Characterization of dynamic solid phase DNA extraction from blood with magnetically controlled silica beads

A novel solid phase extraction technique is described where DNA is bound and eluted from magnetic silica beads in a manner where efficiency is dependent on the magnetic manipulation of the beads and not on the flow of solution through a packed bed. The utility of this technique in the isolation of reasonably pure, PCR-amplifiable DNA from complex samples is shown by isolating DNA from whole human blood, and subsequently amplifying a fragment of the beta-globin gene. By effectively controlling the movement of the solid phase in the presence of a static sample, the issues associated with reproducibly packing a solid phase in a microchannel and maintaining consistent flow rates are eliminated. The technique described here is rapid, simple, and efficient, allowing for recovery of more than 60% of DNA from 0.6 mu L of blood at a concentration which is suitable for PCR amplification. In addition, the technique presented here requires inexpensive, common laboratory equipment, making it easily adopted for both clinical point-of-care applications and on-site forensic sample analysis.

Heavy Metal Contamination in Phrynops geoffroanus (Schweigger, 1812) (Testudines: Chelidae) in a River Basin, So Paulo, Brazil

The Piracicaba River basin is considered the most disturbed river basin in the state of So Paulo. Considerable amounts of agricultural residues are seasonally drained into the river, and the region is also highly urbanized and industrialized with an incipient sewage treatment system. The presence of heavy metals has been previously reported for the water and riverbed in Piracicaba river basin. In this study we evaluated 13 heavy metals in the blood of 37 Geoffroy`s side-necked turtles, Phrynops geoffroanus, from Piracicaba River and Piracicamirim Creek, one of its tributaries. Blood levels of As, Co, Cr, Se and Pb varied among sites, whereas Sn varied between males and females. However, no obvious pathology was detected. Serum level of Cu (2,194 ng g(-1)) and Pb (1,150 ng g(-1)) found in this study are the highest ever described for any reptile; however, no clinical symptoms have been detected in the present study. There is no information about the time scale of such contamination, which could be currently subclinical and yet lead to a breakdown in the population reproductive success in a few years. Based on the present study, legal enforcement is urged in order to locate and extirpate heavy metal sources in the Piracicaba River basin. In addition, monitoring should include humans and commercial fish consumed in local markets.

Arsenic and Chromium in Brazilian Agricultural Supplies

Arsenic (As) and chromium (Cr) contents were measured in agricultural supplies used at different farms in Sao Paulo State, Brazil. The highest mass fractions of As were found in thermophosphates, reaching levels of 4 mg/kg. The highest mass fractions of Cr (21 g/kg) were found in calcium magnesium silicate, while the thermophosphates also presented high values reaching approximately 1 g/kg. The levels of As were within Brazilian guidelines, but the values of Cr in thermophosphates exceeded the levels permitted in Brazil. The As content in fertilizers may be considered safe (5 mg/kg) in terms of environmental pollution. However, the Cr content in calcium magnesium silicate following continuous use may constitute a significant problem in Brazil due to potentially increasing levels of this metal in soils.

Radicicol improves regeneration of skeletal muscle previously damaged by crotoxin in mice

This work investigates the influence of heat shock proteins (HSPs) on necrosis and subsequent skeletal muscle regeneration induced by crotoxin (CTX), the major component of Crotalus durissus terrificus venom. Mice were treated with radicicol, a HSP inductor, followed by an intramuscular injection of CTX into the gastrocnemius muscle. Treated groups were sacrificed 1, 10 and 21 days after CTX injection. Muscle histological sections were stained with toluidine blue and assayed for acid phosphatase or immunostained with either neuronal cell adhesion molecule (NCAM) or neonatal myosin heavy chain (MHCn). Muscle samples were also submitted to Western blotting analysis. The results show that CTX alone and CTX combined with radicicol induced a similar degree of myofiber necrosis. CTX-injured muscles treated with radicicol had increased cross-sectional areas at 10 and 21 days post-lesion compared with untreated CTX-injured muscles. Additionally, radicicol significantly increased the number of NCAM-positive satellite cells in the gastrocnemius at one day post-CTX injury. CTX-injured Muscles treated with radicicol contained more MHCn-positive regenerating myofibers compared with untreated CTX-injured muscles. These results suggest that HSPs contribute to the regeneration of myofibers damaged by CTX. Additionally, further studies should investigate the potential therapeutic effects of radicicol in skeletal muscles affected by Crotalus venom. (C) 2008 Elsevier Ltd. All rights reserved.

