Protein Kinase C and Extracellular Signal-Regulated Kinase Regulate Movement,Attachment, Pairing and Egg Release in Schistosoma mansoni

Protein kinases C (PKCs) and extracellular signal-regulated kinases (ERKs) are evolutionary conserved cell signalling enzymes that coordinate cell function. Here we have employed biochemical approaches using ‘smart’ antibodies and functional screening to unravel the importance of these enzymes to Schistosoma mansoni physiology. Various PKC and ERK isotypes were detected, and were differentially phosphorylated (activated) throughout the various S. mansoni life stages, suggesting isotype-specific roles and differences in signalling complexity during parasite development. Functional kinase mapping in adult worms revealed that activated PKC and ERK were particularly associated with the adult male tegument, musculature and oesophagus and occasionally with the oesophageal gland; other structures possessing detectable activated PKC and/or ERK included the Mehlis'' gland, ootype, lumen of the vitellaria, seminal receptacle and excretory ducts. Pharmacological modulation of PKC and ERK activity in adult worms using GF109203X, U0126, or PMA, resulted in significant physiological disturbance commensurate with these proteins occupying a central position in signalling pathways associated with schistosome muscular activity, neuromuscular coordination, reproductive function, attachment and pairing. Increased activation of ERK and PKC was also detected in worms following praziquantel treatment, with increased signalling associated with the tegument and excretory system and activated ERK localizing to previously unseen structures, including the cephalic ganglia. These findings support roles for PKC and ERK in S. mansoni homeostasis, and identify these kinase groups as potential targets for chemotherapeutic treatments against human schistosomiasis, a neglected tropical disease of enormous public health significance.     

Selection of nest sites by wood mice Apodemus sylvaticus in a Mediterranean agro-forest landscape

One of the most important structures for an individual’s survival is a refuge, especially for species subject to significant predatory pressure or living in environments affected by severe climatic conditions. We studied how wood mice (Apodemus sylvaticus), inhabiting a Mediterranean environment, use nests and the factors that could be determining this use. Radio-tracking of 16 mice allowed us to detect 24 diurnal nests, which were used with a high fidelity rate (1.75 nests/animal). Nest sites that had been identified using radio-tracking were matched with 24 randomly selected non-nest sites (located within an area defined by the sum of all individual home ranges), and all sites were characterized according to their macro- and micro-habitat parameters. Using a generalized linear model, we tested three hypotheses: (1) a nest’s use is influenced by its proximity to food patches; (2) the degree of sheltering provided by each site influences its use; and (3) nest use is constrained by a combination of food- and shelter-related features. Females had higher nest fidelity than males, and the degree of sheltering provided by each site had a positive significant influence on its use. Nests offering higher protection (e.g., under thick vegetation), good drainage (e.g., sleep slopes), and lower human and predatory disturbance (far from orchards) were most often used. The proximity to food patches seemed to negatively influence nest use. Results indicate the need to preserve patches of natural dense vegetation near riparian habitats, which is in agreement with the focus of mammalian protection plans in Mediterranean agricultural landscapes. These patches are important areas for Apodemus sylvaticus, an acorn disperser species, which is preyed on by raptors and mammalian carnivores.     

Cardiovascular risk assessment of dyslipidemic children: analysis of biomarkers to identify monogenic dyslipidemia

The distinction between a monogenic dyslipidemia and a polygenic/environmental dyslipidemia is important for the cardiovascular risk assessment, counseling, and treatment of these patients. The present work aims to perform the cardiovascular risk assessment of dyslipidemic children to identify useful biomarkers for clinical criteria improvement in clinical settings. Main cardiovascular risk factors were analyzed in a cohort of 237 unrelated children with clinical diagnosis of familial hypercholesterolemia (FH). About 40% carried at least two cardiovascular risk factors and 37.6% had FH, presenting mutations in LDLR and APOB. FH children showed significant elevated atherogenic markers and lower concentration of antiatherogenic particles. Children without a molecular diagnosis of FH had higher levels of TGs, apoC2, apoC3, and higher frequency of BMI and overweight/obesity, suggesting that environmental factors can be the underlying cause of their hypercholesterolem≥ia. An apoB/apoA1 ratio ≥0.68 was identified as the best biomarker (area under the curve = 0.835) to differentiate FH from other dyslipidemias. The inclusion in clinical criteria of a higher cut-off point for LDL cholesterol or an apoB/apoA1 ratio ≥0.68 optimized the criteria sensitivity and specificity. The correct identification, at an early age, of all children at-risk is of great importance so that specific interventions can be implemented. apoB/apoA1 can improve the identification of FH patients.     

