from African children before and after administration of praziquantel.; We quantify responses to praziquantel as egg reduction rates (ERRs), using different frameworks to estimate ERRs among population strata, as average responses, and within strata, as individual responses. We compare our model-based average ERRs to corresponding model-free estimates, using as reference the World Health Organization (WHO) 90 % threshold of optimal efficacy. We estimate distributions of individual responses and summarize the variation among these responses as the fraction of ERRs falling below the WHO threshold.; Generic models for evaluating responses to anthelminthics deepen our understanding of variation among populations, sub-populations and individuals. We discuss the future application of statistical modelling approaches for monitoring and evaluation of PCT programmes targeting human helminthiases in the context of the WHO 2020 control and elimination goals

Modulation of the Arginase Pathway in the Context of Microbial Pathogenesis: A Metabolic Enzyme Moonlighting as an Immune Modulator

Arginine is a crucial amino acid that serves to modulate the cellular immune response during infection. Arginine is also a common substrate for both inducible nitric oxide synthase (iNOS) and arginase. The generation of nitric oxide from arginine is responsible for efficient immune response and cytotoxicity of host cells to kill the invading pathogens. On the other hand, the conversion of arginine to ornithine and urea via the arginase pathway can support the growth of bacterial and parasitic pathogens. The competition between iNOS and arginase for arginine can thus contribute to the outcome of several parasitic and bacterial infections. There are two isoforms of vertebrate arginase, both of which catalyze the conversion of arginine to ornithine and urea, but they differ with regard to tissue distribution and subcellular localization. In the case of infection with Mycobacterium, Leishmania, Trypanosoma, Helicobacter, Schistosoma, and Salmonella spp., arginase isoforms have been shown to modulate the pathology of infection by various means. Despite the existence of a considerable body of evidence about mammalian arginine metabolism and its role in immunology, the critical choice to divert the host arginine pool by pathogenic organisms as a survival strategy is still a mystery in infection biology

Increased Secreted Amyloid Precursor Protein-α (sAPPα) in Severe Autism: Proposal of a Specific, Anabolic Pathway and Putative Biomarker

Autism is a neurodevelopmental disorder characterized by deficits in verbal communication, social interactions, and the presence of repetitive, stereotyped and compulsive behaviors. Excessive early brain growth is found commonly in some patients and may contribute to disease phenotype. Reports of increased levels of brain-derived neurotrophic factor (BDNF) and other neurotrophic-like factors in autistic neonates suggest that enhanced anabolic activity in CNS mediates this overgrowth effect. We have shown previously that in a subset of patients with severe autism and aggression, plasma levels of the secreted amyloid-β (Aβ) precursor protein-alpha form (sAPPα) were significantly elevated relative to controls and patients with mild-to-moderate autism. Here we further tested the hypothesis that levels of sAPPα and sAPPβ (proteolytic cleavage products of APP by α- and β-secretase, respectively) are deranged in autism and may contribute to an anabolic environment leading to brain overgrowth. We measured plasma levels of sAPPα, sAPPβ, Aβ peptides and BDNF by corresponding ELISA in a well characterized set of subjects. We included for analysis 18 control, 6 mild-to-moderate, and 15 severely autistic patient plasma samples. We have observed that sAPPα levels are increased and BDNF levels decreased in the plasma of patients with severe autism as compared to controls. Further, we show that Aβ1-40, Aβ1-42, and sAPPβ levels are significantly decreased in the plasma of patients with severe autism. These findings do not extend to patients with mild-to-moderate autism, providing a biochemical correlate of phenotypic severity. Taken together, this study provides evidence that sAPPα levels are generally elevated in severe autism and suggests that these patients may have aberrant non-amyloidogenic processing of APP

Activity and Interactions of Liposomal Antibiotics in Presence of Polyanions and Sputum of Patients with Cystic Fibrosis

