Cathepsin L1, the major protease involved in liver fluke (Fasciola hepatica) virulence.

The secretion and activation of the major cathepsin L1 cysteine protease involved in the virulence of the helminth pathogen Fasciola hepatica was investigated. Only the fully processed and active mature enzyme can be detected in medium in which adult F. hepatica are cultured. However, immunocytochemical studies revealed that the inactive procathepsin L1 is packaged in secretory vesicles of epithelial cells that line the parasite gut. These observations suggest that processing and activation of procathepsin L1 occurs following secretion from these cells into the acidic gut lumen. Expression of the 37-kDa procathepsin L1 in Pichia pastoris showed that an intermolecular processing event within a conserved GXNXFXD motif in the propeptide generates an active 30-kDa intermediate form. Further activation of the enzyme was initiated by decreasing the pH to 5.0 and involved the progressive processing of the 37 and 30-kDa forms to other intermediates and finally to a fully mature 24.5 kDa cathepsin L with an additional 1 or 2 amino acids. An active site mutant procathepsin L, constructed by replacing the Cys26 with Gly26, failed to autoprocess. However, [Gly26]procathepsin L was processed by exogenous wild-type cathepsin L to a mature enzyme plus 10 amino acids attached to the N terminus. This exogenous processing occurred without the formation of a 30-kDa intermediate form. The results indicate that activation of procathepsin L1 by removal of the propeptide can occur by different pathways, and that this takes place within the parasite gut where the protease functions in food digestion and from where it is liberated as an active enzyme for additional extracorporeal roles.

ATR-dependent radiation-induced gammaH2AX foci in bystander primary human astrocytes and glioma cells.

Radiotherapy is an important treatment for patients suffering from high-grade malignant gliomas. Non-targeted (bystander) effects may influence these cells' response to radiation and the investigation of these effects may therefore provide new insights into mechanisms of radiosensitivity and responses to radiotherapy as well as define new targets for therapeutic approaches. Normal primary human astrocytes (NHA) and T98G glioma cells were irradiated with helium ions using the Gray Cancer Institute microbeam facility targeting individual cells. Irradiated NHA and T98G glioma cells generated signals that induced gammaH2AX foci in neighbouring non-targeted bystander cells up to 48 h after irradiation. gammaH2AX bystander foci were also observed in co-cultures targeting either NHA or T98G cells and in medium transfer experiments. Dimethyl sulphoxide, Filipin and anti-transforming growth factor (TGF)-beta 1 could suppress gammaH2AX foci in bystander cells, confirming that reactive oxygen species (ROS) and membrane-mediated signals are involved in the bystander signalling pathways. Also, TGF-beta 1 induced gammaH2AX in an ROS-dependent manner similar to bystander foci. ROS and membrane signalling-dependent differences in bystander foci induction between T98G glioma cells and normal human astrocytes have been observed. Inhibition of ataxia telangiectasia mutated (ATM) protein and DNA-PK could not suppress the induction of bystander gammaH2AX foci whereas the mutation of ATM- and rad3-related (ATR) abrogated bystander foci induction. Furthermore, ATR-dependent bystander foci induction was restricted to S-phase cells. These observations may provide additional therapeutic targets for the exploitation of the bystander effect.

Apoptosis is initiated in human keratinocytes exposed to signalling factors from microbeam irradiated cells.

PURPOSE: There is now no doubt that bystander signalling from irradiated cells occurs and causes a variety of responses in cells not targeted by the ionizing track. However, the mechanisms underlying these processes are unknown and the relevance to radiotherapy and risk assessment remains controversial. Previous research by our laboratory has shown bystander effects in a human keratinocyte cell line, HPV-G cells, exposed to medium from gamma irradiated HPV-G cells. The aim of this work was to investigate if similar mechanisms to those identified in medium transfer experiments occurred in these HPV-G cells when they are in the vicinity of microbeam irradiated cells. Demonstration of a commonality of mechanisms would support the idea that the process is not artifactual. MATERIALS AND METHODS: HPV-G cells were plated as two separate populations on mylar dishes. One population was directly irradiated using a charged particle microbeam (1 - 10 protons). The other population was not irradiated. Bystander factor-induced apoptosis was investigated in both populations following treatment by monitoring the levels of reactive oxygen species and mitochondrial membrane potential using fluorescent probes. Expression of the anti-apoptotic protein, bcl-2, and cytochrome c were determined, as well as apoptosis levels. RESULTS: Microbeam irradiation induced increases in reactive oxygen species and decreases in mitochondrial membrane potential at 6 h post-exposure, increased expression of bcl-2 and cytochrome c release at 6.5 h and increased apoptosis at 24 h. CONCLUSION: This study shows that similar bystander signalling pathways leading to apoptosis are induced following microbeam irradiation and following medium transfer. This demonstrates that the mechanisms involved are common across different radiation qualities and conditions and indicates that they may be relevant in vivo.

