Design and functional characterization of Salmo salar TLR5 agonist peptides derived from high mobility group B1 acidic tail.

Toll-like receptor 5 (TLR5) responds to the monomeric form of flagellin and induces the MyD88-depending signaling pathway, activating proinflammatory transcription factors such as NF-κB and the consequent induction of cytokines. On the other hand, HMGB1 is a highly conserved non-histone chromosomal protein shown to interact with and activate TLR5. The present work aimed to design and characterize TLR5 agonist peptides derived from the acidic tail of Salmo salar HMGB1 based on the structural knowledge of the TLR5 surface using global molecular docking platforms. Peptide binding poses complexed on TLR5 ectodomain model from each algorithm were filtrated based on docking scoring functions and predicted theoretical binding affinity of the complex. Circular dichroism spectra were recorded for each peptide selected for synthesis. Only intrinsically disordered peptides (6W, 11W, and SsOri) were selected for experimental functional assay. The functional characterization of the peptides was performed by NF-κB activation assays, RT-qPCR gene expression assays, and Piscirickettsia salmonis challenge in SHK-1 cells. The 6W and 11W peptides increased the nuclear translation of p65 and phosphorylation. In addition, the peptides induced the expression of genes related to the TLR5 pathway activation, pro- and anti-inflammatory response, and differentiation and activation of T lymphocytes towards phenotypes such as TH1, TH17, and TH2. Finally, it was shown that the 11W peptide protects immune cells against infection with P. salmonis bacteria. Overall, the results indicate the usefulness of novel peptides as potential immunostimulants in salmonids.

Agonistic effect of peptides derived from a truncated HMGB1 acidic tail sequence in TLR5 from Salmo salar.

Based on the structural knowledge of TLR5 surface and using blind docking platforms, peptides derived from a truncated HMGB1 acidic tail from Salmo salar was designed as TLR5 agonistic. Additionally, a template peptide with the native N-terminal of the acidic tail sequence as a reference was included (SsOri). Peptide binding poses complexed on TLR5 ectodomain model from each algorithm were filtrated based on docking scoring functions and predicted theoretical binding affinity of the complex. The best peptides, termed 6WK and 5LWK, were selected for chemical synthesis and experimental functional assay. The agonist activity by immunoblotting and immunocytochemistry was determined following the NF-κBp65 phosphorylation (p-NF-κBp65) and the nuclear translocation of the NF-κBp65 subunit from the cytosol, respectively. HeLa cells stably expressing a S. salar TLR5 chimeric form (TLR5c7) showed increased p-NF-κBp65 levels regarding extracts from flagellin-treated cells. No statistically significant differences (p > 0.05) were found in the detected p-NF-κBp65 levels between cellular extracts treated with peptides or flagellin by one-way ANOVA. The image analysis of NF-κBp65 immunolabeled cells obtained by confocal microscopy showed increased nuclear NF-κBp65 co-localization in cells both 5LWK and flagellin stimulated, while 6WK and SsOri showed less effect on p65 nuclear translocation (p < 0.05). Also, an increased transcript expression profile of proinflammatory cytokines such as TNFα, IL-1ß, and IL-8 in HKL cells isolated from Salmo salar was evidenced in 5LWK - stimulated by RT-PCR analysis. Overall, the result indicates the usefulness of novel peptides as a potential immunostimulant in S. salar.

Mortality caused by venomous animals in Venezuela (2000-2009): A new epidemiological pattern / Mortalidad causada por animales venenosos en Venezuela (2000-2009): nuevo patrón epidemiológico.

