The RNA-dependent RNA polymerase of the infectious pancreatic necrosis virus is linked to viral mRNA acting as a cap substitute.

The infectious pancreatic necrosis virus (IPNV) is responsible for significant economic losses in the aquaculture industry. It is an unenveloped virus with an icosahedral capsid. Its viral genome comprises two dsRNA segments, A and B. Segment A contains a small ORF, which encodes VP5, and a large ORF, which encodes a polyprotein that generates the structural proteins and the viral protease. Segment B encodes the RNA-dependent RNA polymerase (RdRp), called VP1 in this free form, or Vpg when it covalently attaches to the viral RNA. The viral genome does not have cap or poly(A). Instead, each 5' end is linked to the Vpg. Recently, we demonstrated that mRNA-A contains an internal ribosome entry site (IRES) to command polyprotein synthesis. However, the presence of Vpg on IPNV mRNAs and its impact on cellular translation has not been investigated. This research demonstrates that IPNV mRNAs are linked to Vpg and that this protein inhibits cap-dependent translation on infected cells. Also, it is demonstrated that Vpg interacts with eIF4E and that rapamycin treatment partially diminishes the viral protein synthesis. In addition, we determined that an IRES does not command translation of IPNV mRNA-B. We show that VPg serves as a cap substitute during the initiation of IPNV translation, contributing to understanding the replicative cycle of Birnaviruses. Our results indicate that the viral protein VP1/Vpg is multifunctional, having a significant role during IPNV RNA synthesis as the RdRp and the primer for IPNV RNA synthesis and translation as the viral protein genome, acting as a cap substitute.

Analysis by real-time PCR of five transport and conservation mediums of nasopharyngeal swab samples to COVID-19 diagnosis in Santiago of Chile.

Due to the COVID-19 pandemic, many transport kits have been manufactured to preserve and transport nasopharyngeal swab samples (NPSs) from patients. However, there is no information on the performance of the different virus transport media (VTM) used in COVID-19 diagnosis in the population of Santiago de Chile. We compared the RT-qPCR amplification profile of five different viral transport kit mediums, including DNA/RNA Shield™, NAT, VTM-N, Ezmedlab™, and phosphate-buffered saline (PBS), for NPSs from Central Metropolitan Health Service, Santiago, Chile. The DNA/RNA Shield™ medium showed a better performance in terms of Cq and RFU values for the internal reference RNase P and viral ORF1ab probes. By contrast, the PBS transport medium registered higher Cq values for the viral and reference gene, compared to the other VTM. DNA/RNA Shield™ shows higher relative fluorescence units (RFUs) and lower Cq values for the reference gene. Collectively, our results suggest that the PBS medium could compromise the sample diagnosis because of its lower RT-qPCR performance. The NAT, Ezmedlab and VTM-N, and DNA/RNA Shield™ media show acceptable RT-qPCR parameters and, consequently, seem suitable for use in COVID-19 diagnosis.

Characteristics and in-hospital morbidity trends associated with oral and oropharyngeal cancers in Brazil.

BACKGROUND: Brazil experienced an expansion of the population's access to oral health policies after the creation of the Unified Health System (SUS, Sistema Único de Saúde). Through public policies, the consolidation of Primary Health Care (PHC) and the incorporation of dental care into primary and hospital care took place. The objective of this study was to identify epidemiological aspects, including the temporal trend, of hospital morbidity from oral and oropharyngeal cancer in Brazil, considering hospitalizations for this neoplasm in a hospital network linked to the public care system. METHODS: Observational study based on information on hospital admissions for oral cancer throughout Brazil. The research used data from the Brazilian Cancer Registry Information System. For the temporal series analysis, generalized linear regression model was used with the Prais-Winsten method. RESULTS: Of the 121,971 patients hospitalized with oral and oropharyngeal cancers, 76.40% were male and 23.60% were female, resulting in a M:F ratio of 3.24:1. Regarding the anatomical region of involvement among hospitalized patients with oral cavity neoplastic lesions, there was a predominance in non-specific places in the mouth, such as the floor of the mouth, soft and hard palate, among others (32.68%), followed by lesions in the region of tongue (28.89%). In this population, the predominant age group was between the fifth decade (31.09%) and sixth decade of life (24.99%); men presented neoplastic lesions of oral and oropharyngeal cancers at an earlier age than women. In all regions of the country, the staging of cases diagnosed in the tertiary health network accredited to the José Alencar Gomes da Silva National Cancer Institute (INCA) was late, with higher tendency for metastasis. The temporal trend of the adjusted in-hospital morbidity rates showed to be increasing in the Northeast, South and Midwest regions for the male gender. For females, they were increasing in the Northeast and South regions. CONCLUSIONS: It is concluded that the distribution of in-hospital morbidity rates of oral and oropharyngeal cancers in the country is irregular. There is a greater number of cases identified by the study in male patients and in the Southeast and South regions; with an increasing tendency of this coefficient in both genders.

