Transmission Route Used By Parasitic Lasidium Larvae of The Freshwater Mussel Anodontites Trapesialis on Guppies Poecilia Reticulata During Short Cohabitation.

We describe for the first time the transmission route employed by the parasitic larvae of the freshwater mussel Anodontites trapesialis (Lamarck, 1819) during cohabitation on the guppy Poecilia reticulata Peter, 1859. The freshwater mussel and fish-host were employed as a model system to investigate the infection and parasite establishment. Laboratory experiments included video recording and histopathological analysis of the infection. In vivo video observations demonstrated that lasidium larvae could be transmitted to guppies during direct contact. A series of histology samples and photography suggest that this larva attaches and colonizes possibly on-site on the outer surface of the fish, causing cellular inflammation in the epidermis layer with cellular hyperplasia in the zone of parasite attachment. An evident hyaline layer, cellular hypertrophy, and a large number of undifferentiated proliferating cells were observed. Hemorrhagic tissue and swelling were observed in the epidermis and dermic zone. The total number of larvae per male and female guppy was 525 ± 86 and 494 ± 167, respectively. No parasitic preference was detected for male versus female parasitized fish.

Development of an ecotoxicological protocol for the deep-sea fauna using the hydrothermal vent shrimp Rimicaris exoculata.

In light of deep-sea mining industry development, particularly interested in massive-sulphide deposits enriched in metals with high commercial value, efforts are increasing to better understand potential environmental impacts to local fauna. The aim of this study was to assess the natural background levels of biomarkers in the hydrothermal vent shrimp Rimicaris exoculata and their responses to copper exposure at in situ pressure (30MPa) as well as the effects of depressurization and pressurization of the high-pressure aquarium IPOCAMP. R. exoculata were collected from the chimney walls of the hydrothermal vent site TAG (Mid Atlantic Ridge) at 3630m depth during the BICOSE cruise in 2014. Tissue metal accumulation was quantified in different tissues (gills, hepatopancreas and muscle) and a battery of biomarkers was measured: metal exposure (metallothioneins), oxidative stress (catalase, superoxide dismutase, glutathione-S-transferase and glutathione peroxidase) and oxidative damage (lipid peroxidation). Data show a higher concentration of Cu in the hepatopancreas and a slight increase in the gills after incubations (for both exposed groups). Significant induction of metallothioneins was observed in the gills of shrimps exposed to 4µM of Cu compared to the control group. Moreover, activities of enzymes were detected for the in situ group, showing a background protection against metal toxicity. Results suggest that the proposed method, including a physiologically critical step of pressurizing and depressurizing the test chamber to enable the seawater exchange during exposure to contaminants, is not affecting metal accumulation and biomarkers response and may prove a useful method to assess toxicity of contaminants in deep-sea species.

Botulinum toxin type A as an adjunct in postoperative pain management in dogs undergoing radical mastectomy.

The aim of this randomised placebo-controlled, observer-blinded study was to evaluate the analgesic effects of botulinum toxin type A (BoNT-A) as an adjunct for postoperative pain control in dogs. Sixteen dogs undergoing bilateral radical mastectomy for treatment of mammary tumours were enrolled. Twenty-four hours before surgery, the subjects were distributed into two groups of eight dogs each: 7 iu/kg BoNT-A (BoNT-A) or saline (Control) was administered subcutaneously in each mammary gland. Following sedation with intramuscular 0.03 mg/kg acepromazine and 0.3 mg/kg morphine, anaesthesia was induced intravenously with 4 mg/kg propofol and maintained with isoflurane/O2. Postoperative analgesia was evaluated for 72 hours after extubation using the Visual Analogue Scale (VAS) and modified Glasgow Composite Measure Pain Scale (modified-GCMPS). Rescue analgesia was provided with intramuscular morphine (0.5 mg/kg). Data were analysed using analysis of variance, Tukey's test, Mann-Whitney U test and Friedman test (P<0.05). The pain scores were significantly lower in the BoNT-A than in the Control from 8 hours to 60 hours and from 12 hours to 60 hours after extubation, based on the VAS and modified-GCMPS, respectively. Rescue analgesia was required by significantly more dogs in the Control (7/8) compared with the BoNT-A (2/8) (P=0.022). Pre-emptive BoNT-A appears to be effective as an adjuvant for postoperative pain management in dogs undergoing bilateral radical mastectomy.