BACKGROUND:To compare the effectiveness of liposomal tobramycin or polymyxin B against Pseudomonas aeruginosa in the Cystic Fibrosis (CF) sputum and its inhibition by common polyanionic components such as DNA, F-actin, lipopolysaccharides (LPS), and lipoteichoic acid (LTA). METHODOLOGY:Liposomal formulations were prepared from a mixture of 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) or 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) and Cholesterol (Chol), respectively. Stability of the formulations in different biological milieus and antibacterial activities compared to conventional forms in the presence of the aforementioned inhibitory factors or CF sputum were evaluated. RESULTS:The formulations were stable in all conditions tested with no significant differences compared to the controls. Inhibition of antibiotic formulations by DNA/F-actin and LPS/LTA was concentration dependent. DNA/F-actin (125 to 1000 mg/L) and LPS/LTA (1 to 1000 mg/L) inhibited conventional tobramycin bioactivity, whereas, liposome-entrapped tobramycin was inhibited at higher concentrations--DNA/F-actin (500 to 1000 mg/L) and LPS/LTA (100 to 1000 mg/L). Neither polymyxin B formulation was inactivated by DNA/F-actin, but LPS/LTA (1 to 1000 mg/L) inhibited the drug in conventional form completely and higher concentrations of the inhibitors (100 to 1000 mg/L) was required to inhibit the liposome-entrapped polymyxin B. Co-incubation with inhibitory factors (1000 mg/L) increased conventional (16-fold) and liposomal (4-fold) tobramycin minimum bactericidal concentrations (MBCs), while both polymyxin B formulations were inhibited 64-fold. CONCLUSIONS:Liposome-entrapment reduced antibiotic inhibition up to 100-fold and the CFU of endogenous P. aeruginosa in sputum by 4-fold compared to the conventional antibiotic, suggesting their potential applications in CF lung infections

Confirmed adult dengue deaths in Singapore: 5-year multi-center retrospective study

10.1186/1471-2334-11-123BMC Infectious Diseases11-BIDM

HAD hydrolase function unveiled by substrate screening: enzymatic characterization of Arabidopsis thaliana subclass I phosphosugar phosphatise AtSgpp