Low nanomolar concentration of mercury chloride increases vascular reactivity to phenylephrine and local angiotensin production in rats

Exposure to mercury at nanomolar level affects cardiac function but its effects on vascular reactivity have yet to be investigated. Pressor responses to phenylephrine (PHE) were investigated in perfused rat tail arteries before and after treatment with 6 nM HgCl2 during 1 h,,in the presence (E+) and absence (E-) of endothelium, after L-NAME (10(-4) M), indomethacin (10(-5) M), enalaprilate (1 mu M), tempol (1 mu M) and deferoxamine (300 mu M) treatments. HgCl2 increased sensitivity (pD(2)) without modifying the maximum response (Em) to PHE, but the pD(2) increase was abolished after endothelial damage. L-NAME treatment increased pD(2) and Emax. However, in the presence of HgCl2, this increase was smaller, and it did not modify Emax. After indomethacin treatment, the increase of pD(2) induced by HgCl2 was maintained. Enalaprilate, tempol and deferoxamine reversed the increase of pD(2) evoked by HgCl2. HgCl2 increased the angiotensin converting enzyme (ACE) activity explaining the result obtained with enalaprilate. Results suggest that at nanomolar concentrations HgCl2 increase the vascular reactivity to PHE. This response is endothelium mediated and involves the reduction of NO bioavailability and the action of reactive oxygen species. The local ACE participates in mercury actions and depends on the angiotensin 11 generation. (c) 2007 Elsevier Inc. All rights reserved.

Laboratory measurements of biomarkers and individual performances in Chironomus xanthus to evaluate pesticide contamination of sediments in a river of southeastern Brazil

This study aimed at evaluating biomarkers, individual and population responses in the native Chironomus xanthus to assess the toxicity of pesticide-contaminated sediments from the Monjolinho River (Southeast Brazil). We measured cholinesterase (ChE) and glutathione S-transferase activities (GST), as biomarkers and survival, individual growth and adult emergence, as individual performances. There was no response of the ChE activity and a tendency to decreased GST activity in contaminated sites, but this was generally not statistically significant. Therefore, there was no association of the biomarker responses with exposure to sediment containing pesticides. In contrast, ash free dry mass was significantly increased and male emergence was decreased in C. xanthus exposed to the same sediments. In conclusion, the selected biomarkers were not sensitive and specific enough to detect and anticipate effects of pesticide contamination at the levels measured in the study area. Nevertheless, individual performances alterations pointed to potential pollution problems and possible ecological consequences. (C) 2010 Elsevier Inc. All rights reserved.

Suitability of Daphnia similis as an alternative organism in ecotoxicological tests: implications for metal toxicity

The acute toxicity of metals to Daphnia similis was determined and compared to other daphnid species to evaluate the suitability of this organism in ecotoxicology bioassays. To verify the performance D. similis in toxicity tests, we also investigated the effect of Pseudokirchneriella subcapitata at 1 x 10(5) and 1 x 10(6) cells ml(-1) on Cd and Cr acute toxicity to the cladoceran. Daphnid neonates were exposed to a range of chromium and cadmium concentrations in the absence and presence of the algal cells. Metal speciation calculations using MINEQL(+) showed that total dissolved metal concentrations in zooplankton culture corresponded to 96.2% free Cd and 100% free Cr concentrations. Initial total dissolved metal concentrations were used for 48 h-LC(50) determination. LC(50) for D. similis was 5.15 x 10(-7) mol l(-1) dissolved Cd without algal cells, whereas with 1 x 10(5) cells ml(-1), it was significantly higher (7.15 x 10(-7) mol l(-1) dissolved Cd). For Cr, the 48 h-LC(50) value of 9.17 x 10(-7) mol l(-1) obtained for the cladoceran in tests with 1 x 10(6) cells ml(-1) of P. subcapitata was also significantly higher than that obtained in tests without algal cells (5.28 x 10(-7) mol l(-1) dissolved Cr). The presence of algal cells reduced the toxicity of metals to D. similis, as observed in other studies that investigated the effects of food on metal toxicity to standard cladocerans. Comparing our results to those of literature, we observed that D. similis is as sensitive to metals as other standardized Daphnia species and may serve as a potential test species in ecotoxicological evaluations.