Inclusion of oral health teams in primary health care promotes early diagnosis of oral and oropharyngeal cancers: a nationwide study.

BACKGROUND: Oral and oropharyngeal cancers are considered important public health problems worldwide. This study aims to analyze the association between late diagnosis of oral and oropharyngeal cancers in Brazil and the contextual indicators of socioeconomic variables and coverage of Primary Health Care (PHC), and to assess the temporal trend of late diagnosis. METHODS: In this cross-sectional observational study, secondary data were evaluated with a time series analysis. All Brazilian cities that reported at least one case of oral and oropharyngeal cancers each year in the period between 2000 and 2013 were included; and the staging was analyzed by calculating the ratio risk for late diagnosis for each municipality. The association between staging and socioeconomic variables and offer of PHC was calculated using multiple linear regression. The time trend of the risk ratio for late-stage diagnosis was calculated using the Prais-Winsten method. RESULTS: One hundred and sixty Brazilian municipalities had at least one annual case of oral and oropharyngeal cancers notified to the INCA hospital system between 2000 and 2013. The adjusted model showed that the higher the Gini value (greater social inequality) and the lower the HDI value (less human development) was, the higher was the number of tumors diagnosed at a late stage, considering the size of the tumor. A greater risk for late diagnosis was identified, as early as at the stage of lymph node involvement, when there was a higher level of social inequality and lower level of coverage by Oral Health Teams (OHT) in PHC. The greater the social inequality, the greater was the risk of late diagnosis, as early as in the stage of metastasis. CONCLUSIONS: We concluded that, during the evaluated period, there was an increase in the number of cases diagnosed at the most advanced stage. Furthermore, there was association between higher levels of social inequality and an increase in the proportion of late diagnosis of oral and oropharyngeal cancers. In addition, the inclusion of Oral Health Teams in Primary Health Care promoted the early diagnosis of these types of cancers.

The Atlantic salmon interleukin 4/13 receptor family: Structure, tissue distribution and modulation of gene expression.

Interleukin (IL)-4 and IL-13 play a central role in T helper 2 immune response in mammals. The cell signalling is mediated by the type I heterodimeric receptor containing the IL-4Rα and γC chains, and the type II receptors formed by IL-4Rα and IL-13Rα1. In salmonid species, three paralogues of the IL-4 and IL-13 cytokines have been reported, il-4/13a, il-4/13b1 and il-4/13b2. In regard to receptors, two paralogues of each IL-4/13 receptor chains have been identified in rainbow trout while five genes named γc1, il-4rα, il-13rα1a, il-13rα1b, and il-13rα2 have identified in Atlantic salmon. Since Atlantic salmon is an important farmed fish species, the aim of this work was to get new insights into distribution, structure and expression regulation of the IL-4/13 receptors in salmon. By using qRT-PCR, it was shown that all γc1, il-4rα, il-13rα1a, il-13rα1b, and il-13rα2 receptor chains were expressed in lymphoid and non-lymphoid tissues of healthy salmon, nonetheless γC expression was higher in lymphoid than non-lymphoid tissues. The in silico structural analysis and homology modelling of the predicted receptor proteins showed that domains and most motifs present in the superior vertebrate chains are conserved in salmon suggesting a conserved role for these receptor chains. Only IL-13Rα1B is a receptor chain with a unique structure that seem not to be present in higher vertebrates but in fish species. In order to determine the regulatory role of IL-4/13 on the expression of receptor chains, Atlantic salmon il-4/13A gene was synthetized and cloned in pET15b. The recombinant IL-4/13A was produced in E. coli and the activity of the purified cytokine was confirmed in vitro. The regulatory role of IL-4/13A on the expression of their potential receptors was tested in salmon receiving the recombinant cytokine and effects were compared with those of the control group. The results showed that IL-4/13A induced the expression of its own gene and GATA-3, in the head kidney of fish but not in the spleen, while IL-10 increased in both lymphoid organs indicating a regulatory role of this cytokine on the induction of Th2 responses in salmon. IFN-γ and MHC class II were also later induced in head kidney. In regard to the expression of the receptor chains, IL-4/13A upregulated the expression of γC, IL-13Rα1A and IL-13Rα2A in the spleen but not in the head kidney of salmon, indicating a role on the modulation of cell signalling for the Th2 response. Furthermore, Piscirickettsia salmonis infection of Atlantic salmon occurred with an increase of γC and IL-13Rα1A suggesting a potential role of the IL-4/13 system in bacterial immunity or pathogenesis. This study contributes to a better understanding of the IL-4/13A system in salmon, which as a key axis for Th2 response may be involved not only in pathogen elimination but also in adaptive immune repair that seems critical tolerance to infectious diseases.