Introduction: Injuries by venomous animals frequently occur in impoverished communities with limited access to health services. They are considered neglected diseases that stand out as important causes of morbidity and mortality in various countries, including Venezuela. Objective: To assess mortalities resulting from contact with venomous animals in Venezuela from 2000 to 2009 (X20-X29 series). Materials and Methods: The data were obtained from the annual mortality records of the Venezuelan Ministry of Health. Results: From 2000-2009, 759 fatalities were recorded with the greatest number taking place in 2009. Snakebites (n=323; 42.6%) accounted for the largest percentage of envenomation-related deaths in that period, followed by hymenopteran stings (n=170; 22.4%), centipede bites (n=106; 14.0%), and scorpion stings (n=76; 10.0%). The median value of envenomation-related deaths per 100,000 inhabitants (period 2000-2009) was 0.285: 0.120 corresponded to snakebites, 0.065 to hymenopteran stings, 0.035 to centipede bites, and 0.025 to scorpion stings. Conclusions: Taking into account previous records of animal envenomations in Venezuela, we provided evidence for a shift in the pattern of mortality. Deaths due to centipede bites have increased, making it the third leading cause of envenomation-related mortality in Venezuela. Scorpionism, on the other hand, has declined to the fourth most common cause of fatal envenomations in the country.

Introducción. Los accidentes causados por animales venenosos ocurren con mucha frecuencia en comunidades pobres con acceso limitado a los servicios de salud. Se les consideran enfermedades desatendidas y son una de las causas importantes de morbimortalidad en varias naciones del mundo, incluida Venezuela. Objetivo. Evaluar la mortalidad por contacto traumático con animales venenosos (serie X20-X29) en Venezuela en el periodo de 2000 a 2009. Materiales y métodos. Los datos se obtuvieron de los anuarios de mortalidad del Ministerio de Salud. Resultados. Se registraron 759 decesos, la mayoría de ellos en el 2009. La primera causa fue la mordedura de serpientes (n=323; 42,6 %), seguida por la picadura de himenópteros (n=170; 22,4 %), la mordedura de centípedos (n=106; 14,0 %) y la picadura de escorpiones (n=76; 10,0 %). La mediana de la tasa de mortalidad general para el periodo fue de 0,285 fallecidos por 100.000 habitantes, en tanto que, por grupo específico, fue de 0,120 para ofidios, de 0,065 para himenópteros, de 0,035 para centípedos y de 0,025 para escorpiones. Conclusión. Al comparar estos datos con los antecedentes históricos, se evidenció la modificación del patrón de mortalidad en el país caracterizada por un aumento significativo de los decesos por centípedos, tercera causa de muerte, lo que reubica la picadura de escorpiones como la cuarta causa de mortalidad.

Mortalidad causada por animales venenosos en Venezuela (2000-2009): nuevo patrón epidemiológico / Mortality caused by venomous animals in Venezuela (2000-2009): A new epidemiological pattern

Resumen | Introducción. Los accidentes causados por animales venenosos ocurren con mucha frecuencia en comunidades pobres con acceso limitado a los servicios de salud. Se les consideran enfermedades desatendidas y son una de las causas importantes de morbimortalidad en varias naciones del mundo, incluida Venezuela. Objetivo. Evaluar la mortalidad por contacto traumático con animales venenosos (serie X20-X29) en Venezuela en el periodo de 2000 a 2009. Materiales y métodos. Los datos se obtuvieron de los anuarios de mortalidad del Ministerio de Salud. Resultados. Se registraron 759 decesos, la mayoría de ellos en el 2009. La primera causa fue la mordedura de serpientes (n=323; 42,6 %), seguida por la picadura de himenópteros (n=170; 22,4 %), la mordedura de centípedos (n=106; 14,0 %) y la picadura de escorpiones (n=76; 10,0 %). La mediana de la tasa de mortalidad general para el periodo fue de 0,285 fallecidos por 100.000 habitantes, en tanto que, por grupo específico, fue de 0,120 para ofidios, de 0,065 para himenópteros, de 0,035 para centípedos y de 0,025 para escorpiones. Conclusión. Al comparar estos datos con los antecedentes históricos, se evidenció la modificación del patrón de mortalidad en el país caracterizada por un aumento significativo de los decesos por centípedos, tercera causa de muerte, lo que reubica la picadura de escorpiones como la cuarta causa de mortalidad.