Comparação entre dois protocolos de tratamento de ceratoconjuntivite seca experimentalmente induzida em coelhos / Comparison of two treatment protocols of experimentally induced keratoconjunctivitis sicca in rabbits

O objetivo deste estudo foi avaliar e comparar a eficácia de dois protocolos de tratamento de ceratoconjuntivite seca (CCS) experimentalmente induzida em coelhos: uma formulação oftálmica tópica composta por álcool polivinílico 1,4%, adicionado com acetilcisteína 10% e pilocarpina 1% (AAP), e outro protocolo com o uso do óleo de semente de linhaça (OL) tópico em forma de colírio, durante 12 semanas. Foram utilizados 15 coelhos machos, adultos, da raça Nova Zelândia, alocados aleatoriamente em três grupos: grupo C (controle), grupo AAP (formulação oftálmica) e grupo L (OL tópica). Os animais foram avaliados semanalmente pelo teste lacrimal de Schirmer, teste de fluoresceína e teste de Rosa Bengala; uma vez por mês, pelo exame de citologia esfoliativa ocular; ao final do experimento, pela análise histopatológica da córnea e conjuntiva. Os resultados demonstraram que houve um aumento maior na produção lacrimal quando utilizada a formulação oftálmica, e uma resolução mais rápida das úlceras de córnea, bem como diminuição no número de células desvitalizadas quando utilizado o óleo de semente de linhaça, além de aumento no número de células caliciformes em ambos os grupos de tratamento. A associação desses dois protocolos pode ser no futuro uma alternativa no tratamento da CCS.

The objective of this study was to evaluate and compare the effectiveness of two treatment protocol of experimentally induced keratoconjunctivitis sicca (KCS) in rabbits, a topical ophthalmic formulation composed by 1.4% povinilic alcohol added with 10% acetylcysteine and 1% pilocarpine (AAP) and another protocol with the topical use of the linseed seed oil (LO) in eye drop form f or 12 weeks. Fifteen male New Zealand white rabbits were aleatory allocated in 3 groups: Group C (Control), Group AAP (ophthalmic formulation) and Group L (LO topical). The animals were evaluated weekly using the Schirmer's tear test, fluorescein test and Rose Bengal test monthly for ocular cytology, and at the end of the experiment for histopathological analysis of cornea and conjunctive. The results demonstrated that there was a larger increase in the tear production when the ophthalmic formulation was us ed and a faster rapid resolution of corneal ulcers and decrease in the number of devitalized cells when linseed seed oil was used, besides an increase in the number of caliciform cells in both treatment groups. The association of those two protocols can be a future alternative in the treatment of KCS.

In Vivo Effects of Cagaita (Eugenia dysenterica, DC.) Leaf Extracts on Diarrhea Treatment.

Eugenia dysenterica is a plant typically found in the Cerrado biome and commonly used in popular medicine due to its pharmacological properties, which include antidiarrheal, skin healing, and antimicrobial activities. The effects of ethanolic extract, aqueous extract and infusion of E. dysenterica leaves on intestinal motility and antidiarrheal activity were evaluated using ricin oil-induced diarrhea in rats. At doses of 400 and 800 mg·Kg(-1), the ethanolic extract decreased intestinal motility while the other extracts showed no significant effects. Moreover, serum levels of chloride, magnesium, and phosphorus were also measured in rats. Histopathologic and enzymatic analyses were also performed to investigate any toxic effect. Animals treated with infusion, ethanolic extract, ricin oil, and loperamide presented morphological alterations in the small intestine, such as mucosa lesion, epithelial layer damage, and partial loss and/or morphological change of villi. Furthermore, the liver showed congestion and hydropic degeneration. Serum levels of alanine aminotransferase increased significantly in all treatments, but none rose above reference values. In summary, our results suggest that compounds present in leaves of E. dysenterica may have therapeutic benefits on recovery from diarrhea despite their toxic effects.

Identification of E. dysenterica laxative peptide: a novel strategy in the treatment of chronic constipation and irritable bowel syndrome.

