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Understanding the factors that cause heterogeneity among gene trees can increase the accuracy of species trees. Discordant signals across the genome are commonly produced by incomplete lineage sorting (ILS) and introgression, which in turn can result in reticulate evolution. Species tree inference using the multispecies coalescent is designed to deal with ILS and is robust to low levels of introgression, but extensive introgression violates the fundamental assumption that relationships are strictly bifurcating. In this study, we explore the phylogenomics of the iconic Liolaemus subgenus of South American lizards, a group of over 100 species mostly distributed in and around the Andes mountains. Using mitochondrial DNA (mtDNA) and genome-wide restriction site-associated DNA sequencing (RADseq; nDNA hereafter), we inferred a time-calibrated mtDNA gene tree, nDNA species trees, and phylogenetic networks. We found high levels of discordance between mtDNA and nDNA, which we attribute in part to extensive ILS resulting from rapid diversification. These data also reveal extensive and deep introgression, which combined with rapid diversification, explain the high level of phylogenetic discordance. We discuss these findings in the context of Andean orogeny and glacial cycles that fragmented, expanded, and contracted species distributions. Finally, we use the new phylogeny to resolve long-standing taxonomic issues in one of the most studied lizard groups in the New World.[Andes; ddRADSeq; introgression; lizards; mtDNA; reptiles; SNPs.].

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Nanoparticles based on chitosan modified with epigallocatechin gallate (EGCG) were synthetized by nanoprecipitation (EGCG-g-chitosan-P). Chitosan was modified by free-radical-induced grafting, which was verified by Fourier transform infrared (FTIR). Furthermore, the morphology, particle size, polydispersity index, and zeta potential of the nanoparticles were investigated. The grafting degree of EGCG, reactive oxygen species (ROS) production, antibacterial and antioxidant activities of EGCG-g-chitosan-P were evaluated and compared with those of pure EGCG and chitosan nanoparticles (Chitosan-P). FTIR results confirmed the modification of the chitosan with EGCG. The EGCG-g-chitosan-P showed spherical shapes and smoother surfaces than those of Chitosan-P. EGCG content of the grafted chitosan nanoparticles was 330 µg/g. Minimal inhibitory concentration (MIC) of EGCG-g-chitosan-P (15.6 µg/mL) was lower than Chitosan-P (31.2 µg/mL) and EGCG (500 µg/mL) against Pseudomonas fluorescens (p < 0.05). Additionally, EGCG-g-chitosan-P and Chitosan-P presented higher Staphylococcus aureus growth inhibition (100%) than EGCG at the lowest concentration tested. The nanoparticles produced an increase of ROS (p < 0.05) in both bacterial species assayed. Furthermore, EGCG-g-chitosan-P exhibited higher antioxidant activity than that of Chitosan-P (p < 0.05) in 2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and ferric-reducing antioxidant power assays. Based on the above results, EGCG-g-chitosan-P shows the potential for food packaging and biomedical applications.

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Vibrio parahaemolyticus (Vp), a typical microorganism inhabiting marine ecosystems, uses pathogenic virulence molecules such as hemolysins to cause bacterial infections of both human and marine animals. The thermolabile hemolysin VpTLH lyses human erythrocytes by a phospholipase B/A2 enzymatic activity in egg-yolk lecithin. However, few studies have been characterized the biochemical properties and the use of VpTLH as a molecular target for natural compounds as an alternative to control Vp infection. Here, we evaluated the biochemical and inhibition parameters of the recombinant VpTLH using enzymatic and hemolytic assays and determined the molecular interactions by in silico docking analysis. The highest enzymatic activity was at pH 8 and 50 °C, and it was inactivated by 20 min at 60 °C with Tm = 50.9 °C. Additionally, the flavonoids quercetin, epigallocatechin gallate, and morin inhibited the VpTLH activity with IC50 values of 4.5 µM, 6.3 µM, and 9.9 µM, respectively; while phenolics acids were not effective inhibitors for this enzyme. Boltzmann and Arrhenius equation analysis indicate that VpTLH is a thermolabile enzyme. The inhibition of both enzymatic and hemolytic activities by flavonoids agrees with molecular docking, suggesting that flavonoids could interact with the active site's amino acids. Future research is necessary to evaluate the antibacterial activity of flavonoids against Vp in vivo.

