Characterization of cholinesterases in plasma of three Portuguese native bird species: application to biomonitoring

Over the last decades the inhibition of plasma cholinesterase (ChE) activity has been widely used as a biomarker to diagnose organophosphate and carbamate exposure. Plasma ChE activity is a useful and non-invasive method to monitor bird exposure to anticholinesterase compounds; nonetheless several studies had shown that the ChE form(s) present in avian plasma may vary greatly among species. In order to support further biomonitoring studies and provide reference data for wildlife risk-assessment, plasma cholinesterase of the northern gannet (Morus bassanus), the white stork (Ciconia ciconia) and the grey heron (Ardea cinerea) were characterized using three substrates (acetylthiocholine iodide, propionylthiocholine iodide, and S-butyrylthiocholine iodide) and three ChE inhibitors (eserine sulphate, BW284C51, and iso-OMPA). Additionally, the range of ChE activity that may be considered as basal levels for non-exposed individuals was determined. The results suggest that in the plasma of the three species studied the main cholinesterase form present is butyrylcholinesterase (BChE). Plasma BChE activity in non-exposed individuals was 0.48±0.11 SD U/ml, 0.39±0.12 SD U/ml, 0.15±0.04 SD U/ml in the northern gannet, white stork and grey heron, respectively. These results are crucial for the further use of plasma BChE activity in these bird species as a contamination bioindicator of anti-cholinesterase agents in both wetland and marine environments. Our findings also underscore the importance of plasma ChE characterization before its use as a biomarker in biomonitoring studies with birds.     

Proteasome inhibition and ROS generation by 4-nerolidylcatechol induces melanoma cell death

Induction of apoptotic cell death in response to chemotherapy and other external stimuli has proved extremely difficult in melanoma, leading to tumor progression, metastasis formation and resistance to therapy. A promising approach for cancer chemotherapy is the inhibition of proteasomal activity, as the half‐life of the majority of cellular proteins is under proteasomal control and inhibitors have been shown to induce cell death programs in a wide variety of tumor cell types. 4‐Nerolidylcatechol (4‐NC) is a potent antioxidant whose cytotoxic potential has already been demonstrated in melanoma tumor cell lines. Furthermore, 4‐NC was able to induce the accumulation of ubiquitinated proteins, including classic targets of this process such as Mcl‐1. As shown for other proteasomal inhibitors in melanoma, the cytotoxic action of 4‐NC is time‐dependent upon the pro‐apoptotic protein Noxa, which is able to bind and neutralize Mcl‐1. We demonstrate the role of 4‐NC as a potent inducer of ROS and p53. The use of an artificial skin model containing melanoma also provided evidence that 4‐NC prevented melanoma proliferation in a 3D model that more closely resembles normal human skin.     

Human papillomavirus in invasive cervical cancer and cervical intraepithelial neoplasia 2 and 3 in Venezuela: A cross-sectional study

BackgroundThis study investigated the distribution of human papillomavirus (HPV) types in invasive cervical cancer (ICC), cervical intraepithelial neoplasia 2 (CIN2) and cervical intraepithelial neoplasia 3 (CIN3) in Venezuela.MethodsParaffin-embedded samples from 329 women from 29 medical centers of the 24 states of Venezuela were analyzed to determine the distribution of HPV types for ICC, CIN2, and CIN3, the prevalence of single and multiple infection, and the association of HPV types with severity of lesion, comparing CIN2 versus CIN3+ (CIN3 and ICC). The samples were analyzed with the polymerase chain reaction (PCR) followed by reverse hybridization for the identification of HPV types.ResultsHPV was identified in 95/96 ICC specimens (98.9%), in 142/149 CIN3 (95.3%) and in 78/84 CIN2 samples (92.8%). The most common types for ICC and CIN3 were: HPV16, 18, 31, and 33, and for CIN2 were HPV16, 31, 51, 52, and 18. HPV single infection was found in 82.1% of ICC cases, in 79.4% of CIN2 cases, and in 77.4% of CIN3 cases. HPV16 was identified as a single infection more frequently in women with CIN3+ than in those with CIN2 (68.6% versus 46.7%, P = 0.002), and HPV16 or HPV18 types were more prevalent in CIN3+ than in CIN2 (73.4% versus 50%, P = 0.0006).Conclusionthis is the first study of the distribution of HPV types in ICC, CIN2, and CIN3 conducted throughout the territory of Venezuela. HPV16 and HPV18 were the most frequent HPV types identified in single and multiple infections in both ICC and CIN3 groups, and are associated with severity of lesion. The knowledge of the distribution of HPV types would allow organization of an HPV-DNA-based screening test, and consideration of the implementation of prophylactic vaccination in Venezuela.     