Plants have contributed over the years to the discovery of various pharmacological products. Amongst the enormous diversity of herbs with remarkable medicinal use and further pharmacological potential, here in this report we evaluated pulp extracts from Eugenia dysenterica fruits and further identified the active principle involved in such laxative activity in rats. For protein isolation, fruits were macerated with an extraction solution following precipitation with (NH(4))(2)SO(4) (100%). After dialysis, the peptide was applied onto a reversed-phase semi-preparative HPLC column, and the major fraction was eluted with 26% and 66% acetonitrile. The evaluation of molecular masses by MALDI-TOF and Tris/Tricine SDS-PAGE of HPLC fractions showed the presence of a major peptide with approximately 7 kDa. The N-terminal amino acid peptide sequence was determined and showed no similarity to other proteins deposited in the Data Bank. Peptide from E. dysenterica was able to enhance rats' intestinal motility by approximately 20.8%, probably being responsible for laxative activity. Moreover, these proteins were non-toxic to mammals, as observed in histopathology and hemolytic analyses. In conclusion, results here reported indicate that, in the near future, proteins synthesized by E. dysenterica fruits could be utilized in the development of novel biotechnological pharmaceutics with laxative properties for use in chronic constipation and irritable bowel syndrome treatment.

Purification and characterization of a liver-derived beta-N-Acetylhexosaminidase from marine mammal Sotalia fluviatilis.

A beta-N-Acetylhexosaminidase (EC 3.2.1.52) was purified from hepatic extracts of Sotalia fluviatilis, order Cetacea. The protein was purified by using ammonium sulfate fractionation and four subsequent chromatographies (Biogel A 1.5 m, Chitin, Deae-Biogel and hydroxyapatite resins). After these purification steps, the enzyme was purified 380.5-fold with an 8.4% yield. The molecular mass (10 kDa) was estimated by SDS-PAGE and MALDI-TOF analysis. A Km of 2.72 mM and Vmax 9.5 x 10(-6) micromol/(min x mg) were found for this enzyme, determined by p-nitrophenyl-beta-D: -hexosaminide substrate digestion. Optimal pH and temperature for beta-N-Acetylhexosaminidase activity were 5.0 and 60 degrees C, respectively. Enzyme activity was inhibited by sodium selenate (Na(2)SeO(4)), mercuric chloride (HgCl(2)) and sodium dodecyl sulfate (C(12)H(25)SO(4)Na), and activated by zinc, calcium, barium and lithium ions. Characterization of the beta-N-Acetylhexosaminidase in Sotalia fluviatilis can be a basis for physiological studies in this species.

Proregion of Acanthoscelides obtectus cysteine proteinase: a novel peptide with enhanced selectivity toward endogenous enzymes.

Acanthoscelides obtectus is a devastating storage insect pest capable of causing severe bean crop losses. In order to maintain their own development, insect pest larvae feed continuously, synthesizing efficient digestive enzymes. Among them, cysteine proteinases (CPs) are commonly produced as inactive precursors (procysteines), requiring a cleavage of the peptide proregion to become active. The proregion fits tightly into the active site of procysteines, efficiently preventing their activity. In this report, a CP cDNA (cpao) was isolated from A. obtectus midgut larvae. In silico studies indicated that the complete CP sequence contains a hydrophobic signal peptide, a prodomain and a conserved catalytic region. Moreover, the encoding cDNA contains 963bp translating into a 321 residue protein, CPAo, which was expressed in E. coli, fused with thioredoxin. Enzymatic assays using the recombinant protein revealed that the enzyme was catalytically active, being able to cleave the synthetic substrate Z-Phe-Arg-7-AMC. Additionally, this report also focuses the cpao propeptide (PCPAo) subcloning and expression. The expressed propeptide efficiently inhibited CPAo, as well as digestive CP of other bean bruchids. Little or no activity was found against proteolytic enzymes of two other coleopterans: Rhyzopertha dominica and Anthonomus grandis. The data reported here indicate the possibility of endogenous propeptides as a novel strategy on bruchids control, which could be applicable to bean improvement programs.

Combined analysis of supernatant-based feeding bioassays and PCR as a first-tier screening strategy for Vip -derived activities in Bacillus thuringiensis strains effective against tropical fall armyworm.