Short, medium and long term load forecasting model and virtual load forecaster based on radial basis neural networks

Artificial neural networks (ANNs) can be easily applied to short-term load forecasting (STLF) models for electric power distribution applications. However, they are not typically used in medium and long term load forecasting (MLTLF) electric power models because of the difficulties associated with collecting and processing the necessary data. Virtual instrument (VI) techniques can be applied to electric power load forecasting but this is rarely reported in the literature. In this paper, we investigate the modelling and design of a VI for short, medium and long term load forecasting using ANNs. Three ANN models were built for STLF of electric power. These networks were trained using historical load data and also considering weather data which is known to have a significant affect of the use of electric power (such as wind speed, precipitation, atmospheric pressure, temperature and humidity). In order to do this a V-shape temperature processing model is proposed. With regards MLTLF, a model was developed using radial basis function neural networks (RBFNN). Results indicate that the forecasting model based on the RBFNN has a high accuracy and stability. Finally, a virtual load forecaster which integrates the VI and the RBFNN is presented.

Stimulation of phosphate uptake and polyphosphate accumulation by activated sludge microorganisms in response to sulfite addition

Enhanced phosphate removal from wastewaters is dependent on the synthesis and intracellular accumulation of polyphosphate by sludge microorganisms. However the role played by polyphosphate in microbial metabolism and the factors that trigger its formation remain poorly-understood. Many examples of the accumulation of the biopolymer by environmental microorganisms are documented; these include a recent report of the presence of large polyphosphate inclusions in sulfur-oxidizing marine bacteria. To investigate whether any link might exist outside the marine environment between the presence of reduced sulfur compounds and enhanced levels of microbial phosphate uptake and polyphosphate accumulation, activated sludge cultures were grown under laboratory conditions in media that contained sulfite, thiosulfate, hydrosulfite or tetrathionate. Only in the presence of sulfite was there any evidence of a stimulatory effect; in medium that contained 0.5 mM sodium sulfite some 17% more phosphate was removed by the sludge, whilst there was an almost two-fold increase in intracellular polyphosphate levels. No indications of sulfite toxicity were observed.

Hopanoid Production Is Required for Low-pH Tolerance, Antimicrobial Resistance, and Motility in Burkholderia cenocepacia

Hopanoids are pentacyclic triterpenoids that are thought to be bacterial surrogates for eukaryotic sterols, such as cholesterol, acting to stabilize membranes and to regulate their fluidity and permeability. To date, very few studies have evaluated the role of hopanoids in bacterial physiology. The synthesis of hopanoids depends on the enzyme squalene-hopene cyclase (Shc), which converts the linear squalene into the basic hopene structure. Deletion of the 2 genes encoding Shc enzymes in Burkholderia cenocepacia K56-2, BCAM2831 and BCAS0167, resulted in a strain that was unable to produce hopanoids, as demonstrated by gas chromatography and mass spectrometry. Complementation of the Delta shc mutant with only BCAM2831 was sufficient to restore hopanoid production to wild-type levels, while introducing a copy of BCAS0167 alone into the Delta shc mutant produced only very small amounts of the hopanoid peak. The Delta shc mutant grew as well as the wild type in medium buffered to pH 7 and demonstrated no defect in its ability to survive and replicate within macrophages, despite transmission electron microscopy (TEM) revealing defects in the organization of the cell envelope. The Delta shc mutant displayed increased sensitivity to low pH, detergent, and various antibiotics, including polymyxin B and erythromycin. Loss of hopanoid production also resulted in severe defects in both swimming and swarming motility. This suggests that hopanoid production plays an important role in the physiology of B. cenocepacia.