Abstract | Introduction: Injuries by venomous animals frequently occur in impoverished communities with limited access to health services. They are considered neglected diseases that stand out as important causes of morbidity and mortality in various countries, including Venezuela. Objective: To assess mortalities resulting from contact with venomous animals in Venezuela from 2000 to 2009 (X20-X29 series). Materials and Methods: The data were obtained from the annual mortality records of the Venezuelan Ministry of Health. Results: From 2000-2009, 759 fatalities were recorded with the greatest number taking place in 2009. Snakebites (n=323; 42.6%) accounted for the largest percentage of envenomation-related deaths in that period, followed by hymenopteran stings (n=170; 22.4%), centipede bites (n=106; 14.0%), and scorpion stings (n=76; 10.0%). The median value of envenomation-related deaths per 100,000 inhabitants (period 2000-2009) was 0.285: 0.120 corresponded to snakebites, 0.065 to hymenopteran stings, 0.035 to centipede bites, and 0.025 to scorpion stings. Conclusions: Taking into account previous records of animal envenomations in Venezuela, we provided evidence for a shift in the pattern of mortality. Deaths due to centipede bites have increased, making it the third leading cause of envenomation-related mortality in Venezuela. Scorpionism, on the other hand, has declined to the fourth most common cause of fatal envenomations in the country.

Correction: The γ33 subunit of R-phycoerythrin from Gracilaria chilensis has a typical double linked phycourobilin similar to ß subunit.

[This corrects the article DOI: 10.1371/journal.pone.0195656.].

The γ33 subunit of R-phycoerythrin from Gracilaria chilensis has a typical double linked phycourobilin similar to ß subunit.

Phycobilisomes (PBS) are accessory light harvesting protein complexes formed mainly by phycobiliproteins (PBPs). The PBPs absorb light that is efficiently transferred to Photosystems due to chromophores covalently bound to specific cysteine residues. Besides phycobiliproteins (PE), the PBS contains linker proteins responsible for assembly and stabilization of the whole complex and the tuning of energy transfer steps between chromophores. The linker (γ33) from Gracilaria chilensis, is a chromophorylated rod linker associated to (αß)6 hexamers of R-phycoerythrin (R-PE). Its role in the energy transfer process is not clear yet. Structural studies as well as the composition and location of the chromophores are essential to understand their involvement in the energy transfer process in PBS. To achieve this, the coding gene of γ33 was cloned and sequenced. The sequence was analyzed by informatics tools, to obtain preliminary information which leaded the next experiments. The protein was purified from R-phycoerythrin, and the sequence confirmed by mass spectrometry. The coding sequence analysis revealed a protein of 318 aminoacid residues containing a chloroplastidial transit peptide (cTP) of 39 aminoacids at the N-terminus. The conservation of cysteines revealed possible chromophorylation sites. Using α and ß R-PE subunits as spectroscopic probes in denaturation assays, we deduced a double bonded phycourobilin (PUB) on γ33 subunit that were confirmed between Cys62 and Cys73 (DL-PUB62/73) by mass spectrometry. The cysteines involved in the double link are located in a helical region, in a conformation that reminds the position of the DL-PUB50/61 in the ß subunit of R-PE. The position of single linked PUB at Cys95 and a single linked PEB at Cys172 were also confirmed. Spectroscopic studies show the presence of both types of chromophores and that there are not energy transfer by FRET among them.

[Growth and biochemical composition of thalassiosira pseudonana (Thalassiosirales: Thalassiosiraceae) cultivated in semicontinuous system at different culture media and irradiances]. / Crecimiento y composición bioquímica de Thalassiosira pseudonana (Thalassiosirales: Thalassiosiraceae) bajo cultivo semi-continuo en diferentes medios y niveles de irradiancias.