DNA damage, RAD9 and fertility/infertility of Echinococcus granulosus hydatid cysts

Hydatidosis, caused by the larval stage of the platyhelminth parasite Echinococcus granulosus, affects human and animal health. Hydatid fertile cysts are formed in intermediate hosts (human and herbivores) producing protoscoleces, the infective form to canines, at their germinal layers. Infertile cysts are also formed, but they are unable to produce protoscoleces. The molecular mechanisms involved in hydatid cysts fertility/infertility are unknown. Nevertheless, previous work from our laboratory has suggested that apoptosis is involved in hydatid cyst infertility and death. On the other hand, fertile hydatid cysts can resist oxidative damage due to reactive oxygen and nitrogen species. On these foundations, we have postulated that when oxidative damage of DNA in the germinal layers exceeds the capability of DNA repair mechanisms, apoptosis is triggered and hydatid cysts infertility occurs. We describe a much higher percentage of nuclei with oxidative DNA damage in dead protoscoleces and in the germinal layer of infertile cysts than in fertile cysts, suggesting that DNA repair mechanisms are active in fertile cysts. rad9, a conserved gene, plays a key role in cell cycle checkpoint modulation and DNA repair. We found that RAD9 of E. granulosus (EgRAD9) is expressed at the mRNA and protein levels. As it was found in other eukaryotes, EgRAD9 is hyperphosphorylated in response to DNA damage. Our results suggest that molecules involved in DNA repair in the germinal layer of fertile hydatid cysts and in protoscoleces, such as EgRAD9, may allow preserving the fertility of hydatid cysts in the presence of ROS and RNS.     

Contribution of redox status to hepatitis C virus E2 envelope protein function and antigenicity

Disulfide bonding contributes to the function and antigenicity of many viral envelope glycoproteins. We assessed here its significance for the hepatitis C virus E2 envelope protein and a counterpart deleted for hypervariable region-1 (HVR1). All 18 cysteine residues of the antigens were involved in disulfides. Chemical reduction of up to half of these disulfides was compatible with anti-E2 monoclonal antibody reaction, CD81 receptor binding, and viral entry, whereas complete reduction abrogated these properties. The addition of 5,5'-dithiobis-2-nitrobenzoic acid had no effect on viral entry. Thus, E2 function is only weakly dependent on its redox status, and cell entry does not require redox catalysts, in contrast to a number of enveloped viruses. Because E2 is a major neutralizing antibody target, we examined the effect of disulfide bonding on E2 antigenicity. We show that reduction of three disulfides, as well as deletion of HVR1, improved antibody binding for half of the patient sera tested, whereas it had no effect on the remainder. Small scale immunization of mice with reduced E2 antigens greatly improved serum reactivity with reduced forms of E2 when compared with immunization using native E2, whereas deletion of HVR1 only marginally affected the ability of the serum to bind the redox intermediates. Immunization with reduced E2 also showed an improved neutralizing antibody response, suggesting that potential epitopes are masked on the disulfide-bonded antigen and that mild reduction may increase the breadth of the antibody response. Although E2 function is surprisingly independent of its redox status, its disulfide bonds mask antigenic domains. E2 redox manipulation may contribute to improved vaccine design.     

Kinetic modelling and simulation of laccase catalyzed degradation of reactive textile dyes

A kinetic model based on Michaelis-Menten equation was developed to simulate the dye decolourisation of Reactive Black 5 (RB5), Reactive Blue 114 (RB114), Reactive Yellow 15 (RY15), Reactive Red 239 (RR239) and Reactive Red 180 (RR180) dyes by commercial laccase. The unusual kinetic behavior of some of these reactions suggests that the kinetic model must consider the activation of the laccase-mediator system. Several reactions at different concentrations of each dye were performed in batch reactors and time courses were obtained. A LSODE code to solve the differential equation obtained from the batch reactor was combined with an optimization Fortran program to obtain the theoretical time courses. The time courses obtained from the developed program were compared with the experimentally obtained ones to estimate the kinetic constants that minimized the difference between them. The close correlation between the predicted and the experimental results seems to support the reliability of the established models.     