Effect of yeast (Xanthophyllomyces dendrorhous) and plant (Saint John's wort, lemon balm, and rosemary) extract based functional diets on antioxidant and immune status of Atlantic salmon (Salmo salar) subjected to crowding stress.

Salmon farming may face stress due to the intensive culture conditions with negative impacts on overall performance. In this aspect, functional feed improves not only the basic nutritional requirements but also the health status and fish growth. However, to date no studies have been carried out to evaluate the effect of functional diets in salmon subjected to crowding stress. Thus, the aim of this study was to evaluate the effect of yeast extract (Xanthophyllomyces dendrorhous; diet A) and the combination of plant extracts (common Saint John's wort, lemon balm, and rosemary; diet B) on the antioxidant and immune status of Atlantic salmon grown under normal cultured conditions and then subjected to crowding stress. Fish were fed with functional diets during 30 days (12 kg/m3) and then subjected to crowding stress (20 kg/m3) for 10 days. The lipid peroxidation in gut showed that both diets induced a marked decrease on oxidative damage when fish were subjected to crowding stress. The protein carbonylation in muscle displayed at day 30 a marked decrease in both functional diets that was more marked on the stress condition. The expression of immune markers (IFNγ, CD4, IL-10, TGF-ß, IgMmb, IgMsec, T-Bet, and GATA-3) indicated the upregulation of those associated to humoral-like response (CD4, IL-10, GATA-3) when fish were subjected to crowding stress. These results were confirmed with the expression of secreted IgM. Altogether, these functional diets improved the antioxidant status and increased the expression of genes related to Th2-like response suggesting a protective role on fish subjected to crowding stress.

Expression of an Mg2+-dependent HIV-1 RNase H construct for drug screening.

A single polypeptide of the HIV-1 reverse transcriptase that reconstituted Mg(2+)-dependent RNase H activity has been made. Using molecular modeling, the construct was designed to encode the p51 subunit joined by a linker to the thumb (T), connection (C), and RNase H (R) domains of p66. This p51-G-TCR construct was purified from the soluble fraction of an Escherichia coli strain, MIC2067(DE3), lacking endogenous RNase HI and HII. The p51-G-TCR RNase H construct displayed Mg(2+)-dependent activity using a fluorescent nonspecific assay and showed the same cleavage pattern as HIV-1 reverse transcriptase (RT) on substrates that mimic the tRNA removal required for second-strand transfer reactions. The mutant E706Q (E478Q in RT) was purified under similar conditions and was not active. The RNase H of the p51-G-TCR RNase H construct and wild type HIV-1 RT had similar K(m)s for an RNA-DNA hybrid substrate and showed similar inhibition kinetics to two known inhibitors of the HIV-1 RT RNase H.

The Analysis of Live-Attenuated Piscirickettsia salmonis Vaccine Reveals the Short-Term Upregulation of Innate and Adaptive Immune Genes in Atlantic Salmon (Salmo salar): An In Situ Open-Sea Cages Study.

Piscirickettsia salmonis, the etiological agent of the Salmon Rickettsial Septicemia (SRS), is one the most serious health problems for the Chilean salmon industry. Typical antimicrobial strategies used against P. salmonis include antibiotics and vaccines, but these applications have largely failed. A few years ago, the first attenuated-live vaccine against SRS (ALPHA JECT LiVac® SRS vaccine) was released to the market. However, there is no data about the agents involved in the activation of the immune response induced under field conditions. Therefore, in this study we evaluated the expression profile of a set of gene markers related to innate and adaptive immunity in the context of a cellular response in Atlantic salmon (Salmo salar) reared under productive farm conditions and immunized with a live-attenuated vaccine against P. salmonis. We analyzed the expression at zero, 5-, 15- and 45-days post-vaccination (dpv). Our results reveal that the administration of the attenuated live SRS LiVac vaccine induces a short-term upregulation of the cellular-mediated immune response at 5 dpv modulated by the upregulation of ifnα, ifnγ, and the cd4 and cd8α T cell surface markers. In addition, we also registered the upregulation of il-10 and tgfß. Altogether, the results suggest that a balanced activation of the immune response took place only at early times post-vaccination (5 dpv). The scope of this short-term upregulation of the cellular-mediated immune response against a natural outbreak in fish subjected to productive farm conditions deserves further research.

