Physicochemical and bioinformatic characterization of Oreochromis niloticus vitellogenin as an endocrine disruption biomarker.

Aquatic biota is increasingly being exposed to chemical pollutants due to human activities and the relationship between the level of environmental pollution and fish reproduction is a continuously ongoing issue. The vitellogenin (Vtg) protein synthesis can be induced in the liver of juvenile and male fish after stimulation of the estrogen receptor and therefore, Vtg has been used as a biomarker of xenoestrogen exposure in several fish species. The current study reported the first physicochemical characterization of Vtg from Oreochromis niloticus. Adult male fish were exposed to 17α-ethinylestradiol for Vtg induction. Purified vitellogenin from plasma showed low stability at 25 and 4 °C in saline conditions, and good stability in acidic (low pH) or in heated conditions. The 3D modeling provided useful information on the structure of O. niloticus Vtg showing conserved structural features. According to bioinformatics and experimental results, there are important structural differences between the two chemical forms of Vtg (VtgAb and VtgC) in a phylogenetic context. The present results add information about the development of ecotoxicological immunoassays to study the endocrine disruption in O. niloticus improving the Vtg performance as a biomarker of reproduction in fish.

BDE-99 (2,2',4,4',5 - pentain polybrominated diphenyl ether) induces toxic effects in Oreochromis niloticus after sub-chronic and oral exposure.

PBDEs are toxic, lipophilic, hydrophobic, and persistent artificial chemicals, characterized by high physical and chemical stability. Although PBDEs are known to disturb hormone signaling, many effects of 2,2',4,4',5 - pentain polybrominated diphenyl ethers (BDE-99) in fish remain unclear. The current study investigates the effects of BDE-99 in Oreochromis niloticus where sixty-four juvenile fish were orally exposed to 0.294, 2.94, 29.4 ng g-1 of BDE-99, every 10 days, during 80 days. The results showed histopathological findings in liver and kidney, increasing acetylcholinesterase activity in muscle, disturbs in the antioxidant system in liver and brain and decreasing the plasmatic levels of vitellogenin in females. According to multivariate analysis (IBR), the higher doses are related to the interaction of oxidative and non-oxidative enzymes. The present study provided evidence of deleterious effects after sub-chronic exposure of BDE 99 to O. niloticus, increasing the knowledge about its risk of exposure in fish.

Assessing the antigenicity of different VP3 regions of infectious bursal disease virus in chickens from South Brazil.

BACKGROUND: Infectious bursal disease (IBD), also known as Gumboro disease, is a viral infection that causes mortality and immunosuppression in chickens (Gallus gallus). VP2 and VP3 are the major structural viral capsid components and are the most immunogenic proteins of IBD virus (IBDV). Reliable diagnostic tests using VP2 and VP3 produced in heterologous systems are important tools to control this infection. One advantage of an IBD diagnostic based on VP3, over those that use VP2, is that VP3 has linear epitopes, enabling its production in bacteria. RESULTS: We tested the suitability of recombinant VP3 (rVP3) as a diagnostic reagent in an enzyme-linked immunosorbent assay (ELISA). Compared with a commercial test, rVP3 ELISA showed high sensitivity and specificity as a diagnostic tool for vaccinated animals. In addition, rVP3, but not the commercial ELISA, was able to detect antibodies in nonvaccinated chickens, probably developed against circulating IBDV strains. It was possible the assessment of VP3 regions antigenicity using chicken antisera. CONCLUSIONS: The full-length recombinant VP3 can be used to assess post vaccination immunological status of chickens and its production is feasible and inexpensive. The evaluation of VP3 regions as candidates for general use in the diagnosis of IBD in chickens should be conducted with caution. Our work was the first to identify several regions of VP3 recognized by chicken antibodies.

Synthetic fish metallothionein design as a potential tool for monitoring toxic metals in water.

The diversity of aquatic ecosystems impacted by toxic metals is widely distributed throughout the world. The application of metallothionein (MT) as an early warning sign of metal exposure in freshwater fish is important in biomonitoring, but a more accessible, sensitive, safe, and efficient new methodological strategy is necessary. On this way, a fish MT synthetic gene from Oreochromis aureos was expressed in Escherichia coli to produce polyclonal antibodies against the protein. In the validation assays, these antibodies were able to detect hepatic MT from freshwater fishes Oreochromis niloticus, Pimelodus maculatus, Prochilodus lineatus, and Salminus brasiliensis showing a potential tool for toxic metals biomarker in biomonitoring of aquatic ecosystems. The current results showed the applicability of this molecule in quantitative immunoassays as a sensor for monitoring aquatic environments impacted by toxic metals. Due to the lack of methods focusing on metal pollution diagnostics in aquatic ecosystems, the current proposal revealed a promising tool to applications in biomonitoring programs of water resources, mainly in Brazil where the mining activity is very developed.

Identification of the major allergenic proteins from silkworm moth (Bombyx mori) involved in respiratory allergic diseases

INTRODUCTION AND OBJECTIVES: Moths are a significant source of indoor and outdoor aeroallergens. High prevalence of IgE-mediated sensitization was demonstrated in a group of patients with allergic respiratory diseases. There are no studies on adult stage of these moth species allergens involved in allergic respiratory reactions - the aim of this study. MATERIAL AND METHODS: 36 participants were included in an experimental study, submitted to skin prick test with Bombyx mori wing extract and six other common allergens, as well as Western blot analysis with incubated nitrocellulose membrane impregnated with silkworm moth extract and human IgE-antibody. The participants were divided into 3 groups: 1) 21 allergic patients whose skin prick test was positive to Bombyx mori wing extract, 2) eight allergic patients whose skin prick test was positive to mite and negative to Bombyx mori extract 3) seven negative non-allergic subjects. RESULTS: Among the 21 participants from group 1, 19 serum samples reacted to Bombyx mori extract by Western blot. All of them reacted to a protein at 80 kDa and five other proteins (66, 50, 45, 37 and 30 kDa) were identified in more than 50% of the individuals tested, considered as major allergenic proteins. Sera from seven out of eight patients sensitized to house dust mite demonstrated IgE-reactivity to Bombyx mori extract by Western blot analysis. Serum samples from healthy participants did not react at all. CONCLUSION: Six major reactive proteins by immunoblot analysis from moth’s wings sensitized patients can be potential allergens. The one at 80 kDa is the major protein, seen in all IgE-reactive patients from group 1 and in none from group 2, yet to be identified. Future studies should be conducted to better characterize these proteins

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Identification of the major allergenic proteins from silkworm moth (Bombyx mori) involved in respiratory allergic diseases.