Can we trust mass spectrometry for determination of arsenic peptides in plants:comparison of LC-ICP-MS and LC-ES-MS/ICP-MS with XANES/EXAFS in analysis of Thunbergia alata

The weakest step in the analytical procedure for speciation analysis is extraction from a biological material into an aqueous solution which undergoes HPLC separation and then simultaneous online detection by elemental and molecular mass spectrometry (ICP-MS/ES-MS). This paper describes a study to determine the speciation of arsenic and, in particular, the arsenite phytochelatin complexes in the root from an ornamental garden plant Thunbergia alata exposed to 1 mg As L(-1) as arsenate. The approach of formic acid extraction followed by HPLC-ES-MS/ICP-MS identified different As(III)-PC complexes in the extract of this plant and made their quantification via sulfur (m/z 32) and arsenic (m/z 75) possible. Although sulfur sensitivity could be significantly increased when xenon was used as collision gas in ICP-qMS, or when HR-ICP-MS was used in medium resolution, the As:S ratio gave misleading results in the identification of As(III)-PC complexes due to the relatively low resolution of the chromatography system in relation to the variety of As-peptides in plants. Hence only the parallel use of ES-MS/ICP-MS was able to prove the occurrence of such arsenite phytochelatin complexes. Between 55 and 64% of the arsenic was bound to the sulfur of peptides mainly as As(III)(PC(2))(2), As(III)(PC(3)) and As(III)(PC(4)). XANES (X-ray absorption near-edge spectroscopy) measurement, using the freshly exposed plant root directly, confirmed that most of the arsenic is trivalent and binds to S of peptides (53% As-S) while 38% occurred as arsenite and only 9% unchanged as arsenate. EXAFS data confirmed that As-S and As-O bonds occur in the plants. This study confirms, for the first time, that As-peptides can be extracted by formic acid and chromatographically separated on a reversed-phase column without significant decomposition or de-novo synthesis during the extraction step.

Effects of mixture of nanoparticles under different salinity in the clams Ruditapes philippinarum and Ruditapes decussatus

Dissertação de mestrado, Biologia Marinha, Faculdade de Ciências e Tecnologia, Universidadde do Algarve, 2015

Partial volume correction using bimodal contrast agents in PET-MRI

Tese de mestrado integrado em Engenharia Biomédica e Biofísica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2014

Alcohol drinking, the metabolic syndrome and diabetes in a population with high mean alcohol consumption.

AIMS: To investigate the relationship of alcohol consumption with the metabolic syndrome and diabetes in a population-based study with high mean alcohol consumption. Few data exist on these conditions in high-risk drinkers. METHODS: In 6172 adults aged 35-75 years, alcohol consumption was categorized as 0, 1-6, 7-13, 14-20, 21-27, 28-34 and ≥ 35 drinks/week or as non-drinkers (0), low-risk (1-13), medium-to-high-risk (14-34) and very-high-risk (≥ 35) drinkers. Alcohol consumption was objectively confirmed by biochemical tests. In multivariate analysis, we assessed the relationship of alcohol consumption with adjusted prevalence of the metabolic syndrome, diabetes and insulin resistance, determined with the homeostasis model assessment of insulin resistance (HOMA-IR). RESULTS: Seventy-three per cent of participants consumed alcohol, 16% were medium-to-high-risk drinkers and 2% very-high-risk drinkers. In multivariate analysis, the prevalence of the metabolic syndrome, diabetes and mean HOMA-IR decreased with low-risk drinking and increased with high-risk drinking. Adjusted prevalence of the metabolic syndrome was 24% in non-drinkers, 19% in low-risk (P<0.001 vs. non-drinkers), 20% in medium-to-high-risk and 29% in very-high-risk drinkers (P=0.005 vs. low-risk). Adjusted prevalence of diabetes was 6.0% in non-drinkers, 3.6% in low-risk (P<0.001 vs. non-drinkers), 3.8% in medium-to-high-risk and 6.7% in very-high-risk drinkers (P=0.046 vs. low-risk). Adjusted HOMA-IR was 2.47 in non-drinkers, 2.14 in low-risk (P<0.001 vs. non-drinkers), 2.27 in medium-to-high-risk and 2.53 in very-high-risk drinkers (P=0.04 vs. low-risk). These relationships did not differ according to beverage types. CONCLUSIONS: Alcohol has a U-shaped relationship with the metabolic syndrome, diabetes and HOMA-IR, without differences between beverage types.