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Lysozymes play a key role in innate immune response to bacterial pathogens, catalyzing the hydrolysis of the peptidoglycan layer of bacterial cell walls. In this study, the genes encoding the c-type (TmLyzc) and g-type (TmLyzg) lysozymes from Totoaba macdonaldi were cloned and characterized. The cDNA sequences of TmLyzg and TmLyzc were 582 and 432 bp, encoding polypeptides of 193 and 143 amino acids, respectively. Amino acid sequences of these lysozymes shared high identity (60-90%) with their counterparts of other teleosts and showed conserved functional-structural signatures of the lysozyme superfamily. Phylogenetic analysis indicated a close relationship with their vertebrate homologues but distinct evolutionary paths for each lysozyme. Expression analysis by qRT-PCR revealed that TmLyzc was expressed in stomach and pyloric caeca, while TmLyzg was highly expressed in stomach and heart. These results suggest that both lysozymes play important roles in defense of totoaba against bacterial infections or as digestive enzyme.

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Trypsins (E.C. 3.4.21.4) are digestive enzymes that catalyze the hydrolysis of peptide bonds containing arginine and lysine residues. Some trypsins from fish species are active at temperatures just above freezing, and for that are called cold-adapted enzymes, having many biotechnological applications. In this work, we characterized a recombinant trypsin-III from Monterey sardine (Sardinops caeruleus) and studied the role of a single residue on its cold-adapted features. The A236N mutant from sardine trypsin-III showed higher activation energy for the enzyme-catalyzed reaction, it was more active at higher temperatures, and exhibited a higher thermal stability than the wild-type enzyme, suggesting a key role of this residue. The thermodynamic activation parameters revealed an increase in the activation enthalpy for the A236N mutant, suggesting the existence of more intramolecular contacts during the activation step. Molecular models for both enzymes suggest that a hydrogen-bond involving N236 may contact the C-terminal α-helix to the vicinity of the active site, thus affecting the biochemical and thermodynamic properties of the enzyme.

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BACKGROUND: The diagnosis of active Toxocara canis infections in humans is challenging. Larval stages of T. canis do not replicate in human tissues and disease may result from infection with a single T. canis larva. Recently, we developed a nanobody-based electrochemical magnetosensor assay with superior sensitivity to detect T. canis excretory-secretory (TES) antigens. Here, we evaluate the performance of the assay in children from an Ecuadorian birth cohort that followed children to five years of age. METHODS: Samples were selected based on the presence of peripheral blood eosinophilia and relative eosinophil counts. The samples were analyzed by the nanobody-based electrochemical magnetosensor assay, which utilizes a bivalent biotinylated nanobody as capturing agent on the surface of streptavidin pre-coated paramagnetic beads. Detection was performed by a different nanobody chemically labelled with horseradish peroxidase. RESULTS: Of 87 samples tested, 33 (38%) scored positive for TES antigen recognition by the electrochemical magnetosensor assay. The average concentration of TES antigen in serum was 2.1 ng/ml (SD = 1.1). The positive result in the electrochemical assay was associated with eosinophilia > 19% (P = 0.001). Parasitological data were available for 57 samples. There was no significant association between positivity by the electrochemical assay and the presence of other soil-transmitted helminth infections. CONCLUSIONS: Our nanobody-based electrochemical assay provides highly sensitive quantification of TES antigens in serum and has potential as a valuable tool for the diagnosis of active human toxocariasis.

