Medusins: a new class of antimicrobial peptides from the skin secretions of phyllomedusine frogs

Natural drug discovery represents an area of research with vast potential. The investigation into the use of naturally-occurring peptides as potential therapeutic agents provides a new “chemical space” for the procurement of drug leads. Intensive and systematic studies on the broad-spectrum antimicrobial peptides found in amphibian skin secretions are of particular interest in the quest for new antibiotics to treat multiple drug-resistant bacterial infections. Here we report the molecular cloning of the biosynthetic precursor-encoding cDNAs and respective mature peptides representing a novel group of antimicrobial peptides from the skin secretions of representative species of phyllomedusine leaf frogs: the Central American red-eyed leaf frog (Agalychnis callidryas), the South American orange-legged leaf frog (Phyllomedusa hypochondrialis) and the Giant Mexican leaf frog, (Pachymedusa dacnicolor). Each novel peptide possessed the highly-conserved sequence, LGMIPL/VAISAISA/SLSKLamide, and each exhibited activity against the Gram-positive bacterium, Staphylococcus aureus and the yeast, Candida albicans, but all were devoid of haemolytic effects at concentrations up to and including the MICs for both organisms. The novel peptide group were named medusins, derived from the name of the hylid frog sub-family, Phyllomedusinae, to which all species investigated belong. These data clearly demonstrate that comparative studies of the skin secretions of phyllomedusine frogs can continue to produce novel peptides that have the potential to be leads in the development of new and effective antimicrobials.

A Novel Investigation of a Blister-Like Syndrome in Aquarium Echinopora lamellosa

This study investigates potential causes of a novel blister-like syndrome in the plating coral Echinopora lamellosa. Visual inspections of this novel coral syndrome showed no obvious signs of macroparasites and the blisters themselves manifested as fluid-filled sacs on the surface of the coral, which rose from the coenosarc between the coral polyps. Histological analysis of the blisters showed that there was no associated necrosis with the epidermal or gastrodermal tissues. The only difference between blistered areas and apparently healthy tissues was the presence of proliferated growth (possible mucosal cell hyperplasia) directly at the blister interface (area between where the edge of the blister joined apparently healthy tissue). No bacterial aggregates were identified in any histological samples, nor any sign of tissue necrosis identified. We conclude, that the blister formations are not apparently caused by a specific microbial infection, but instead may be the result of irritation following growth anomalies of the epidermis. However, future work should be conducted to search for other potential casual agents, including viruses. © 2014 Smith et al.

The Host Defence Peptide LL-37 is Susceptible to Proteolytic Degradation by Wound Fluid Isolated from Foot Ulcers of Diabetic Patients

The pleiotropic effects of host defence peptides (HDPs), including the ability to kill microorganisms, enhance re-epithelialisation and increase angiogenesis, indicates a role for these important peptides as potential therapeutic agents in the treatment of chronic, non-healing wounds. However, the maintenance of peptide integrity, through resistance to degradation by the array of proteinases present at the wound site, is a prerequisite for clinical success. In this study we explored the degradation of exogenous LL-37, one such HDP, by wound fluid from diabetic foot ulcers to determine its susceptibility to proteolytic degradation. Our results suggest that LL-37 is unstable in the diabetic foot ulcer microenvironment. Following overnight treatment with wound fluid, LL-37 was completely degraded. Analysis of cleavage sites suggested potential involvement of both host- and bacterial-derived proteinases. The degradation products were shown to retain some antibacterial activity against Pseudomonas aeruginosa but were inactive against Staphylococcus aureus. In conclusion, our data suggest that stabilising selected peptide bonds within the sequence of LL-37 would represent an avenue for future research prior to clinical studies to address its potential as an exogenously-applied therapeutic in diabetic wounds. 