[EN] This work presents the isolation and the biochemical characterization of the Arabidopsis thaliana gene AtSgpp. This gene shows homology with the Arabidopsis low molecular weight phosphatases AtGpp1 and AtGpp2 and the yeast counterpart GPP1 and GPP2, which have a high specificity for dl-glycerol-3-phosphate. In addition, it exhibits homology with DOG1 and DOG2 that dephosphorylate 2-deoxy-d-glucose-6-phosphate. Using a comparative genomic approach, we identified the AtSgpp gene as a conceptual translated haloacid dehalogenase-like hydrolase HAD protein. AtSgpp (locus tag At2g38740), encodes a protein with a predicted Mw of 26.7 kDa and a pI of 4.6. Its sequence motifs and expected structure revealed that AtSgpp belongs to the HAD hydrolases subfamily I, with the C1-type cap domain. In the presence of Mg2+ ions, the enzyme has a phosphatase activity over a wide range of phosphosugars substrates (pH optima at 7.0 and K (m) in the range of 3.6-7.7 mM). AtSgpp promiscuity is preferentially detectable on d-ribose-5-phosphate, 2-deoxy-d-ribose-5-phosphate, 2-deoxy-d-glucose-6-phosphate, d-mannose-6-phosphate, d-fructose-1-phosphate, d-glucose-6-phosphate, dl-glycerol-3-phosphate, and d-fructose-6-phosphate, as substrates. AtSgpp is ubiquitously expressed throughout development in most plant organs, mainly in sepal and guard cell. Interestingly, expression is affected by abiotic and biotic stresses, being the greatest under Pi starvation and cyclopentenone oxylipins induction. Based on both, substrate lax specificity and gene expression, the physiological function of AtSgpp in housekeeping detoxification, modulation of sugar-phosphate balance and Pi homeostasis, is provisionally assigned.We acknowledge Professors Montserrat Pages (CSIC Barcelona, Spain), Thomas Kupke (University of Heidelberg, Germany) and Manuel Hernandez (University Polytechnic of Valencia, Spain) for their warm support. We also thank the advice and provision of plasmid pSBETa by Dr. Florence Vignols and Yves Meyer (University of Perpignan, France); the computer software helps by Ramon Nogales-Rangel and Alexis Gonzalez-Policarpo; Eugenio Grau-Ferrando for kind advice and help for sequencing. This work was funded by the 10 month research contract MEC-FEDER to J.A.C.-M., 10 month research contract JAE-DOC to I.M.-S. and by the research project BIO2006-10138 from the MEC-FEDER of Spain to F.A.C.-M. In memoriam of Dr. Mari Cruz Cutanda-Perez.Caparrós Martín, JA.; Mccarthy Suarez, I.; Culiañez Macia, FA. (2013). HAD hydrolase function unveiled by substrate screening: enzymatic characterization of Arabidopsis thaliana subclass I phosphosugar phosphatise AtSgpp. Planta. 237(4):943-954. https://doi.org/10.1007/s00425-012-1809-5S9439542374Allen KN, Dunaway-Mariano D (2004) Phosphoryl group transfer: evolution of a catalytic scaffold. Trends Biochem Sci 29:495–503Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410Ames BN (1966) Assay of inorganic phosphate, total phosphate, and phosphatases. Methods Enzymol 8:115–118Böhmer M, Schroeder JI (2011) Quantitative transcriptomic analysis of abscisic acid-induced and reactive oxygen species-dependent expression changes and proteomic profiling in Arabidopsis suspension cells. Plant J 67:105–118Bradford MM (1976) A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254Burroughs AM, Allen KN, Dunaway-Mariano D, Aravind L (2006) Evolutionary genomics of the HAD superfamily: understanding the structural adaptations and catalytic diversity in a superfamily of phosphoesterases and allied enzymes. J Mol Biol 361:1003–1034Caparrós-Martín JA, Reiland S, Köchert K, Cutanda MC, Culiáñez-Macia FA (2007) Arabidopsis thaliana AtGpp 1 and AtGpp2: two novel low molecular weight phosphatases involved in plant glycerol metabolism. Plant Mol Biol 63:505–517Collet JF, Stroobant V, Pirard M, Delpierre G, Van Schaftingen E (1998) A new class of phosphotransferases phosphorylated on an aspartate residue in an amino-terminal DXDX(T/V) motif. J Biol Chem 273:14107–14112Corpet F, Servantm F, Gouzy J, Kahn D (2000) ProDom and ProDom-CG: tools for protein domain analysis and whole genome comparisons. Nucleic Acids Res 28:267–269Cutanda MC (2003) Effect of altering levels of hexoses phosphate in carbohydrate metabolism and glucose signalling in yeast and plants. PhD thesis, Polytechnic University of Valencia, Valencia, SpainHiggins D, Thompson J, Gibson T, Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680Koonin EV, Tatusov RL (1994) Computer analysis of bacterial haloacid dehalogenases defines a large superfamily of hydrolases with diverse specificity. Application of an iterative approach to database search. J Mol Biol 244:125–132Kupke T, Caparrós-Martín JA, Malquichagua Salazar KJ, Culiàñez-Macià FA (2009) Biochemical and physiological characterization of Arabidopsis thaliana AtCoAse: a Nudix CoA hydrolyzing protein that improves plant development. Physiol Plant 135:365–378Kuznetsova E, Proudfoot M, Sanders SA, Reinking J, Savchenko A, Arrowsmith CH, Edwards AM, Yakunin AF (2005) Enzyme genomics: application of general enzymatic screens to discover new enzymes. FEMS Microbiol