Estudo comparativo da retenção das bandas ortodônticas Grip Tite, convencionais e microjateadas / Comparison of the retention of Grip Tite, conventionals and sandblasted, conventionals orthodontic bands

O objetivo deste trabalho foi avaliar in litro a força necessária para a remoção das bandas ortodônticas Grip Tite (TP Orthodontics, LaPorte/lndiana, EUA), convencionais (Abzil Lancer/São José do Rio Preto, SP) e convencionais com a superfície interna microjateada por óxido de alumínio. Em adição, examinar visualmente a quantidade de cimento remanescente nas superfícies internas das bandas e analisar por meio de microscopia eletrônica de varredura (MEV) as superfícies com melhores características para a retenção. Os resultados obtidos mostraram que a média de carga do grupo de bandas convencionais microjateadas foi significantemente maior que as dos grupos de bandas convencionais e bandas Grip Tite. Quanto a quantidade de cimento remanescente, nas bandas microjateadas foi encontrada mais de 75% de cimento residual aderido as suas superfícies, enquanto nos grupos convencional e Grip Tite foi observado menos de 25% de cimento residual, sem diferença significativa entre estes dois grupos. Os resultados foram confirmados pela MEV, onde foram visualizadas melhores características de superfície para a retenção do cimento nas bandas microjateadas.

The purpose of this study was to evaluate in vitro, the necessary force for the removal of Grip Tite (TP Orthodontics/LaPorte, Indiana), Conventionals (Abzil Lancer/São José do Rio Preto, SP) and Sandblasted Conventionals orthodontics molar bands, us well as, to analyze and to classify, the amount of residual cement in the internal surface of these bands. After that, the surface with better characteristics for the retention was checked using scanning electron microscopy ( SEM). The obtained results showed that the load average of the Sandblasted bands was significantly higher than the conventional and Grip Tite bands. Observing the amount of remaining cement in the bands, Sandblasted bunds indicated the presence of more than 75% of residual cement in the bands, while in the Conventional and Grip Tite groups showed less than 25% of residual cement in the bands, without significantdifference between these two groups. The results were confirmed by the SEM, where the surface with better. Characteristics for the retention of the cement was observed in the bands where the sandblasted process was carried through.

Estudo comparativo da retenção das bandas ortodônticas Grip Tite, convencional e convencional com a superfície interna microjateada por óxido de alumínio / Retention of Grip Tite bands, conventional orthodontic bands and sandblasted bands

O objetivo do presente trabalho foi avaliar in vitro, por meio de um ensaio mecânico, a força necessária para a remoção das bandas ortodônticas Grip Tite (C), Convencional (A) e banda Convencional com a superfície interna microjateada por óxido de alumínio (B). Também analisar visualmente a quantidade de cimento residual, e classificar usando uma escala de incrementos a 25%. Após, verificar, por meio de MEV, a superfície com melhores características para a retenção. Os resultados obtidos mostraram que a média de carga do Grupo B, bandas microjateadas internamente por óxido de alumínio, foi significantemente maior que as médias dos Grupos A, bandas Convencionais, e C, bandas Grip Tite. Quanto a quantidade de cimento remanescente nas bandas, o Grupo B apresentou 100% das bandas com índice 4, indicando a presença de mais de 75% de cimento residual nas bandas, enquanto que nos Grupos A e C, houve a predominância do índice 1, indicativo de menos de 25% de cimento residual nas bandas, sem diferença significativa entre esses dois grupos. Os resultados foram confirmados pela MEV, onde observou-se uma superfície com melhores características para a retenção do cimento nas bandas onde foi realizado o processo de microjateamento. Conclui-se que o processo de microjateamento por óxido de alumínio, na superfície interna das bandas convencionais, aumenta a força necessária para a remoção das bandas e aumenta a quantidade de cimento residual preso à superfície da banda. Quando analisada pela MEV, essa superfície apresenta-se como a mais áspera e rugosa, com a maior quantidade de cimento de ionômero de vidro aderido à banda

The purpose of this study was to evaluate in vitro, the necessary force for the removal of Grip Tite, Conventional and Sandblasted orthodontics molar bands, as well as, to analyze and to classify the amount of residual cement in the internal surface of these bands. After that, checking through scanning electron microscopy, the surface with better characteristics for the retention. The obtained results showed that the load average of the group B, Sandblasted bands, was significantly higher than the group A, Conventional bands, and C, Grip Tite bands. Observing the amount of remaining cement in the bands, group B presented 100% of the bands with index 4, indicating the presence of more than 75% of residual cement in the bands, while in the groups A and C, had the predominance of index 1, indicative of less than 25% of residual cement in the bands, without significant difference between these two groups. The results were confirmed by the scanning electronic microscopy, where the surface with better characteristics for the retention of the cement was observed, in the bands where the sandblasted process was carried through. It was concluded that the process of sandblasted, in the internal surface of the conventional bands, increases the necessary force the removal of the bands and increases the amount of residual cement in the internal surface of the band. When analyzed by the scanning electronic microscopy, this surface appeared rougher and corrugated, with great amount of glass ionomer cement adhered to the band