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Species delimitation in Phymaturus has been a difficult task due to the highly conserved morphological and ecological features present in this genus. Almost all species of Phymaturus have been described without DNA data or lacking statistical analyses which makes even more difficult to compare species. Although two molecular phylogenetic studies have been recently published, here we provide the first multilocus phylogenetic reconstruction including all Chilean species, with samples from all type localities and some previously unsampled populations. We also estimate pairwise distances among the Chilean species of Phymaturus (P. vociferator and P. mallimaccii clades) and compare our results with the P. payuniae clade, where previous studies have used multiple lines of evidence. Additionally, we performed univariate and multivariate morphological analyses and skeletal comparisons (clavicle) for the species of the P. vociferator clade. As a result of this integrative approach, we describe a new species.

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BACKGROUND: Species of Schellackia Reichenow, 1919 have been described from the blood of reptiles distributed worldwide. Recently, Schellackia spp. detected in European and Asian lizards have been molecularly characterised. However, parasites detected in American lizard hosts remain uncharacterised. Thus, phylogenetic affinities between the Old and New World parasite species are unknown. METHODS: In the present study, we characterised morphologically and molecularly the hemococcidian parasites (sporozoites) that infect three lizard hosts from North America and two from South America. RESULTS: In total, we generated 12 new 18S rRNA gene sequences of hemococcidian parasites infecting New World lizard hosts. By the microscopic examination of the smears we identified Schellackia golvani Rogier & Landau, 1975 (ex Anolis carolinensis Voigt) and Schellackia occidentalis Bonorris & Ball, 1955 (ex Uta stansburiana Baird & Girard and Sceloporus occidentalis Baird & Girard) in some samples, but the phylogenetic analysis indicated that all 18S rDNA sequences are distant from Schellackia species found in Old World lizards. In fact, the hemococcidian parasites detected in the New World lizards (including S. occidentalis and S. golvani) were closely related to the genus Lankesterella Labbé, 1899. Consequently, we suggest these two species to be included within the genus Lankesterella. CONCLUSIONS: Life history traits of hemococcidian parasites such as the type of host blood cells infected, host species or number of refractile bodies are not valid diagnostic characteristics to differentiate the parasites between the genera Schellackia and Lankesterella. Indeed, lankesterellid parasites with a different number of refractile bodies had a close phylogenetic origin. Based on the phylogenetic results we provide a systematic revision of the North American hemococcidians. Our recommendation is to include the species formerly described in the genus Schellackia that infect American lizards into Lankesterella (Lankesterellidae) as Lankesterella golvani (Rogier & Landau, 1975) n. comb and L. occidentalis (Bonorris & Ball, 1955) n. comb.

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Nucleotide phosphorylation is a key step in DNA replication and viral infections, since suitable levels of nucleotide triphosphates pool are required for this process. Deoxythymidine monophosphate (dTMP) is produced either by de novo or salvage pathways, which is further phosphorylated to deoxythymidine triphosphate (dTTP). Thymidyne monophosphate kinase (TMK) is the enzyme in the junction of both pathways, which phosphorylates dTMP to yield deoxythymidine diphosphate (dTDP) using adenosine triphosphate (ATP) as a phosphate donor. White spot syndrome virus (WSSV) genome contains an open reading frame (ORF454) that encodes a thymidine kinase and TMK domains in a single polypeptide. We overexpressed the TMK ORF454 domain (TMKwssv) and its specific activity was measured with dTMP and dTDP as phosphate acceptors. We found that TMKwssv can phosphorylate dTMP to yield dTDP and also is able to use dTDP as a substrate to produce dTTP. Kinetic parameters K M and k cat were calculated for dTMP (110 µM, 3.6 s(-1)), dTDP (251 µM, 0.9 s(-1)) and ATP (92 µM, 3.2 s(-1)) substrates, and TMKwssv showed a sequential ordered bi-bi reaction mechanism. The binding constants K d for dTMP (1.9 µM) and dTDP (10 µM) to TMKwssv were determined by Isothermal Titration Calorimetry. The affinity of the nucleotidic analog stavudine monophosphate was in the same order of magnitude (K d 3.6 µM) to the canonical substrate dTMP. These results suggest that nucleotide analogues such as stavudine could be a suitable antiviral strategy for the WSSV-associated disease.

