Transient inactivation of myostatin induces muscle hypertrophy and overcompensatory growth in zebrafish via inactivation of the SMAD signaling pathway.

Myostatin (MSTN) is the main negative regulator of muscle growth and development in vertebrates. In fish, little is known about the molecular mechanisms behind how MSTN inactivation triggers skeletal muscle enhancement, particularly regarding the signaling pathways involved in this process. Moreover, there have not been reports on the biotechnological applications of MSTN and its signal transduction. In this context, zebrafish underwent compensatory growth using fasting and refeeding trials, and MSTN activity was inactivated with dominant negative LAPD76A recombinant proteins during the refeeding period, when a rapid, compensatory muscle growth was observed. Treated fish displayed an overcompensation of growth characterized by higher muscle hypertrophy and growth performance than constantly fed, control fish. Treatment with LAPD76A recombinant proteins triggered inactivation of the SMAD signaling pathway in skeletal muscle, the main signal transduction used by MSTN to achieve its biological actions. Therefore, transient inactivation of MSTN during the compensatory growth of zebrafish led to a decrease in the SMAD signaling pathway in muscle, triggering muscle hypertrophy and finally improving growth performance, thus, zebrafish achieved an overcompensation of growth. The present study shows an attractive strategy for improving muscle growth in a fish species by mixing a classical strategy, such as compensatory growth, and a biotechnological approach, such as the use of recombinant proteins for inhibiting the biological actions of MSTN. The mix of both strategies may represent a method that could be applied in order to improve growth in commercial fish of interest for aquaculture.

Temporal and spatial expression pattern of the myostatin gene during larval and juvenile stages of the Chilean flounder (Paralichthys adspersus).

The full length cDNA sequence of the myostatin gene was cloned from a teleostean fish, the Chilean flounder (Paralichthys adspersus) through RT-PCR amplification coupled with the RACE approach to complete the 5'- and 3'-region. The deduced amino acid sequence encodes a protein of 377 amino acid residues, including the structural domains responsible for its biological activity. Amino acid sequence comparison revealed high sequence conservation, and confirmed that the isolated sequence corresponds to the MSTN1 gene. Gene expression analysis showed that cfMSTN mRNA is present in a wide variety of tissues in juvenile fish. In addition, we assessed the spatial expression pattern of the MSTN mRNA during embryos and larval stages through whole mount in situ hybridization. No expression was observed in embryos, whereas in larvae of 8 and 9 days post fertilization, the notochord, somites, intestine and some discrete territories in the head, such as brain and eye, were positive for MSTN mRNA. Our results contribute to the knowledge of the MSTN system in larval and juvenile stages; in particular the strong expression observed in the notochord suggests that MSTN, in synchronization with positive growth signals, may play an important role in the control of the development of larvae somites.

MAC-ELISA and ELISA inhibition methods for detection of antibodies after yellow fever vaccination.

The IgM antibody capture ELISA (MAC-ELISA) and ELISA inhibition methods for the detection of antibodies against dengue virus were modified to detect antibodies against yellow fever virus. Tests were carried out in 21 persons vaccinated with 17D and compared with the Plaque reduction neutralizing test. Of 17 naive subjects vaccinated, 16 (94%) seroconverted using the MAC-ELISA test and 14 (82%) seroconverted (or >/=fourfold titer increase) in the ELISA inhibition method. Cross-reactivity was evaluated by both tests and resulted in a high specificity to IgM antibodies against yellow fever, when all the samples from vaccinated individuals were negative by MAC-ELISA using dengue antigen. However, 10.7% of the positive dengue sera from the Santiago de Cuba epidemic cross-reacted by MAC-ELISA using yellow fever antigen. ELISA inhibition method showed high cross-reactivity when the 21 sera pairs were worked with yellow fever and dengue antigens. The MAC-ELISA and ELISA inhibition methods have become indispensable tools in our laboratory in order to maintain a surveillance system for dengue and dengue hemorrhagic fever. They are relatively rapid, simple, and they do not require sophisticated equipment. Both MAC-ELISA and ELISA inhibition methods for yellow fever could be useful for diagnosis, surveillance and yellow fever vaccine evaluation.