Microbe-mediated recovery of salt marshes contaminated with oil hydrocarbons

Salt marshes are highly productive intertidal habitats that serve as nursery grounds for many commercially and economically important species. Because of their location and physical and biological characteristics, salt marshes are considered to be particularly vulnerable to anthropogenic inputs of oil hydrocarbons. Sediment contamination with oil is especially dangerous for salt marsh vegetation, since low molecular weight aromatic hydrocarbons can affect plants at all stages of development. However, the use of vegetation for bioremediation (phytoremediation), by removal or sequestration of contaminants, has been intensively studied. Phytoremediation is an efficient, inexpensive and environmental friendly approach for the removal of aromatic hydrocarbons, through direct incorporation by the plant and by the intervention of degrading microbial populations in the rhizosphere (microbe-assisted phytoremediation). Rhizosphere microbial communities are enriched in important catabolic genotypes for degradation of oil hydrocarbons (OH) which may have a potential for detoxification of the sediment surrounding the roots. In addition, since rhizosphere bacterial populations may also internalize into plant tissues (endophytes), rhizocompetent AH degrading populations may be important for in planta AH degradation and detoxification. The present study involved field work and microcosms experiments aiming the characterization of relevant plant-microbe interactions in oilimpacted salt marshes and the understanding of the effect of rhizosphere and endosphere bacteria in the role of salt marsh plants as potential phytoremediation agents. In the field approach, molecular tools were used to assess how plant species- and OH pollution affect sediment bacterial composition [bulk sediment and sediment surrounding the roots (rhizosphere) of Halimione portulacoides and Sarcocornia perennis subsp. perennis] in a temperate estuary (Ria de Aveiro, Portugal) chronically exposed to OH pollution. In addition, the 16S rRNA gene sequences retrieved in this study were used to generate in silico metagenomes and to evaluate the distribution of potential bacterial traits in different microhabitats. Moreover, a combination of culture-dependent and -independent approaches was used to investigate the effect of oil hydrocarbons contamination on the structure and function of endophytic bacterial communities of salt marsh plants.Root systems of H. portulacoides and S. perennis subsp. perennis appear to be able to exert a strong influence on bacterial composition and in silico metagenome analysis showed enrichment of genes involved in the process of polycyclic aromatic hydrocarbon (PAH) degradation in the rhizosphere of halophyte plants. The culturable fraction of endophytic degraders was essentially closely related to known OH-degrading Pseudomonas species and endophytic communities revealed sitespecific effects related to the level of OH contamination in the sediment. In order to determine the effects of oil contamination on plant condition and on the responses in terms of structure and function of the bacterial community associated with plant roots (rhizosphere, endosphere), a microcosms approach was set up. The salt marsh plant Halimione portulacoides was inoculated with a previous isolated Pseudomonas sp. endophytic degrader and the 2-methylnaphthalene was used as model PAH contaminant. The results showed that H. portulacoides health and growth were not affected by the contamination with the tested concentration. Moreover, the decrease of 2-methylnaphthalene at the end of experiment, can suggest that H. portulacoides can be considered as a potential plant for future uses in phytoremedition approaches of contaminated salt marsh. The acceleration of hydrocarbon degradation by inoculation of the plants with the hydrocarbon-degrading Pseudomonas sp. could not, however, be demonstrated, although the effects of inoculation on the structure of the endophytic community observed at the end of the experiment indicate that the strain may be an efficient colonizer of H. portulacoides roots. The results obtained in this work suggest that H. portulacoides tolerates moderate concentrations of 2-methylnaphthalene and can be regarded as a promising agent for phytoremedition approaches in salt marshes contaminated with oil hydrocarbons. Plant/microbe interactions may have an important role in the degradation process, as plants support a diverse endophytic bacterial community, enriched in genetic factors (genes and plasmids) for hydrocarbon degradation.

How the gut links innate and adaptive immunity.