INTRODUCTION AND OBJECTIVES: Moths are a significant source of indoor and outdoor aeroallergens. High prevalence of IgE-mediated sensitization was demonstrated in a group of patients with allergic respiratory diseases. There are no studies on adult stage of these moth species allergens involved in allergic respiratory reactions - the aim of this study. MATERIAL AND METHODS: 36 participants were included in an experimental study, submitted to skin prick test with Bombyx mori wing extract and six other common allergens, as well as Western blot analysis with incubated nitrocellulose membrane impregnated with silkworm moth extract and human IgE-antibody. The participants were divided into 3 groups: 1) 21 allergic patients whose skin prick test was positive to Bombyx mori wing extract, 2) eight allergic patients whose skin prick test was positive to mite and negative to Bombyx mori extract 3) seven negative non-allergic subjects. RESULTS: Among the 21 participants from group 1, 19 serum samples reacted to Bombyx mori extract by Western blot. All of them reacted to a protein at 80 kDa and five other proteins (66, 50, 45, 37 and 30 kDa) were identified in more than 50% of the individuals tested, considered as major allergenic proteins. Sera from seven out of eight patients sensitized to house dust mite demonstrated IgE-reactivity to Bombyx mori extract by Western blot analysis. Serum samples from healthy participants did not react at all. CONCLUSION: Six major reactive proteins by immunoblot analysis from moth's wings sensitized patients can be potential allergens. The one at 80 kDa is the major protein, seen in all IgE-reactive patients from group 1 and in none from group 2, yet to be identified. Future studies should be conducted to better characterize these proteins.

Synthesis and characterization of mead: from the past to the future and development of a new fermentative route.

In ancient times, mead was produced by fermentation of honey in water and presented low quality, undesired by-products, off-flavors, and the production was time consuming. In this study, nine experiments were performed to improve the fermentation and mead characteristics. Distillation was not part of the production process and it was performed in this work to produce a new spirit. The samples were characterized by gas chromatography coupled to mass spectrometry, high performance liquid chromatography, digital densimetry, titration, gravimetric method, pH, and refractometry. The results were compared to commercial beverages and legal limits. The meads presented high ethanol concentration, low by-products, fast fermentation, and high quality. The spirits showed high quality and the concentrations of acetic acid, ethyl acetate, methanol, higher alcohols, and ethyl carbamate were below the limits for safe consumption. In conclusion, it was possible to develop new conditions to produce high quality mead and mead spirit.

In silico analysis of amino acid variation in human respiratory syncytial virus: insights into immunodiagnostics

BACKGROUND The highly contagious nature of human respiratory syncytial virus (HRSV) and the gravity of its infection in newborns and vulnerable adults pose a serious public health problem. Thus, a rapid and sensitive diagnostic test for viral detection that can be implemented upon the first appearance of symptoms is needed. The genetic variation of the virus must be considered for immunodiagnostic purposes. OBJECTIVES To analyse HRSV genetic variation and discuss the possible consequences for capture immunoassay development. METHODS We performed a wide analysis of N, F and G protein variation based on the HRSV sequences currently available in the GenBank database. We also evaluated their similarity with homologous proteins from other viruses. FINDINGS The mean amino acid divergences for the N, F, and G proteins between HRSV-A and HRSV-B were determined to be approximately 4%, 10% and 47%, respectively. Due to their high conservation, assays based on the full-length N and F proteins may not distinguish HRSV from human metapneumovirus and other Mononegavirales viruses, and the full-length G protein would most likely produce false negative results due to its high divergence. MAIN CONCLUSIONS We have identified specific regions in each of these three proteins that have higher potential to produce specific results, and their combined utilisation should be considered for immunoassay development.

In silico analysis of amino acid variation in human respiratory syncytial virus: insights into immunodiagnostics.

BACKGROUND: The highly contagious nature of human respiratory syncytial virus (HRSV) and the gravity of its infection in newborns and vulnerable adults pose a serious public health problem. Thus, a rapid and sensitive diagnostic test for viral detection that can be implemented upon the first appearance of symptoms is needed. The genetic variation of the virus must be considered for immunodiagnostic purposes. OBJECTIVES: To analyse HRSV genetic variation and discuss the possible consequences for capture immunoassay development. METHODS: We performed a wide analysis of N, F and G protein variation based on the HRSV sequences currently available in the GenBank database. We also evaluated their similarity with homologous proteins from other viruses. FINDINGS: The mean amino acid divergences for the N, F, and G proteins between HRSV-A and HRSV-B were determined to be approximately 4%, 10% and 47%, respectively. Due to their high conservation, assays based on the full-length N and F proteins may not distinguish HRSV from human metapneumovirus and other Mononegavirales viruses, and the full-length G protein would most likely produce false negative results due to its high divergence. MAIN CONCLUSIONS: We have identified specific regions in each of these three proteins that have higher potential to produce specific results, and their combined utilisation should be considered for immunoassay development.