The Bactericidal Activity of Carbon Monoxide–Releasing Molecules against Helicobacter pylori

Helicobacter pylori is a pathogen that establishes long life infections responsible for chronic gastric ulcer diseases and a proved risk factor for gastric carcinoma. The therapeutic properties of carbon-monoxide releasing molecules (CORMs) led us to investigate their effect on H. pylori. We show that H. pylori 26695 is susceptible to two widely used CORMs, namely CORM-2 and CORM-3. Also, several H. pylori clinical isolates were killed by CORM-2, including those resistant to metronidazole. Moreover, sub-lethal doses of CORM-2 combined with metronidazole, amoxicillin and clarithromycin was found to potentiate the effect of the antibiotics. We further demonstrate that the mechanisms underpinning the antimicrobial effect of CORMs involve the inhibition of H. pylori respiration and urease activity. In vivo studies done in key cells of the innate immune system, such as macrophages, showed that CORM-2, either alone or when combined with metronidazole, strongly reduces the ability of H. pylori to infect animal cells. Hence, CORMs have the potential to kill antibiotic resistant strains of H. pylori.     

Guardians' Knowledge and Husbandry Practices of Feline Environmental Enrichment

Feline environmental enrichment can prevent numerous disorders including anxiety, stress, obesity, and feline idiopathic cystitis. Despite its easy implementation and low cost, it has received little attention. The main goal of this study was to assess guardians' knowledge concerning feline environmental enrichment and husbandry practices. A questionnaire was given to 130 companion animal guardians at the Lisbon Veterinary Faculty's Teaching Hospital. The applications of 22 environmental enrichment measures related to food/water, litter box, and space/entertainment areas were evaluated. The majority of the households studied (74.6%) had a moderately enriched environment. Hygiene-related measures were those most adopted by guardians, while those requiring guardians' commitment or previous awareness were the least implemented. A rating scale was proposed and applied to assess feline domestic environmental quality.     

Volatile metabolomic signature of bladder cancer cell lines based on gas chromatography–mass spectrometry

Introduction

Recent studies provide a convincing support that the presence of cancer cells in the body leads to the alteration of volatile organic compounds (VOCs) emanating from biological samples, particularly of those closely related with tumoral tissues. Thus, a great interest emerged for the study of cancer volatilome and subsequent attempts to confirm VOCs as potential diagnostic biomarkers.

Objectives

The aim of this study was to determine the volatile metabolomic signature of bladder cancer (BC) cell lines and provide an in vitro proof-of-principle that VOCs emanated into the extracellular medium may discriminate BC cells from normal bladder epithelial cells.

Methods

VOCs in the culture media of three BC cell lines (Scaber, J82, 5637) and one normal bladder cell line (SV-HUC-1) were extracted by headspace-solid phase microextraction and analysed by gas chromatography-mass spectrometry (HS-SPME/GC–MS). Two different pH (pH 2 and 7) were used for VOCs extraction to infer the best pH to be used in in vitro metabolomic studies.

Results

Multivariate analysis revealed a panel of volatile metabolites that discriminated cancerous from normal bladder cells, at both pHs, although a higher number of discriminative VOCs was obtained at neutral pH. Most of the altered metabolites were ketones and alkanes, which were generally increased in BC compared to normal cells, and alcohols, which were significantly decreased in BC cells. Among them, three metabolites, namely 2-pentadecanone, dodecanal and γ-dodecalactone (the latter only tentatively identified), stood out as particularly important metabolites and promising volatile biomarkers for BC detection. Furthermore, our results also showed the potential of VOCs in discriminating BC cell lines according to tumour grade and histological subtype.

Conclusions

We demonstrate that a GC–MS metabolomics-based approach for analysis of VOCs is a valuable strategy for identifying new and specific biomarkers that may improve BC diagnosis. Future studies should entail the validation of volatile signature found for BC cell lines in biofluids from BC patients.

     

Influence of vanadate on glycolysis,intracellular sodium,and pH in perfused rat hearts

Vanadium compounds have been shown to cause a variety of biological and metabolic effects including inhibition of certain enzymes, alteration of contractile function, and as an insulin like regulator of glucose metabolism. However, the influence of vanadium on metabolic and ionic changes in hearts remains to be understood. In this study we have examined the influence of vanadate on glucose metabolism and sodium transport in isolated perfused rat hearts. Hearts were perfused with 10 mM glucose and varying vanadate concentrations (0.7100 M) while changes in high energy phosphates (ATP and phosphocreatine (PCr)), intracellular pH, and intracellular sodium were monitored using 31P and 23Na NMR spectroscopy. Tissue lactate, glycogen, and (Na+, K+)-ATPase activity were also measured using biochemical assays. Under baseline conditions, vanadate increased tissue glycogen levels two fold and reduced (Na+, K+)-ATPase activity. Significant decreases in ATP and PCr were observed in the presence of vanadate, with little change in intracellular pH. These changes under baseline conditions were less severe when the hearts were perfused with glucose, palmitate and b-hydroxybutyrate. During ischemia vanadate did not limit the rise in intracellular sodium, but slowed sodium recovery on reperfusion. The presence of vanadate during ischemia resulted in attenuation of acidosis, and reduced lactate accumulation. Reperfusion in the presence of vanadate resulted in a slower ATP recovery, while intracellular pH and PCr recovery was not affected. These results indicate that vanadate alters glucose utilization and (Na+, K+)-ATPase activity and thereby influences the response of the myocardium to an ischemic insult.