Mucosal surfaces represent the main sites in which environmental microorganisms and antigens interact with the host. Sentinel cells, including epithelial cells, lumenal macrophages, and intraepithelial dendritic cells, continuously sense the environment and coordinate defenses for the protection of mucosal tissues. The mucosal epithelial cells are crucial actors in coordinating defenses. They sense the outside world and respond to environmental signals by releasing chemokines and cytokines that recruit inflammatory and immune cells to control potential infectious agents and to attract cells able to trigger immune responses. Among immune cells, dendritic cells (DC) play a key role in controlling adaptive immune responses, due to their capacity to internalize foreign materials and to present antigens to naive T and B lymphocytes, locally or in draining organized lymphoid tissues. Immune cells recruited in epithelial tissues can, in turn, act upon the epithelial cells and change their phenotype in a process referred to as epithelial metaplasia.

Synthèse et caractérisation physicochimique de peptides de polyglutamines

Neuf maladies neurodégénératives sont le produit de l’expression de gènes mutés, dans lesquels le codon CAG est répété au-delà d’un seuil pathologique. Ceci produit des protéines mutantes dans lesquelles sont insérés des segments de polyglutamines (polyGln), qui perdent leur activité et acquièrent une nouvelle fonction, ce qui est toxique pour le neurone. Ces altérations sont attribuables aux propriétés particulières de la polyGln. En effet, ces dernières possèdent la capacité de s’assembler pour former des corps d’inclusion intracellulaires. Cette propension à l’agrégation de la polyGln rend difficile l’étude de ces pathologies. C’est ainsi que l’utilisation de peptides peut s’avérer une approche avantageuse. Toutefois, la synthèse de polyGln est associée à de nombreuses délétions et nécessite l’ajout de groupements chargés afin de permettre leur purification. Cependant, ce prérequis donne lieu à des interactions électrostatiques qui biaisent la structure et la cinétique d’agrégation de ces peptides, en plus d’interférer avec l’évaluation d’éventuels agents thérapeutiques. L’objectif du projet est de développer un système permettant l’étude de la polyGln en s’affranchissant des effets de charges. Pour ce faire, deux approches ont été explorées, la première utilise la polyGln non chargée et la seconde utilise une structure polyGln-morpholine ayant des charges labiles en fonction du pH. Ces peptides ont été produits en utilisant une approche linéaire de synthèse peptidique sur support solide avec protection maximale des chaînes latérales. La purification a été effectuée par chromatographie de haute performance en phase inverse en milieu acide. Ces stratégies ont permis de produire des peptides de polyGln de grande pureté avec des rendements acceptables. Une procédure de solubilisation des peptides alliant sonication et lyophilisation a été développée afin d’étudier chacun de ces peptides à l’aide de diverses techniques physicochimiques, telles que la diffusion de la lumière, la spectroscopie de résonance magnétique nucléaire, Raman et UV-visible, le dichroïsme circulaire et la microscopie optique polarisée. La polyGln non chargée solubilisée dans le trifluoroéthanol-eau a montré que la taille des particules et la vitesse d’agrégation sont proportionnelles à la fraction volumique en eau. De plus, la structure secondaire en solution est à prédominance alpha et semble être peu sensible à la fraction d’eau jusqu’à un certain seuil (25%) après lequel la structure aléatoire prédomine. L’analyse des agrégats à l’état solide montre des structures hélicoïdales > aléatoires et ont les caractéristiques des fibrilles amyloïdes. Le peptide de polyGln-morpholines a un pKa de 7,3 en milieu aqueux. Il demeure en solution lorsque le pH < pKa et à faible force ionique, alors qu’il s’autoassemble lorsque ces conditions ne sont pas respectées. Ceci suggère que la répulsion électrostatique est responsable de la stabilisation du peptide en solution. La dimension fractale nous indique que le peptide forme des agrégats compacts dont les constituants ont une taille de 2,5 nm, compatibles avec une conformation aléatoire compacte, en coude bêta ou hélicoïdale. Ceci est en accord avec l’étude structurale des peptides en solution qui a montré des espèces aléatoires > bêta > alpha. De plus, en RMN, l’élargissement des signaux du 1Hγ en cours d’agrégation suggère une interaction via les chaînes latérales. Les analyses en phase solide ont plutôt montré une prédominance de structures bêta et alpha. L’inhibition de l’agrégation à pH 8 varie selon rouge de Congo > tréhalose, alors que le peptide liant la polyGln 1 et la thioflavine T ne semble pas avoir d’effet. Ces approches ont donc permis pour la première fois de s’affranchir des effets de charges auparavant inhérents à l’étude de la polyGln en solution et par conséquent d’obtenir des informations inédites quant à la solubilité, la structure et la cinétique d’agrégation. Enfin, le dispositif à charges labiles permet d’évaluer l’efficacité d’éventuels agents thérapeutiques à pH quasi physiologique.