Rev 29:263–279Kuznetsova E, Proudfoo M, Gonzalez CF, Brown G, Omelchenko MV, Borozan I, Carmel L, Wolf YI, Mori H, Savchenko AV, Arrowsmith CH, Koonin EV, Edwards AM, Yakunin AF (2006) Genome-wide analysis of substrate specificities of the Escherichia coli haloacid dehalogenase-like phosphatase family. J Biol Chem 281:36149–36161Lahiri SD, Zhang G, Dai J, Dunaway-Mariano D, Allen KN (2004) Analysis of the substrate specificity loop of the HAD superfamily cap domain. Biochemistry 43:2812–2820Lahiri SD, Zhang G, Dunaway-Mariano D, Allen KN (2006) Diversification of function in the haloacid dehalogenase enzyme superfamily: the role of the cap domain in hydrolytic phosphorus—carbon bond cleavage. Bioorganic Chem 34:394–409Lambert C, Leonard N, De Bolle X, Depiereux E (2002) ESyPred3D: prediction of proteins 3D structures. Bioinformatics 18:1250–1256Lu Z, Dunaway-Mariano D, Allen KN (2005) HAD superfamily phosphotransferase substrate diversification: structure and function analysis of HAD subclass IIB sugar phosphatase BT4131. Biochemistry 44:8684–8696Lu Z, Dunaway-Mariano D, Allen KN (2008) The catalytic scaffold of the haloalkanoic acid dehalogenase enzyme superfamily acts as a mold for the trigonal bipyramidal transition state. Proc Natl Acad Sci USA 105:5687–5692Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborMorais MC, Zhang W, Baker AS, Zhang G, Dunaway-Mariano D, Allen KN (2000) The crystal structure of Bacillus cereus phosphonoacetaldehyde hydrolase: insight into catalysis of phosphorus bond cleavage and catalytic diversification within the HAD enzyme superfamily. Biochemistry 39:10385–10396Morais MC, Zhang G, Zhang W, Olsen DB, Dunaway-Mariano D, Allen KN (2004) X-ray crystallographic and site-directed mutagenesis analysis of the mechanism of Schiff-base formation in phosphonoacetaldehyde hydrolase catalysis. J Biol Chem 279:9353–9361Mueller WS, Hilbert B, Dueckershoff K, Roitsch T, Krischke M, Mueller MJ, Berger S (2008) General detoxification and stress responses are mediated by oxidized lipids through TGA transcription factors in Arabidopsis. Plant Cell 20:768–785Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco cultures. Physiol Plant 15:473–497Norbeck J, Pahlman AK, Akhtar N, Blomberg A, Adler L (1996) Purification and characterization of two isoenzymes of dl-glycerol-3-phosphatase from Saccharomyces cerevisiae. Identification of the corresponding GPP1 and GPP2 genes and evidence for osmotic regulation of Gpp 2p expression by the osmosensing mitogen-activated protein kinase signal transduction pathway. J Biol Chem 271:13875–13881Rández-Gil F, Blasco A, Prieto JA, Sanz P (1995) DOGR1 and DOGR2: two genes from Saccharomyces cerevisiae that confer 2-deoxyglucose resistance when overexpressed. Yeast 11:1233–1240Rao KN, Kumaran D, Seetharaman J, Bonanno JB, Burley SK, Swaminathan S (2006) Crystal structure of trehalose-6-phosphate phosphatase-related protein: biochemical and biological implications. Protein Sci 15:1735–1744Schagger H, von Jagow G (1987) Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379Schenk PM, Baumann S, Mattes R, Steinbiss HH (1995) Improved high-level expression system for eukaryotic genes in Escherichia coli using T7 RNA polymerase and rare ArgtRNAs. Biotechniques 19:196–200Selengut JD (2001) MDP-1 is a new and distinct member of the haloacid dehalogenase family of aspartate-dependent phosphohydrolases. Biochemistry 40:12704–12711Selengut JD, Levine RL (2000) MDP-1: a novel eukaryotic magnesium-dependent phosphatase. Biochemistry 39:8315–8324Shin DH, Roberts A, Jancarik J, Yocota H, Kim R, Wemmer DE, Kim S-H (2003) Crystal structure of a phosphatase with a unique substrate binding domain from Thermotoga maritime. Protein Sci 12:1464–1472Sussman I, Avron M (1981) Characterization and partial puri-fication of dl-glycerol-1-phosphatase from Dunaliella salina. Biochim Biophys Acta 661:199–204The Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815Tremblay LW, Dunaway-Mariano D, Allen KN (2006) Structure and activity analyses of Escherichia coli K-12 NagD provide insight into the evolution of biochemical function in the haloalkanoic acid dehalogenase superfamily. Biochemistry 45:1183–1193Vicient CM, Delseny M (1999) Isolation of total RNA from Arabidopsis thaliana seeds. Anal Biochem 268:412–413Wang W, Cho HS, Kim R, Jancarik J, Yokota H, Nguyen HH, Grigoriev IV, Wemmer DE, Kim S-H (2002) Structural characterization of the reaction pathway in phosphoserine phosphatase: crystallographic “snapshots” of intermediate states. J Mol Biol 319:421–431Zhang G, Mazurkie AS, Dunaway-Mariano D, Allen KN (2002) Kinetic evidence for a substrate-induced fit in phosphonoacetaldehyde hydrolase catalysis. Biochemistry 41:13370–13377Zhang G, Morais MC, Dai J, Zhang W, Dunaway-Mariano D, Allen KN (2004) Investigation of metal Ion binding in phosphonoacetaldehyde hydrolase identifies sequence markers for metal-activated enzymes of the HAD enzyme superfamily. Biochemistry 43:4990–4997Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W (2004) GENEVESTIGATOR: Arabidopsis microarray database and analysis toolbox. Plant Physiol 136:2621–263