Thalassiosira pseudonana is a marine Bacillariophyta commonly used as live feed in mariculture. The growth rate and biochemical composition of microalgae are highly influenced by environmental factors such as, irradiance and nutrient availability. The aim of this study was to investigate the influence of three irradiances (60, 120 and 180 microE/m2.s) and two culture media (Algal and Humus) on growth and biochemical composition of this diatom. The microalga was grown semicontinuously at a daily renewal rate of fresh media of 30%, 37 per thousand salinity, 25 +/- 1 degree C and constant aeration (200 mL/min). The cell densities (cel/mL) and contents of protein, lipid, carbohydrate, chlorophyll a, total carotenoids, and fatty acids, showed significant differences (p < 0.05) between treatments. During steady-state phase, the maximal cell density, and lipid and carbohydrate contents were of 4.62 x 10(6) cel/mL, 20.3 +/- 2.28% and 16.6 +/- 2.43%, respectively, and were achieved in Humus medium at 180 microE/ m2.s. Moreover, highest protein contents (45.0 +/- 5.05%) and total carotenoids (0.5 +/- 0.01%) were obtained in Algal medium at 180 microE/m2.s. Chlorophyll a (0.93 +/- 0.04%) was higher at low irradiances in Algal medium. In both media, the fatty acids unsaturation degree was lower with increasing irradiance, being eicosapentaenoic acid, 20:5 n-3 (EPA) most represented (6.20%) in Algal medium at 60 microE/m2.s. This strain of T. pseudonana showed multiple physiological responses to changes in culture conditions, and may be cultivated with an alternative medium, which reduced the operating costs and allowed a high nutritional biomass production value for animals under culture.

[Epidemiology of ophidism in Venezuela (1996-2004)]. / Epidemiologia del ofidismo en Venezuela (1996-2004).

The data of accidents caused by snakebites in Venezuela, registered at the morbidity statistics of the Direction of Epidemiology and Strategic Analysis of the Ministry of Health and Social Development were analyzed. During the years of 1996-2004, 53,792 snakebites were registered in Venezuela (5,976 cases average per year), with a higher incidence during the year 2004 (7,486 incidents). Zulia reported the highest frequency of all the states (5,975 cases); meanwhile the Midwestern region, constituted by Lara, Portuguesa, Falc6n and Yaracuy states, had a higher morbidity for snake bites. The highest incidence, distributed per states was registered in Cojedes, during the year 2001, with 228.72 cases per 100,000 inhabitants. When it was determined by regions, the highest incidence occurred during the year 2004 at los Llanos with 63.81 per 100,000 inhabitants. The median of the incidence rate for Venezuela during the period was of 21.46 accidents per 100,000 inhabitants. The classification of the endemic areas for ophidism, according to the percentiles 23, 50, 75 and 90, organized the country in: (a) states and regions of very high endemicity, (b) high endemicity, (c) middle, (d) low and (e) very low endemicity. These epidemiological data indicated that the accidents caused by snakes constitute a collective health problem in Venezuela.

Epidemiología del ofidismo en Venezuela (1996-2004) / Epidemiology of ophidism in Venezuela (1996-2004)

Se analizaron los datos de accidentes por serpientes, registrados en las estadísticas de morbilidad de la Dirección de Epidemiología y Análisis Estratégico del Ministerio de Sanidad y Desarrollo Social. En Venezuela, entre los años 1996-2004, se registraron 53.792 mordeduras de serpientes (5.976 casos, en promedio, por año); con mayor incidencia en 2004 (7.486 incidentes). De todos los estados, Zulia reportó la mayor frecuencia (5.975 casos); mientras que la región Centro-Occidental, constituida por los estados Lara, Portuguesa, Falcón y Yaracuy, tuvo mayor morbilidad por mordeduras con 13.426. La mayor tasa por ofidismo, distribuida por estados, se registró en Cojedes, en el año 2001, con 228,72 casos por 100.000 habitantes. Cuando se determinó por regiones la mayor se ubicó, en 2004, en los Llanos, con 63,81 por 100.000 habitantes. La mediana de la tasa de incidencia para Venezuela en el periodo fue de 24,46 accidentes por 100.000 habitantes. La clasificación de las áreas de endemicidad por ofidismo, según los percentiles 25, 50, 75 y 90, ordenó al país en: (a) estados y regiones de muy alta endemicidad, (b) alta endemicidad, (c) mediana, (d) baja y (e) muy baja endemicidad. Las cifras indicaron que los accidentes causados por serpientes constituyen un problema de salud colectiva en Venezuela.