Strategies for revascularizing the ischemic retina

Les rétinopathies ischémiques (RI) sont la cause majeure de cécité chez les personnes âgées de moins de 65 ans. Il existe deux types de RIs soit la rétinopathie du prématuré (ROP) ainsi que la rétinopathie diabétique (RD). Les RIs sont décrites en deux phases soit la phase de vasooblitération, marquée par une perte importante de vaisseaux sanguins, et une phase de néovascularisation secondaire à lʼischémie menant à une croissance pathologique de vaisseaux. Cette seconde phase peut générer des complications cliniques telles quʼun oedème dans lʼhumeur vitré ainsi que le détachement de la rétine chez les patients déjà atteints dʼune RI. Les traitements approuvés pour les RIs visent à réduire la formation des vaisseaux pathologiques ou lʼoedème; mais ceux-ci malheureusement ne règlent pas les problèmes sous-jacents tels que la perte vasculaire et lʼischémie. La rétine est un tissu hautement vascularisé qui contribue à lʼirrigation et à lʼhoméostasie des neurones. Lʼinteraction neurovasculaire, comprenant de neurones, vaisseaux et cellules gliales, contribue au maintien de cette homéostasie. Durant le développement, les neurones et les cellules gliales jouent un rôle important dans la vascularisation de la rétine en sécrétant des facteurs qui stimulent l'angiogenèse. Cependant, nos connaissances sur lʼinteraction neurovasculaire dans les RIs sont limitées. En identifiant les interactions importantes entre les cellules composant cette unité neurovasculaire dans la rétine, nous pourrons viser des cibles qui engendreront une revascularisation seine afin de diminuer les signes pathologiques chez les patients atteints dʼune RI. Les travaux présentés dans cette thèse visent à mieux expliquer cette interaction neurovasculaire en soulignant des concepts importants propres aux RIs. En utilisant un modèle de rétinopathie induite par lʼoxygène chez la souris, qui reproduit les caractéristiques importantes de la ROP (et en certaines instances, la RD), nous identifions quelques molécules clés jouant un rôle significatif dans les RIs soit la sémaphorine 3A (sema3A), lʼIL-1β, ainsi que le récepteur PAR2. Nos résultats démontrent que Sema3A, sécrétée par les cellules ganglionnaires rétiniennes (CGRs) durant une ischémie, empêche la revascularisation normale et que cette expression est induite par lʼIL-1β provenant des microglies activées. En bloquant Sema3A directement ou via lʼinhibition de lʼIL- 1β, nous remarquons une revascularisation seine ainsi quʼune diminution importante des vaisseaux pathologiques. Cela nous indique que Sema3A est impliquée dans la guidance vasculaire et quʼelle contribue à la pathogenèse des RIs. Lʼactivation de façon exogène de PAR2, identifié aussi comme régulateur du récepteur de lʼIL-1β (IL- 1RI) sur les CGRs, se traduit par une diminution séquentielle de lʼIL-1RI et de Sema3A ce qui mène également à une revascularisation seine. En conclusion, ces travaux soulignent lʼimportance de lʼinteraction neurovasculaire ainsi que la guidance vasculaire dans les RIs. Ils renforcent lʼimportance de la communication entre neurone, vaisseau et microglie dans la pathogenèse des RIs. Finalement, nous identifions quelques molécules clés qui pourront servir comme cibles afin de lutter contre lʼischémie qui cause des problèmes vasculaires chez les patients atteints dʼune RI.