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The effect of refrigerated 48h transport and 4 days storage on the quality and shelf life of the whole lion's paw scallop Nodipecten subnodosus gonad was evaluated. Proximal composition, adenosine 5´triphosphate (ATP) and related products, K-value, total volatile bases (TVB-N), trimethylamine (TMA-N), pH, fatty acid profile and microbiological analyses were quantified. Gonad holds a significant composition of essential fatty acids while levels of gonadal ATP were initially low; moreover, K-value of the gonad remained constant. With respect to TVB-N and TMA-N, only the former exceeded allowed limits. The pH level showed no significant variation during storage and, despite the high level of TVB-N, according to the TMA-N as well as microbiological analyses it was demonstrated innocuity after 4 days under the transportation and storage conditions utilized.

Avaliou-se o efeito do transporte em refrigeração por 48 horas e quatro dias de armazenamento sobre a qualidade e vida de prateleira da gônada do bivalve pata de leão, Nodipecten subnodosus. Determinou-se a composição centesimal, a adenosina 5'trifosfato (ATP) e afins, o índice K, bases voláteis totais (TVB-N), trimetilamina (TMA-N), pH, perfil de ácidos graxos e análise microbiológica. A Gônada apresentou uma importante composição de ácidos graxos essenciais e baixos níveis iniciais de ATP, enquanto o índice K manteve-se constante. Quanto a TVB -N e TMA- N, apenas as primeiras ultrapassaram os limites admissíveis. Os valores de pH não mostraram nenhuma mudança significativa durante o armazenamento e, apesar dos altos níveis de TVB -N, de acordo com a análise quantitativa e microbiológica TMA- N, a segurança do produto foi demonstrada após quatro dias sob as condições de transporte e armazenamento utilizado.

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The effect of refrigerated 48h transport and 4 days storage on the quality and shelf life of the whole lion's paw scallop Nodipecten subnodosus gonad was evaluated. Proximal composition, adenosine 5´triphosphate (ATP) and related products, K-value, total volatile bases (TVB-N), trimethylamine (TMA-N), pH, fatty acid profile and microbiological analyses were quantified. Gonad holds a significant composition of essential fatty acids while levels of gonadal ATP were initially low; moreover, K-value of the gonad remained constant. With respect to TVB-N and TMA-N, only the former exceeded allowed limits. The pH level showed no significant variation during storage and, despite the high level of TVB-N, according to the TMA-N as well as microbiological analyses it was demonstrated innocuity after 4 days under the transportation and storage conditions utilized.(AU)

Avaliou-se o efeito do transporte em refrigeração por 48 horas e quatro dias de armazenamento sobre a qualidade e vida de prateleira da gônada do bivalve pata de leão, Nodipecten subnodosus. Determinou-se a composição centesimal, a adenosina 5'trifosfato (ATP) e afins, o índice K, bases voláteis totais (TVB-N), trimetilamina (TMA-N), pH, perfil de ácidos graxos e análise microbiológica. A Gônada apresentou uma importante composição de ácidos graxos essenciais e baixos níveis iniciais de ATP, enquanto o índice K manteve-se constante. Quanto a TVB -N e TMA- N, apenas as primeiras ultrapassaram os limites admissíveis. Os valores de pH não mostraram nenhuma mudança significativa durante o armazenamento e, apesar dos altos níveis de TVB -N, de acordo com a análise quantitativa e microbiológica TMA- N, a segurança do produto foi demonstrada após quatro dias sob as condições de transporte e armazenamento utilizado.(AU)