Ascomycetous yeast species recovered from grapes damaged by honeydew and sour rot

Aims: To identify ascomycetous yeasts recovered from sound and damaged grapes by the presence of honeydew or sour rot. Methods and Results: In sound grapes, the mean yeast counts ranged from 3·20 ± 1·04 log CFU g^?1 to 5·87 ± 0·64 log CFU g^?1. In honeydew grapes, the mean counts ranged from 3·88 ± 0·80 log CFU g^?1 to 6·64 ± 0·77 log CFU g^?1. In sour rot grapes counts varied between 6·34 ± 1·03 and 7·68 ± 0·38 logCFU g^?1. Hanseniaspora uvarum was the most frequent species from sound samples. In both types of damage, the most frequent species were Candida vanderwaltii, H. uvarum and Zygoascus hellenicus. The latter species was recovered in high frequency because of the utilization of the selective medium DBDM (Dekkera/Brettanomyces differential medium). The scarce isolation frequency of the wine spoilage species Zygosaccharomyces bailii (in sour rotten grapes) and Zygosaccharomyces bisporus (in honeydew affected grapes) could only be demonstrated by the use of the selective medium ZDM (Zygosaccharomyces differential medium). Conclusions: The isolation of several species only from damaged grapes indicates that damage constituted the main factor determining yeast diversity. The utilization of selective media is required for eliciting the recovery of potentially wine spoilage species. Significance and Impact of the Study: The impact of damaged grapes in the yeast ecology of grapes has been underestimated.     

Canadians' Representation of Chemical,Biological, Radiological,Nuclear, and Explosive (CBRNE) Terrorism: A Content Analysis

The global threat of terrorism raises questions about preparedness and risk communication in the context of public health and security. Although experts discriminate between chemical, biological, radiological, nuclear, and explosive (CNRNE) terrorist events, little is known about how the Canadian public represents these forms of terrorism. A stratified random sample of 1502 Canadians participated in a telephone survey on CBRNE terrorism. A word association technique was used to assess first words or images that came to mind while thinking about different types of terrorist scenarios. Content analysis of this data revealed a number of potential uncertainties and misconceptions regarding different types of terrorism scenarios. Despite most frequently providing agents in response questions surrounding chemical or biological terrorism, respondents frequently confounded agents of biological and chemical nature. Similarly, different aspects of nuclear events were not consistently distinguished. Most notably, however, a sizable proportion of respondents had difficulty providing word associations to the different types of terrorist events or only provided vague responses that closely mirrored the scenario in question. Finally, images regarding the potential impacts of scenarios were infrequent. Implications for risk communication and preparedness are discussed; in particular, the need to provide the public with more detailed information regarding the nature of different forms of CBRNE terrorism and how to best prepare for a potential event.     

Metabolic regulation of pathways of carbohydrate oxidation in potato (Solanum tuberosum) tubers

In the present article we evaluate the consequence of tuber-specific expression of yeast invertase, on the pathways of carbohydrate oxidation, in potato ( Solanum tuberosum L. cv. Desiree). We analysed the relative rates of glycolysis and the oxidative pentose phosphate pathway that these lines exhibited as well as the relative contributions of the cytochrome and alternative pathways of mitochondrial respiration. Enzymatic and protein abundance analysis revealed concerted upregulation of the glycolytic pathway and of specific enzymes of the tricarboxylic acid cycle and the alternative oxidase but invariant levels of enzymes of the oxidative pentose phosphate pathway and proteins of the cytochrome pathway. When taken together these experiments suggest that the overexpression of a cytosolic invertase (EC 3.2.1.26) results in a general upregulation of carbohydrate oxidation with increased flux through both the glycolytic and oxidative pentose phosphate pathways as well as the cytochrome and alternative pathways of oxidative phosphorylation. Moreover these data suggest that the upregulation of respiration is a consequence of enhanced efficient mitochondrial metabolism.     

Genetics University of Toronto Thrombophilia Study in Women (GUTTSI): genetic and other risk factors for venous thromboembolism in women

Background  

Women may be at increased risk for venous thromboembolism (VTE) as compared with men. We studied the effects of genetic and biochemical markers of thrombophilia in women, in conjunction with other established risk factors for VTE.