The data of accidents caused by snakebites in Venezuela, registered at the morbidity statistics of the Direction of Epidemiology and Strategic Analysis of the Ministry of Health and Social Development were analyzed. During the years of 1996-2004, 53,792 snakebites were registered in Venezuela (5,976 cases average per year), with a higher incidence during the year 2004 (7,486 incidents). Zulia reported the highest frequency of all the states (5,975 cases); meanwhile the Midwestern region, constituted by Lara, Portuguesa, Falcón and Yaracuy states, had a higher morbidity for snake bites. The highest incidence, distributed per states was registered in Cojedes, during the year 2001, with 228.72 cases per 100,000 inhabitants. When it was determined by regions, the highest incidence occurred during the year 2004 at los Llanos with 63.81 per 100,000 inhabitants. The median of the incidence rate for Venezuela during the period was of 21.46 accidents per 100,000 inhabitants. The classification of the endemic areas for ophidism, according to the percentiles 23, 50, 75 and 90, organized the country in: (a) states and regions of very high endemicity, (b) high endemicity, (c) middle, (d) low and (e) very low endemicity. These epidemiological data indicated that the accidents caused by snakes constitute a collective health problem in Venezuela.

Perfil eco-epidemiológico de los accidentes por ofidios en Monagas, Venezuela (2002-2006) / Eco-epidemiological profile of snakebite accidents in Monagas state, Venezuela (2002-2006)

El accidente causado por animales venenosos representa un problema global, especialmente en las áreas subtropicales y tropicales del mundo. En Venezuela, es endémico para varias regiones incluyendo la zona nororiental. Con el objetivo de ampliar y actualizar el perfil eco-epidemiológico del ofidismo en el estado Monagas, se evaluó su comportamiento, en cada municipio, durante 5 años de registro (2002 - 2006). Se contabilizaron 339 accidentes ofídicos (con promedio de 68 anuales). El incidente ocurrió con mayor frecuencia en agricultores, adultos jóvenes y de género masculino, durante sus labores en el campo y en el periodo diurno. La incidencia siguió un patrón estacional bimodal con predominio en épocas de alta y baja pluviosidad. Los envenenamientos botrópicos (28,6%) fueron más frecuentes que los crotálicos (14,5%), con proporción 2:1. El 87,9% recibió sueroterapia específica. La incidencia promedio anual en la entidad se ubicó en 11,30 casos por 100.000 habitantes, siendo Punceres (46,29), Acosta (20,91) y Bolívar (19,52) los municipios con mayor impacto. El estado Monagas presentó un mapa de endemicidad distribuido en municipios de (1) muy alta endemicidad, (2) alta endemicidad, (3) mediana endemicidad, (4) baja endemicidad y (5) muy baja endemicidad. Durante el período analizado no se registró muerte por esta causa en el estado. Los hallazgos sugieren la importancia del accidente por serpientes en Monagas, con especial importancia en la mitad norte de la entidad.

The accidents caused by venomous animals are a global problem, especially in subtropical and tropical regions of the world. In Venezuela, they are endemic in several regions including the northeast. In order to expand and update the ecoepidemiological profile of snake bites in Monagas state, their behavior was assessed in each municipality for 5 years (2002 -2006). There were 339 ophidian accidents (on average 68 per year). The bites occurred more frequently in young adult male farmers, while working in the field and during daytime. The incidence followed a bimodal seasonal pattern with predominance in high and low rainfall periods. Bothropic envenoming (28.6%) were the most frequent followed by Crotalic ones (14.5%), with a 2:1 ratio. 87.9% received specific serum therapy. The annual average incidence in the state was 11.30 cases per 100,000 inhabitants. Punceres (46.29), Acosta (20.91) and Bolivar (19.52) were the municipalities with the highest impact. Monagas state showed an endemicity map with municipalities having (1) very high endemicity, (2) high endemicity, (3) medium endemicity, (4) low endemicity and (5) very low endemicity. In the studied period there were no deaths from this cause in the state. The findings suggest the importance of snake accidents in Monagas, especially in the northern half of the state.

Differences in venom toxicity and antigenicity between females and males Tityus nororientalis (Buthidae) scorpions.