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Solid wastes generated from the seafood industry represent an important environmental pollutant; therefore, utilization of those wastes for the development of processing biochemical tools could be an attractive and clean solution for the seafood industry. This study reports the immobilization of semi-purified acidic proteases from Monterey sardine stomachs onto chitin and chitosan materials extracted from shrimp head waste. Several supports (chitosan beads, chitosan flakes, and partially deacetylated flakes) were activated either with genipin or Na-tripolyphosphate and evaluated as a mean to immobilize acidic proteases. The protein load varied within the 67-91% range on different supports. The immobilization systems based on chitosan beads achieved the highest protein loads but showed the lowest retained catalytic activities. The best catalytic behavior was obtained using partially deacetylated chitin flakes activated either with genipin or Na-tripolyphosphate. According to results, the immobilization matrix structure, as well as acetylation degree of chitin-chitosan used, has considerable influence on the catalytic behavior of immobilized proteases. Partially deacetylated chitin flakes represent a suitable option as support for enzyme immobilization because its preparation requires fewer steps than other supports. Two abundant seafood by-products were used to obtain a catalytic system with enough proteolytic activity to be considered for biotechnological applications in diverse fields.

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Resumen Se describe el caso de una metaplasia ósea en un canino, hembra, de raza Mastín Napolitano, con historia de cojera intermitente del miembro anterior izquierdo. En el examen ortopédico se evidenció una claudicación del miembro afectado, molestia a la flexión y extensión pasiva de la articulación del codo izquierdo. A la palpación se detectó una masa de consistencia sólida a nivel medio-distal del codo, en el examen radiológico del miembro afectado se observó una estructura radiodensa medial a la articulación, en la zona de los músculos flexores del miembro anterior izquierdo sin evidencia de adhesión o contacto con huesos. La estructura fue resectada quirúrgicamente y evaluada histológicamente, diagnosticando una alteración en la reparación tisular de tipo metaplasico, con la formación de tejido óseo. Tanto en el postquirúrgico, como en la reevaluación a los 12 meses, la paciente mostró una marcada mejoría en la claudicación. En el presente reporte, se discute el caso clínico de un paciente con metaplasia ósea a nivel de los músculos flexores del miembro anterior, siendo este el primer caso reportado por tal patología a nivel sudamericano y el segundo caso reportado mundialmente en un perro de raza Mastín Napolitano.

Abstract We describe a case of osseous metaplasia in a canine. A female Neapolitan Mastiff with a history of intermittent left forelimb lameness was evaluated. Orthopedic examination revealed the animal was not standing on the affected limb and had signs of pain on passive flexion and extension of the left elbow joint. A solid mass mass medial of the left elbow was detected on palpation. Radiological examination showed a radiopaque structure medial to the joint, at the level of flexor muscles of the left forelimb, without evident adhesion or contact with bone. The mass was surgically removed and histologically examined. The mass was diagnosed as an altered metaplasic regeneration with formation of bone tissue. The patient showed marked improvement of the lameness few days after surgery and at reevaluation 12 months post-surgery. This report describes a case of osseous metaplasia at the level of the forelimb flexor muscles in a canine patient. To the author's knowledge, this is the first case of such pathology reported in South America, and the second case reported worldwide in a purebred Neapolitan Mastiff.

Resumo Descreve-se o caso de uma metaplasia óssea de uma fêmea canina da raça Mastim Napolitana, com historia de dificuldade intermitente para caminhar no membro anterior esquerdo. No exame ortopédico evidenciou-se uma claudicação do membro afetado, moléstia na flexão e extensão passiva da articulação do cotovelo esquerdo. À palpação detectou-se uma protuberância de consistência solida no nível médio-distal do cotovelo, no exame radiológico do membro afetado observou-se uma estrutura radiodensa medial á articulação, na região dos músculos flexores do membro anterior esquerdo não se encontrou evidencia de adesão ou contato com os ossos. A estrutura foi resectada cirurgicamente e avaliada histologicamente, diagnosticando uma alteração na reparação tissular de tipo metaplásico com a formação de tecido ósseo. Tanto no post-cirúrgico, quanto na reavaliação aos 12 meses, a paciente teve uma marcada melhoria na claudicação. No presente reporte, se discute o caso clínico de uma paciente com metaplasia óssea no nível dos músculos flexores do membro anterior, sendo este o primeiro caso reportado por esta patologia em toda América do Sul e o segundo caso do mundo num cachorro da raça Mastim Napolitano.