Correlations in hot asymmetric nuclear matter

Bulk and single-particle properties of hot hyperonic matter are studied within the Brueckner-Hartree-Fock approximation extended to finite temperature. The bare interaction in the nucleon sector is the Argonne V18 potential supplemented with an effective three-body force to reproduce the saturating properties of nuclear matter. The modern Nijmegen NSC97e potential is employed for the hyperon-nucleon and hyperon-hyperon interactions. The effect of temperature on the in-medium effective interaction is found to be, in general, very small and the single-particle potentials differ by at most 25% for temperatures in the range from 0 to 60 MeV. The bulk properties of infinite matter of baryons, either nuclear isospin symmetric or a Beta-stable composition that includes a nonzero fraction of hyperons, are obtained. It is found that the presence of hyperons can modify the thermodynamical properties of the system in a non-negligible way.

Shifts in resident bird communities associated with cloud forest patch size in Central Veracruz, Mexico

Avian communities in cloud forests have high levels of endemism and are at major risk given the accelerated rate of habitat fragmentation. Nevertheless, the response of these communities to changes in fragment size remains poorly understood. We evaluated species richness, bird community density, community composition, and dominance as indicators of the response to fragment size in a fragmented cloud forest landscape in central Veracruz, Mexico. Medium-sized fragments had statistically higher than expected species richness and more even communities, which may be a reflection of the intermediate disturbance hypothesis, in which medium-sized fragments are exploited by both forest and disturbance-associated species. Bird density also reached higher values in medium-sized fragments, which may indicate a carrying capacity in this habitat. However, large cloud forest fragments had a distinct taxonomic and functional composition, attributable to an increased number of understory insectivore species and canopy frugivores. By comparison, omnivorous species associated with human-altered habitats were more abundant in smaller fragments. Hence, although medium-sized cloud forest fragments had higher species richness and high bird density, large forest tracts maintained a distinct avian community composition, particularly of insectivorous and frugivorous species. Furthermore, the underlying response to fragmentation can only be properly addressed when contrasting several community attributes, such as richness, density, composition, and species dominance. Therefore, cloud forest conservation should aim to preserve the remaining large forest fragments to maintain comprehensive avian communities and avoid local extinctions.

Moisture can be the dominant environmental parameter governing cadaver decomposition in soil

Forensic taphonomy involves the use of decomposition to estimate postmortem interval (PMI) or locate clandestine graves. Yet, cadaver decomposition remains poorly understood, particularly following burial in soil. Presently, we do not know how most edaphic and environmental parameters, including soil moisture, influence the breakdown of cadavers following burial and alter the processes that are used to estimate PMI and locate clandestine graves. To address this, we buried juvenile rat (Rattus rattus) cadavers (∼18 g wet weight) in three contrasting soils from tropical savanna ecosystems located in Pallarenda (sand), Wambiana (medium clay), or Yabulu (loamy sand), Queensland, Australia. These soils were sieved (2 mm), weighed (500 g dry weight), calibrated to a matric potential of -0.01 megapascals (MPa), -0.05 MPa, or -0.3 MPa (wettest to driest) and incubated at 22 °C. Measurements of cadaver decomposition included cadaver mass loss, carbon dioxide-carbon (CO2-C) evolution, microbial biomass carbon (MBC), protease activity, phosphodiesterase activity, ninhydrin-reactive nitrogen (NRN) and soil pH. Cadaver burial resulted in a significant increase in CO2-C evolution, MBC, enzyme activities, NRN and soil pH. Cadaver decomposition in loamy sand and sandy soil was greater at lower matric potentials (wetter soil). However, optimal matric potential for cadaver decomposition in medium clay was exceeded, which resulted in a slower rate of cadaver decomposition in the wettest soil. Slower cadaver decomposition was also observed at high matric potential (-0.3 MPa). Furthermore, wet sandy soil was associated with greater cadaver decomposition than wet fine-textured soil. We conclude that gravesoil moisture content can modify the relationship between temperature and cadaver decomposition and that soil microorganisms can play a significant role in cadaver breakdown. We also conclude that soil NRN is a more reliable indicator of gravesoil than soil pH.