Effects of interactions between algal densities and cadmium concentrations on Ceriodaphnia dubia fecundity and survival

The influence of different densities of the algae Pseudokirchneriella subcapitata on the chronic toxicity of cadmium to Ceriodaphnia dubia was investigated. The importance of algal cells as a source of metal to zooplankton was studied by exposing P. subcapitata cells to free cadmium ions and supplying the algae as food to C. dubia. The results of a bifactorial analysis (metal versus food levels) showed that metal toxicity to zooplankton was dependent on food level. Significant toxic effects on the fecundity and survival of C. dubia were observed at low metal concentrations with high algal density. Algae contaminated with Cd2+ were less toxic to cladoceran than was the Cd2+ in solution. Green algae retained cadmium and released low metal concentration in the test medium. We concluded that algal cells are an important route of exposure to metal and a factor that has an appreciable influence on the expression of metal toxicity to daphnids. (C) 2007 Elsevier Inc. All rights reserved.

Oxidative stress biomarkers of exposure in the blood of cichlid species from a metal-contaminated river

The oxidative stress biomarkers of exposure, such as reduced glutathione (GSH), activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and the levels of lipid peroxidation (LPO), were measured in the blood of three cichlid fish (Oreochromis niloticus, Tilapia rendalli, and Geophagus brasiliensis) taken during two seasons from two sites, unpolluted and polluted by industrial effluents, to evaluate the effectiveness of these biomarkers in assessing the impact of water contamination. The LPO levels in the blood were higher in fish from the metal-contaminated site and the chronic exposure led to significant changes in GPx, CAT, and SOD activities in all three cichlid species. The considerable variation of responses in these cichlids to water contamination evidenced differences in sensitivity to the metal contamination and/or in the potential to respond to it highlighting the importance of using a set of related biomarkers to assess the impact of water contamination. (C) 2007 Elsevier Inc. All rights reserved.

Post-failure behaviour of impulsively loaded circular plates

The dynamic plastic response of a simply supported circular plate is analysed. Emphasis is given to the plate behaviour after it has broken free from the supports due to a local material failure. The theoretical rigid plastic analysis predicts various features of the response such as the time to failure, residual kinetic energy and the critical velocity at failure. The residual kinetic energy of the plate could be significant enough to cause secondary impact damage. It is shown that the shape of the plate changes after breaking free from the supports, which is important for forensic investigations. The solution for various cases were proven to be exact in the context of the upper and lower bounds theorems of the theory of plasticity. (C) 2009 Elsevier Ltd. All rights reserved.

Saxitoxins accumulation by freshwater tilapia (Oreochromis niloticus) for human consumption

The accumulation of saxitoxins (STXs) in fish from freshwater aquaculture was investigated for the first time in the present study. Cyanotoxins have been monitored in liver and muscle samples of Oreochromis miloticus by chromatographic methods, both before and after the deputation process. The results show that tilapia can accumulate STXs. Our findings suggest that deputation with clean water is an alternative process to eliminate STXs from fish and, therefore, improve the safety of tilapia for consumers. (C) 2009 Elsevier Ltd. All rights reserved.