The use of culture-independent tools to characterize bacteria in endo-tracheal aspirates from pre-term infants at risk of bronchopulmonary dysplasia

Although premature infants are increasingly surviving the neonatal period, up to one-third develop bronchopulmonary dysplasia (BPD). Despite evidence that bacterial colonization of the neonatal respiratory tract by certain bacteria may be a risk factor in BPD development, little is known about the role these bacteria play. The aim of this study was to investigate the use of culture-independent molecular profiling methodologies to identify potential etiological agents in neonatal airway secretions. This study used terminal restriction fragment length polymorphism (T-RFLP) and clone sequence analyses to characterize bacterial species in endo-tracheal (ET) aspirates from eight intubated pre-term infants. A wide range of different bacteria was identified in the samples. Forty-seven T-RF band lengths were resolved in the sample set, with a range of 0-15 separate species in each patient. Clone sequence analyses confirmed the identity of individual species detected by T-RFLP. We speculate that the identification of known opportunistic pathogens including S. aureus, Enterobacter sp., Moraxella catarrhalis, Pseudomonas aeruginosa and Streptococcus sp., within the airways of pre-term infants, might be causally related to the subsequent development of BPD. Further, we suggest that culture-independent techniques, such as T-RFLP, hold important potential for the characterization of neonatal conditions, such as BPD.

Pharmacological actions of nobiletin in the modulation of platelet function

Background and Purpose The discovery that flavonoids are capable of inhibiting platelet function has led to their investigation as potential antithrombotic agents. However, despite the range of studies on the antiplatelet properties of flavonoids, little is known about the mechanisms by which flavonoids inhibit platelet function. In this study, we aimed to explore the pharmacological effects of a polymethoxy flavonoid, nobiletin in the modulation of platelet function. Experimental Approach The ability of nobiletin to modulate platelet function was explored by using a range of in vitro and in vivo experimental approaches. Aggregation, dense granule secretion and spreading assays were performed using washed platelets. The fibrinogen binding, α-granule secretion and calcium mobilisation assays were performed using platelet-rich plasma and whole blood was used in impedance aggregometry and thrombus formation experiments. The effect of nobiletin in vivo was assessed by measuring tail bleeding time using C57BL/6 mice. Key Results Nobiletin was shown to supress a range of well-established activatory mechanisms, including platelet aggregation, granule secretion, integrin modulation, calcium mobilisation and thrombus formation. Nobiletin was shown to extend bleeding time in mice and reduce the phosphorylation of Akt and PLCγ2 within the collagen receptor (GPVI) - stimulated pathway, in addition to increasing the levels of cGMP and phosphorylation of VASP, a protein whose activity is associated with inhibitory cyclic nucleotide signalling. Conclusions and Implications This study provides insight into the underlying molecular mechanisms through which nobiletin modulates haemostasis and thrombus formation. Therefore nobiletin may represent a potential antithrombotic agent of dietary origins.

Inhibition of Mycobacterium tuberculosis tyrosine phosphatase PtpA by synthetic chalcones: Kinetics, molecular modeling, toxicity and effect on growth

Tuberculosis (TB) is a major cause of morbidity and mortality throughout the world, and it is estimated that one-third of the world`s population is infected with Mycobacterium tuberculosis. Among a series of tested compounds, we have recently identified five synthetic chalcones which inhibit the activity of M. tuberculosis protein tyrosine phosphatase A (PtpA), an enzyme associated with M. tuberculosis infectivity. Kinetic studies demonstrated that these compounds are reversible competitive inhibitors. In this work we also carried out the analysis of the molecular recognition of these inhibitors on their macromolecular target, PtpA, through molecular modeling. We observed that the predominant determinants responsible for the inhibitory activity of the chalcones are the positions of the two methoxyl groups at the A-ring, that establish hydrogen bonds with the amino acid residues Arg17, His49, and Thr12 in the active site of PtpA, and the substitution of the phenyl ring for a 2-naphthyl group as B-ring, that undergoes p stacking hydrophobic interaction with the Trp48 residue from PtpA. Interestingly, reduction of mycobacterial survival in human macrophages upon inhibitor treatment suggests their potential use as novel therapeutics. The biological activity, synthetic versatility, and low cost are clear advantages of this new class of potential tuberculostatic agents. (C) 2010 Elsevier Ltd. All rights reserved.