AIMS: To assess whether feeding bioassays using culture-supernatant proteins could be combined with PCR into a first-tier screening strategy for Vip3A-like genes efficient against tropical Spodoptera frugiperda. METHODS AND RESULTS: Out of 12 Bacillus thuringiensis strains studied, the total protein concentrated from the culture supernatant of only the strain HD125 yielded a significantly increased armyworm mortality and an intense band of the predicted size for VIP3A protein in SDS-PAGE. However, PCR and sequencing data indicated Vip-like genes are ubiquitous in tropical B. thuringiensis isolates. Interestingly, the HD125 strain was also the only one displaying a single-band amplification pattern and the highest sequence identity to the reported Vip3A(a) gene. CONCLUSIONS: Results suggest the insecticidal effectiveness of putative VIPs in B. thuringiensis isolates can be preliminarily estimated by the use of supernatant-derived total protein in feeding experiments, though only in a limited manner. SIGNIFICANCE AND IMPACT OF THE STUDY: A simple and cost-effective first-tier screening strategy for VIP-derived activities in B. thuringiensis collections can be developed by combining PCR and feeding bioassays. Moreover, the employed primers showed to be useful as a tool for strains differentiation at DNA level, and for characterization and isolation of Vip-like genes in tropical B. thuringiensis germplasm.

Expression and secretion of recombinant ovine beta-lactoglobulin in Saccharomyces cerevisiae and Kluyveromyces lactis.

High expression and secretion of recombinant ovine beta-lactoglobulin has been achieved in the yeast Kluyveromyces lactis. The yield of beta-lactoglobulin is 40-50 mg per litre of culture supernatant and accounts for approx. 72% of the total secreted protein. Constitutive expression is under the control of the Saccharomyces cerevisiae phosphoglycerate kinase promoter from an intronless version of the beta-lactoglobulin gene. Secretion is specified by the ovine protein's own signal sequence. this system, coupled to an efficient and novel recovery protocol, allows 30 mg of pure protein to be isolated from a typical 1 litre culture. The protein is virtually indistinguishable from beta-lactoglobulin conventionally purified from sheep milk by its behaviour in native PAGE and SDS/PAGE, reactivity to antibodies, CD, fluorescence spectroscopy and N-terminal sequencing. Attempts to achieve a similar expression and secretion system in the yeast S. cerevisiae met with only limited success, although it was found that heat-shock treatment modestly increased the yield up to approx. 3-4 mg per litre of culture supernatant. Site-directed mutagenesis showed that secretion in S. cerevisiae depended upon correct formation of the two disulphide bonds present in beta-lactoglobulin.

The Anticarsia gemmatalis nuclear polyhedrosis virus polyhedrin gene region: sequence analysis, gene product and structural comparisons.

The genomic region of the Anticarsia gemmatalis multiple nucleocapsid nuclear polyhedrosis virus (AgMNPV) strain 2D encoding the polyhedrin gene was cloned and mapped, and a 2085 bp SphI-PstI fragment containing the gene was sequenced. The polyhedrin polypeptide of the parental isolate AgMNPV was manually sequenced, and the amino acid sequence obtained agreed with that deduced from the DNA coding region sequence. AgMNPV and Orgyia pseudotsugata MNPV (OpMNPV) are similar in terms of promoter structure and polyhedrin primary sequence, and the polyhedrin gene of both viruses is transcribed in the anti-clockwise direction in relation to their physical maps. The region upstream from the polyhedrin gene of AgMNPV, OpMNPV, Bombyx mori NPV and Autographa californica MNPV (AcMNPV) was compared and this showed that the open reading frame (ORF) common to all four viruses (ORF 5) has sequence homology with the AcMNPV 25K gene. The sequences between ORF 5 and the polyhedrin gene were found to be variable among the polyhedrin gene loci compared. Additionally, conserved elements in the promoters of the very late genes encoding polyhedrin and granulin, and those encoding two p10 proteins were found to share sequence homology and positional similarity with consensus regions in the conserved boxes A and C, responsible for binding transcription factors to eukaryotic 5S ribosomal RNA genes, and to box C of tRNA genes.