Predicción del serotipo del virus del dengue mediante la respuesta de anticuerpos IgM / Prediction of serotypes of dengue virus by response to IgM antibodies

Se realizó un estudio en 4 grupos de muestras de suero procedentes de Cuba, Costa Rica, Nicaragua y Panamá, con el objetivo de determinar el serotipo del virus dengue responsable de una epidemia o brote, aplicando el Elisa de Captura de anticuerpos IgM (MAC-ELISA). Se empleó el estuche diagnóstico Dengue-IgM con cada uno de los serotipos por separado y se calculó un valor índice (densidad óptica de la muestra / valor de corte) en cada caso, con los cuales se realizaron 2 análisis estadísticos; el análisis de varianzas de Fisher y el cálculo de la LSD (Litter significant difference). Los cálculos permitieron determinar que en Cuba y Panamá circuló el serotipo 2 y en Costa Rica el serotipo 1, esto correspondió con los serotipos aislados durante las respectivas epidemias y en el caso de Nicaragua, las respuestas son muy heterogéneas, no se pudo determinar el serotipo responsable, probablemente porque en este país han circulado más de 2 serotipos a la vez

Predicción del serotipo del virus del dengue mediante la respuesta de anticuerpos IgM / Prediction of the serotipo of the virus of the fastidiousness by means of the answer of antibodies IgM

Se realizó un estudio en 4 grupos de muestras de suero procedentes de Cuba, Costa Rica, Nicaragua y Panamá, con el objetivo de determinar el serotipo del virus dengue responsable de una epidemia o brote, aplicando el Elisa de Captura de anticuerpos IgM (MAC-ELISA). Se empleó el estuche diagnóstico Dengue-IgM con cada uno de los serotipos por separado y se calculó un valor índice (densidad óptica de la muestra / valor de corte) en cada caso, con los cuales se realizaron 2 análisis estadísticos; el análisis de varianzas de Fisher y el cálculo de la LSD (Litter significant difference). Los cálculos permitieron determinar que en Cuba y Panamá circuló el serotipo 2 y en Costa Rica el serotipo 1, esto correspondió con los serotipos aislados durante las respectivas epidemias y en el caso de Nicaragua, las respuestas son muy heterogéneas, no se pudo determinar el serotipo responsable, probablemente porque en este país han circulado más de 2 serotipos a la vez(AU)

Immune response to synthetic peptides of dengue prM protein.

The immunological activities of five synthetic peptides of the prM protein of dengue-2 (DEN-2) virus containing B cell epitopes were evaluated in BALB/c mice. Two peptides elicited neutralizing antibodies against all four DEN serotypes. Virus-specific proliferative responses were demonstrated in mice immunized with four of the five peptides, demonstrating the presence of T cell epitopes. Mice immunized with three of the five peptides conjugated with bovine albumin showed statistically significant levels (P<0.05) of protection when challenged with DEN-2 virus. These results could constitute the basis for the establishment of the role of DEN virus pre and M antigens in the development of anti-flaviviral vaccines.

[Prediction of serotypes of dengue virus by response to IgM antibodies]. / Predicción del serotipo del virus del dengue mediante la respuesta de anticuerpos IgM.

A study of 4 groups of serum samples from Cuba, Costa Rica, Nicaragua and Panama was made to determine the serotypes of dengue virus causing epidemics or outbreaks by using IgM antibody-capture ELISA(MAC-ELISA). Dengue-IgM diagnostic kit was independently used in each of the serotypes and an index value was calculated (optical density of the sample/cut-off value) in each case, with which two statistical analyses were made: Fisher's variances and Litter significant difference (LSD) calculation. Such calculations allowed determining that in Cuba and Panama, serotype 2 circulated whereas in Costa Rica, serotype 1 prevailed. These were the isolated serotypes in various epidemics. Regarding Nicaragua, very heterogeneous responses were obtained, the responsible serotype could not be determined since more than two serotypes co-circulated in this country.

Epidemiologic Studies on Dengue in Santiago de Cuba, 1997

A small, isolated outbreak of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) due to dengue virus type 2 (DEN-2) was documented in Santiago de Cuba on the island of Cuba beginning in January 1997.There were 205 DHF/DSS cases, all in persons older than age 15 years. All but three had evidence of a prior dengue infection, with the only known opportunity being the islandwide dengue virus type 1 (DEN-1) epidemic of 1977–1979. Virtually complete clinical and laboratory surveillance of overt disease was achieved. From December 1997 to January 1998, a random, age-stratified serum sample was obtained from 1,151 persons in Lunes miercoles 40 residential clusters in Santiago. Sera were tested for DEN-1 and DEN-2 neutralizing antibodies. The prevalence of DEN-2 antibodies in children age 15 years and under, born after the 1981 DEN-2 epidemic, was taken as the 1997 DEN-2 infection rate. This was adjusted slightly to accommodate observed cases, resulting in an estimated infection rate of 4.3%. Dengue fever and DHF/DSS attack rates were calculated from estimated total primary and secondary DEN-2 infections. Only 3% of 13,116 primary infections were overt. The DHF/DSS attack rate for adults of all ages was 420 per 10,000 secondary DEN-2 infections(AU)