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Biosynthesis of nucleoside triphosphates is critical for bioenergetics and nucleic acid replication, and this is achieved by nucleoside diphosphate kinase (NDK). As an emerging biological model and the global importance of shrimp culture, we have addressed the study of the Pacific whiteleg shrimp (Litopenaeus vannamei) NDK. We demonstrated its activity and affinity towards deoxynucleoside diphosphates. Also, the quaternary structure obtained by gel filtration chromatography showed that shrimp NDK is a trimer. Affinity was in the micro-molar range for dADP, dGDP, dTDP and except for dCDP, which presented no detectable interaction by isothermal titration calorimetry, as described previously for Plasmodium falciparum NDK. This information is particularly important, as this enzyme could be used to test nucleotide analogs that can block white spot syndrome virus (WSSV) viral replication and to study its bioenergetics role during hypoxia and fasting.

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This paper describes and evaluates the mortality associated with the potential stressor effect induced by application of artificial photoperiods in rainbow trout (Oncorhynchus mykiss). After application of artificial photoperiods, high mortalities of trout subjected to this management were recorded (36% in LD 14:10 and 25% in LD 24:0) as compared to controls (7%). The recorded signology related to diseased trout included darkening, lethargy and erratic swimming. Among the morphological changes, skin lesions and ulcerative-type necrosis could be observed. Blood tests, histopathology and isolation of pathogens identified as Flavobacterium psychrophilum, associated with Aeromonas, Pseudomonas and Saprolegnia sp., were performed. The application of artificial photoperiods suggests that fish may be more susceptible to opportunistic diseases; thus, it is advisable to emphasize preventive measures in fish subjected to this management.

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A 4 x 3 factorial study was conducted to evaluate the effect of four experimental diets (a control diet and a 0, 50, and 100% fish meal replacement diet) and the period of time Nile tilapia, Oreochromis niloticus, were fed those diets (0, 20, and 40 days) on the alkaline proteolytic activity of the animals' digestive tract, as well as their potential interaction. Significant differences (at P < 0.05) and a significant interaction were observed among dietary treatments for the alkaline proteolytic activity of tilapia after 40 days of feeding. This study confirmed that, under these experimental conditions, a 50% fish meal replacement formulation elicited the highest alkaline proteolytic activity in the digestive tract of tilapia, which resulted in the highest final weight and specific growth rate (SGR), but further research is needed to establish the relative contribution of the alkaline proteases to the overall proteolytic activity of this omnivorous fish species.

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Trypsin from pyloric caeca of Monterey sardine was purified by fractionation with ammonium sulfate, gel filtration, affinity and ionic exchange chromatography. Fraction 102, obtained from ionic exchange chromatography, generated one band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The molecular mass of the isolated trypsin was 25 kDa and showed esterase-specific activity on Nalpha-p-tosyl-L-arginine methyl ester (TAME) that was 4.5 times greater than amidase-specific activity on N-benzoyl-L-arginine-p-nitroanilide. The purified enzyme was partially inhibited by the serine-protease phenyl-methyl-sulfonyl fluoride (PMSF) inhibitor and fully inhibited by the soybean trypsin inhibitor (SBTI) and benzamidine, but was not inhibited by the metallo-protease inactivator EDTA or the chymotrypsin inhibitor tosyl-L-phenylalanine chloromethyl-ketone. The optimum pH for activity was 8.0 and maximum stability was observed between pH 7 and 8. A marked loss in stability was observed below pH 4 and above pH 11. Activity was optimum at 50 degrees C and lost activity at higher temperatures. The kinetic trypsin constants K(m) and k(cat) were 0.051 mM and 2.12 s(-1), respectively, while the catalytic efficiency (k(cat)/K(m)) was 41 s(-1) mM(-1). General characteristics of the Monterey sardine trypsin resemble those of trypsins from other fish, especially trypsins from the anchovy Engraulis japonica and Engraulis encrasicholus and the sardine Sardinops melanostica.

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