Use of Sugar Cane Vinasse to Mitigate Aluminum Toxicity to Saccharomyces cerevisiae

Owing to its toxicity, aluminum (Al), which is one of the most abundant metals, inhibits the productivity of many cultures and affects the microbial metabolism. The aim of this work was to investigate the capacity of sugar cane vinasse to mitigate the adverse effects of Al on cell growth, viability, and budding, as the likely result of possible chelating action. For this purpose, Fleischmann`s yeast (Saccharomyces cerevisiae) was used in growth tests performed in 125-mL Erlenmeyer flasks containing 30 mL of YED medium (5.0 g/L yeast extract plus 20 g/L glucose) supplemented with the selected amounts of either vinasse or Al in the form of AlCl(3) center dot A H(2)O. Without vinasse, the addition of increasing levels of Al up to 54 mg/L reduced the specific growth rate by 18%, whereas no significant reduction was observed in its presence. The toxic effect of Al on S. cerevisiae growth and the mitigating effect of sugar cane vinasse were quantified by the exponential model of Ciftci et al. (Biotechnol Bioeng 25:2007-2023, 1983). The cell viability decreased from 97.7% at the start to 84.0% at the end of runs without vinasse and to 92.3% with vinasse. On the other hand, the cell budding increased from 7.62% at the start to 8.84% at the end of runs without vinasse and to 17.8% with vinasse. These results demonstrate the ability of this raw material to stimulate cell growth and mitigate the toxic effect of Al.

Bait and Habitat Preferences, and Temporal Variability of Social Wasps (Hymenoptera: Vespidae) Attracted to Vertebrate Carrion

In addition to feeding on carrion tissues and fluids, social wasps can also prey on immature and adult carrion flies, thereby reducing their populations and retarding the decomposition process of carcasses. In this study, we report on the occurrence and behavior of social wasps attracted to vertebrate carrion. The collections were made monthly from September 2006 to October 2007 in three environments (rural, urban, and forest) in six municipalities of southeast Brazil, using baited bottle traps. We collected Agelaia pallipes (Olivier, 1791) (n = 143), Agelaia vicina (Saussure, 1854) (n = 106), Agelaia multipicta (Haliday, 1836) (n = 18), and Polybia paulista Ihering, 1896 (n = 3). The wasps were observed feeding directly on the baits and preying on adult insects collected in the traps. Bait and habitat associations, temporal variability of social wasps, and possible forensic implications of their actions are discussed.

Bioinsecticidal activity of Talisia esculenta reserve protein on growth and serine digestive enzymes during larval development of Anticarsia gemmatalis

Plants synthesize a variety of molecules to defend themselves against an attack by insects. Talisin is a reserve protein from Talisia esculenta seeds, the first to be characterized from the family Sapindaceae. In this study, the insecticidal activity of Talisin was tested by incorporating the reserve protein into an artificial diet fed to the velvetbean caterpillar Anticarsia gemmatalis, the major pest of soybean crops in Brazil. At 1.5% (w/w) of the dietary protein, Talisin affected larval growth, pupal weight, development and mortality, adult fertility and longevity, and produced malformations in pupae and adult insects. Talisin inhibited the trypsin-like activity of larval midgut homogenates. The trypsin activity in Talisin-fed larvae was sensitive to Talisin, indicating that no novel protease-resistant to Talisin was induced in Talisin-fed larvae. Affinity chromatography showed that Talisin bound to midgut proteinases of the insect A. gemmatalis, but was resistant to enzymatic digestion by these larval proteinases. The transformation of genes coding for this reserve protein could be useful for developing insect resistant crops. (C) 2010 Elsevier Inc. All rights reserved.

Breeding of Ornidia obesa (Diptera: Syrphidae: Eristalinae) on Pig Carcasses in Brazil

On Mho obesa F. (Diptera: Syrphidae) is usually neglected in forensic entomology, although adults are rather frequent on vertebrate carrion. In this study, conducted in southeastern Brazil in 2008, we used two pig carcasses, one killed by cocaine overdose and the other by shooting, to evaluate mainly the possible influences of the type of death on the larval development of O. obesa in the pig remains. We recorded the breeding of 218 adult specimens of this syrphid fly from the carcass killed by shooting, and none from the carcass killed by cocaine. These observations may open a new perspective for the use of O. obesa in forensic studies, considering its breeding preferences and its complete development on vertebrate carrion.