Structural basis for selective inhibition of purine nucleoside phosphorylase from Schistosoma mansoni: Kinetic and structural studies

Selectivity plays a crucial role in the design of enzyme inhibitors as novel antiparasitic agents, particularly in cases where the target enzyme is also present in the human host. Purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) is an attractive target for the discovery of potential antischistosomal agents. In the present work, kinetic studies were carried out in order to determine the inhibitory potency, mode of action and enzyme selectivity of a series of inhibitors of SmPNP. In addition, crystallographic studies provided important structural insights for rational inhibitor design, revealing consistent structural differences in the binding mode of the inhibitors in the active sites of the SmPNP and human PNP (HsPNP) structures. The molecular information gathered in this work should be useful for future medicinal chemistry efforts in the design of new inhibitors of SmPNP having increased affinity and selectivity. (C) 2010 Elsevier Ltd. All rights reserved.

Avaliação do efeito do composto tipo heparina isolado do caranguejo Chaceon fenneri na hemostasia e na morte celular

Heparin is a pharmaceutical animal widely used in medicine due to its potent anticoagulant effect. Furthermore, it has the ability to inhibit the proliferation, invasion and adhesion of cancer cells to vascular endothelium. However, its clinical applicability can be compromised by side effects such as bleeding. Thus, the search for natural compounds with low bleeding risk and possible therapeutic applicability has been targeted by several research groups. From this perspective, this study aims to evaluate the hemorrhagic and anticoagulant activities and citotoxic effect for different tumor cell lines (HeLa, B16-F10, HepG2, HS-5,) and fibroblast cells (3T3) of the Heparin-like from the crab Chaceon fenneri (HEP-like). The HEP-like was purified after proteolysis, ion-exchange chromatography, fractionation with acetone and characterized by electrophoresis (agarose gel) and enzymatic degradation. Hep-like showed eletroforetic behavior similar to mammalian heparin, and high trisulfated /Nacetylated disaccharides ratio. In addition, HEP-like presented low in vitro anticoagulant activity using aPTT and a minor hemorrhagic effect when compared to mammalian heparin. Furthermore, the HEP-like showed significant cytotoxic effect (p<0.001) on HeLa, HepG2 and B16-F10 tumor cells with IC50 values of 1000 ug/mL, after incubation for 72 hours. To assess the influence of heparin-like on the cell cycle in HeLa cells, analysis was performed by flow cytometry. The results of this analysis showed that HEP-like influence on the cell cycle increasing S phase and decreasing phase G2. Thus, these properties of HEP-like make these compounds potential therapeutic agents

Atividades antinociceptiva e anti-inflamatória de uma fração rica em heterogalactana sulfatada extraída da alga codium isthmocladum (vickers 1905)