Venom from male and female specimens of the medically important Venezuelan scorpion Tityus nororientalis have been compared. Males showed a significantly higher venom yield (2.39mg/individual) compared to female scorpions (0.98mg/individual). Female venom was significantly more toxic than that of males, with a median lethal dose (LD(50)) in C57BL/6 mice of 9.46 µg venom protein/gm body weight [95% confidence interval (8.91-9.94)] whereas LD(50) for males was 13.36(12.58-14.03) µg/gm. Mass spectral analyses by MALDI-TOF revealed differences in venom composition between males and females. From a clinical standpoint, the time course of toxicity course indicated a tendency, in the case of the female venom, to elicit the earlier occurrence of severe signs such as sialorrhea, dyspnea (bradypnea/apnea) and exophthalmus particularly in the late toxicity phase. Female venom was significantly less efficient than male venom to inhibit the binding of anti-T. discrepans antibodies to immobilized T. discrepans venom in ELISA assays, suggesting sex-related differences in the bioactive surfaces of T. nororientalis toxins. These results indicate that males and females of T. nororientalis produce venoms with different composition and activity which may have epidemiological implications.

Calidad microbiológica de la ostra Crassostrea rhizophorae y aguas de extracción estado Sucre Venezuela / Microbiological quality of the oyster Crassostrea rhizophorae and extraction waters Sucre state Venezuela

La ostra Crassostrea rhizophorae es consumida comúnmente en diversas playas del estado Sucre, sin conocer la carga microbiana que éstas presentan. Por tal motivo se evaluó la calidad microbiológica de la misma y de muestras de agua proveniente de tres estaciones del banco natural de Laguna Grande del Obispo, con el propósito de verificar el cumplimiento de los requisitos microbiológicos establecidos por la Legislación Sanitaria Venezolana y la Administración de Alimentos y Drogas de los Estados Unidos (FDA). Se realizaron muestreos mensuales entre agosto 2005 y julio 2006, determinándose el NMP/100 mL de coliformes totales (CT) y coliformes fecales (CF) en muestras de agua, mientras que en los bivalvos se cuantificó el NMP de CT, CF y Escherichia coli (EC) por gramo de ostra, además de la detección de Salmonella spp. Los resultados obtenidos de CT y CF en muestras de agua de las tres estaciones, cumplieron en la mayoría de los meses, con las exigencias permitidas (70 NMP/100 mL de CT y 14 NMP/100 mL de CF). Durante los meses de muestreo y entre las estaciones analizadas, se observaron variaciones considerables en los valores de CF y EC que estuvieron entre 0 y 2,4 × 10(4) NMP/g de ostra. Los límites permitidos de CF y EC (230 NMP/g de ostra) fueron excedidos por los CF en todas las estaciones durante agosto y diciembre 2005, y también en marzo, mayo y junio 2006, mientras que los de EC se excedieron sólo en diciembre 2005, y marzo y mayo 2006. Salmonella spp. fue detectada en la primera y segunda estación para el mes de septiembre 2005, en la tercera estación para el mes de diciembre del mismo año y en la segunda y tercera estación, para los meses de mayo y julio 2006. Los resultados obtenidos reflejan un alto riesgo de consumir estos moluscos sin haber sido previamente sometidos a una depuración.

In beaches of Sucre State commonly the oyster Crassostrea rhizophorae is consumed without knowing the microbial load microbial that may be presenting. For such motive, the microbiology quality of C. rhizophorae and water samples of three stations from natural bank in Laguna Grande of Obispo was evaluated, to check the compliance of microbiological requirements established by the Food and Drugs Administration of United States (FDA) and Sanitary Venezuelan Legislation. Monthly samplings between August 2005 and July 2006 was realized, determining the MPN/100 mL of coliforms totals (TC) and faecal coliforms (FC) in water samples, whereas in bivalves MPN of TC, FC and Escherichia coli (EC) was quantified for gram of oyster, further to detection Salmonella spp. The results obtained of TC and FC, in water samples of the three stations, fulfilled in majority of the months, with the requirements mentioned above. During the months sampling and between analyzed stations, considerable variations was observed in values of FC and EC that were between 0 and 2.4 × 10(4) MPN/g oyster. The limits allowed for FC and EC (230 MPN/g oyster) were exceeded by the FC on all stations during August, December 2005, and also in March, May and June 2006, whereas EC exceded only in December 2005, and March and May 2006. Salmonella spp. was detected on first and second station in September 2005, on the third station for December of the same year, and second and third station for May and July 2006. The obtained results reflect high risk of consuming these mollusks without having being before submitted to depuration.