Sulfated polysaccharides comprise a complex group of macromolecules with a range of several biological activities, including antiviral activity, anticoagulant, antiproliferative, antiherpética, antitumor, anti-inflammatory and antioxidant. These anionic polymers are widely distributed in tissues of vertebrates, invertebrates and algae. Seaweeds are the most abundant sources of sulfated polysaccharides in nature. The green algal sulfated polysaccharides are homo or heteropolysaccharides comprised of galactose, glucose, arabinose and/or glucuronic acid. They are described as anticoagulant, anti-inflammatory, antiviral, anti-angiogenic, antitumor compounds. However, there are few studies about elucidation and evaluation of biological/pharmacological effects of sulfated polysaccharides obtained from green algae, for example, there is only one paper reporting the antinociceptive activity of sulfated polysaccharides of these algae. Therefore this study aimed to obtain sulfated polysaccharides of green seaweed Codium isthmocladum and evaluates them as potential antinociceptive agents. Thus, in this study, the total extract of polysaccharides of green alga C. isthmocladum was obtained by proteolytic digestion, followed by fractionation resulting in five fractions (F0.3, F0.5, F0.7, F0.9 and F1.2) by sequential precipitation with acetone. Using the test of abdominal contractions we observed that the fraction F0.9 was the most potent antinociceptive aompound. F0.9 consists mainly of a sulfated heterogalactana. More specific tests showed that Fo.9 effect is dose and time dependent, reaching a maximum at 90 after administration (10 mg / kg of animal). F0.9 is associated with TRPV1 and TRPA1 receptors and inhibits painful sensation in animals. Furthermore, F0.9 inhibits the migration of lymphocytes induced peritonitis test. On the other hand, stimulates the release of NO and TNF-α. These results suggest that F0.9 has the potential to be used as a source of sulfated galactan antinociceptive and anti-inflammatory

DNA damage and aberrant crypt foci as putative biomarkers to evaluate the chemopreventive effect of annatto (Bixa orellana L.) in rat colon carcinogenesis

Chemoprevention opens new perspectives in the prevention of cancer and other degenerative diseases. Use of target-organ biological models at the histological and genetic levels can markedly facilitate the identification of such potential chemopreventive agents. Colon cancer is one of the highest incidence rates throughout the world and some evidences have indicated carotenoids as possible agents that decrease the risk of colorectal cancer. In the present study, we evaluate the activity of annatto (Bixa orellaria L.), a natural food colorant rich in carotenoid, on the formation of aberrant crypt foci (ACF) induced by dimethy1hydrazine (DMH) in rat colon. Further, we investigate, the effect of annatto on DMH-induced DNA damage, by the comet assay. Male Wistar rats were given s.c. injections of DMH (40 mg/kg body wt.) twice a week for 2 weeks to induce ACE They also received experimental diets containing annatto at 20, 200 or 1000 ppm for five 5 weeks before (pre-treatment), or 10 weeks after (post-treatment) DMH treatment. In both protocols the rats were sacrificed on week 15th. For the comet assay, the animals were fed with the same experimental diets for 2 weeks. Four hours before the sacrifice, the animals received an s.c. injection of DMH (40 mg/kg body wt.). Under such conditions, dietary administration of 1000 ppm annatto neither induce DNA damage in blood and colon cells nor aberrant crypt foci in rat distal colon. Conversely, annatto was successful in inhibiting the number of crypts/colon (animal), but not in the incidence of DMH-induced ACF, mainly when administered after DMH. However, no antigenotoxic effect was observed in colon cells. These findings suggest possible chemopreventive effects of annatto through their modulation of the cryptal cell proliferation but not at the initiation stage of colon carcinogenesis. (c) 2005 Elsevier B.V. All rights reserved.

Modelling the lethargic crab disease.

The lethargic crab disease (LCD) is an emergent infirmity that has decimated native populations of the mangrove land crab (Ucides cordatus, Decapoda: Ocypodidae) along the Brazilian coast. Several potential etiological agents have been linked with LCD, but only in 2005 was it proved that it is caused by an ascomycete fungus. This is the first attempt to develop a mathematical model to describe the epidemiological dynamics of LCD. The model presents four possible scenarios, namely, the trivial equilibrium, the disease-free equilibrium, endemic equilibrium, and limit cycles arising from a Hopf bifurcation. The threshold values depend on the basic reproductive number of crabs and fungi, and on the infection rate. These scenarios depend on both the biological assumptions and the temporal evolution of the disease. Numerical simulations corroborate the analytical results and illustrate the different temporal dynamics of the crab and fungus populations.