Analysis of Kojic Acid Derivatives as Competitive Inhibitors of Tyrosinase: A Molecular Modeling Approach.

Tyrosinases belong to the functional copper-containing proteins family, and their structure contains two copper atoms, in the active site, which are coordinated by three histidine residues. The biosynthesis of melanin in melanocytes has two stages depending on the actions of the natural substrates L-DOPA and L-tyrosine. The dysregulation of tyrosinase is involved in skin cancer initiation. In the present study, using molecular modeling tools, we analyzed the inhibition activity of tyrosinase activity using kojic acid (KA) derivatives designed from aromatic aldehydes and malononitrile. All derivatives showed conformational affinity to the enzyme active site, and a favorable distance to chelate the copper ion, which is essential for enzyme function. Molecular dynamics simulations revealed that the derivatives formed promising complexes, presenting stable conformations with deviations between 0.2 and 0.35 Å. In addition, the investigated KA derivatives showed favorable binding free energies. The most stable KA derivatives showed the following binding free energies: -17.65 kcal mol-1 (D6), -18.07 kcal mol-1 (D2), -18.13 (D5) kcal mol-1, and -10.31 kcal mol-1 (D4). Our results suggest that these derivatives could be potent competitive inhibitors of the natural substrates of L-DOPA (-12.84 kcal mol-1) and L-tyrosine (-9.04 kcal mol-1) in melanogenesis.

Comparative analysis of the transcriptome of the Amazonian fish species Colossoma macropomum (tambaqui) and hybrid tambacu by next generation sequencing.

BACKGROUND: The C. macropomum is a characiform fish from the Amazon basin that has been hybridized with other pacu species to produce commercial hybrids, such as the tambacu. However, little is known of the functional genomics of the parental species or these hybrid forms. The transcriptome of C. macropomum and tambacu were sequenced using 454 Roche platform (pyrosequencing) techniques to characterize the domains of Gene Ontology (GO) and to evaluate the levels of gene expression in the two organisms. RESULTS: The 8,188,945 reads were assembled into 400,845 contigs. A total of 58,322 contigs were annotated with a predominance of biological processes for both organisms, as determined by Gene Ontology (GO). Similar numbers of metabolic pathways were identified in both the C. macropomum and the tambacu, with the metabolism category presenting the largest number of transcripts. The BUSCO analysis indicated that our assembly was more than 40% complete. We identified 21,986 genes for the two fishes. The P and Log2FC values indicated significant differences in the levels of gene expression, with a total of 600 up-regulated genes. CONCLUSION: In spite of the lack of a reference genome, the functional annotation was successful, and confirmed a considerable difference in the specificity and levels of gene expression between the two organisms. This report provides a comprehensive baseline for the genetic management of these commercially important fishes, in particular for the identification of specific genes that may represent markers involved in the immunity, growth, and fertility of these organisms, with potential practical applications in aquaculture management.

De novo transcriptome based on next-generation sequencing reveals candidate genes with sex-specific expression in Arapaima gigas (Schinz, 1822), an ancient Amazonian freshwater fish.

BACKGROUND: The Arapaima (Arapaima gigas) is one of the world's largest freshwater bony fish, and is found in the rivers of the Amazon basin. This species is a potential aquaculture resource, although reproductive management in captivity is limited in particular due to the lack of external sexual dimorphism. In this study, using the 454 Roche platform (pyrosequencing) techniques, we evaluated a major portion of the transcriptome of this important Amazonian species. RESULTS: Four libraries obtained from the liver and skin tissue of juvenile specimens (representing males and females separately) were sequenced, yielding 5,453,919 high-quality reads. The de novo transcriptome assembly resulted in 175,792 contigs, with 51,057 significant blast hits. A total of 38,586 transcripts were mapped by Gene Ontology using Blast2GO. We identified 20,219 genes in the total transcriptome (9,551 in the liver and 16,818 in the skin). The gene expression analyses indicated 105 genes in the liver and 204 in the skin with differentiated expression profiles, with 95 being over-expressed in the females and 214 in the males. The log2 Fold Change and heatmap based on Reads Per Kilobase per Million mapped reads (RPKM) revealed that the gene expression in the skin is highly differentiated between male and female arapaima, while the levels of expression in the liver are similar between the sexes. CONCLUSION: Transcriptome analysis based on pyrosequencing proved to be a reliable tool for the identification of genes with differentiated expression profiles between male and female arapaima. These results provide useful insights into the molecular pathways of sexual dimorphism in this important Amazonian species, and for comparative analyses with other teleosts.

Genomic characterization and evolution of Tacaiuma orthobunyavirus (Peribunyaviridae family) isolated in Brazil.

Tacaiuma virus (TCMV) is antigenically characterized as a member of the Anopheles A complex in the Orthobunyavirus genus, Peribunyaviridae family (Bunyavirales order). Clinically, the TCMV infection is characterized by acute febrile illness with myalgia and arthralgia lasting three to five days. However, the genomic and evolutionary aspect of this virus has not been elucidated. In this study, we described the complete coding sequences of three segments of two TCMV strains isolated in Brazil and three complete coding sequences of the small segment of three TCMV strains. All the strains sequenced in this study showed the typical genomic organization of orthobunyaviruses that infect vertebrates, except for the absence of the open reading frame that encodes the well-described non-structural small protein. This study presents the genomic and evolutionary characterization of TCMV strains and would be helpful for diagnostic purposes and epidemiology.

New Virus Genome Sequences of the Guama Serogroup (Genus Orthobunyavirus, Family Bunyaviridae), Isolated in the Brazilian Amazon Region.

This is the first announcement of two nearly complete viral genome sequences belonging to the Guama serogroup (genus Orthobunyavirus, family Bunyaviridae) isolated in the Brazilian Amazon region: Mirim virus (MIRV; BEAN7722) and Ananindeua virus (ANUV; BEAN109303).

Identification and characterization of the expression profile of the microRNAs in the Amazon species Colossoma macropomum by next generation sequencing.

Colossoma macropomum is a resistant species native of Amazonas and Orinoco river basins. It is regarded as the second largest finfish of Solimões and Amazon rivers, representing a major fishery resource in Amazonas and an important species in tropical aquaculture. MicroRNAs are non-coding endogenous riboregulators of nearly 22 nucleotides that play a key role in post-transcriptional gene regulation of several organisms. We analyzed samples of liver and skin from specimens of C. macropomum using next generation sequencing. The dataset was evaluated using computational programs to check the quality of sequences, identification of miRNAs, as well as to evaluate the expression levels of these microRNAs and interaction of target genes. We identified 279 conserved miRNAs, being 257 from liver and 272 from skin, with several miRNAs shared between tissues, with divergence in the number of reads. The strands miR-5p and miR-3p were observed in 72 miRNAs, some of them presenting a higher number of 3p reads. The functional annotation of the most expressed miRNAs resulted in 27 pathways for the liver and skin mainly related to the "biological processes" domain. Based on the identified pathways, we visualized a large gene network, suggesting the regulation of selected miRNA over this interactive dataset. We were able to identify and characterize the expression levels of miRNAs in two tissues of great activity in C. macropomum, which stands out as the beginning of several studies that can be carried out to elucidate the influence of miRNAs in this species and their applicability as biotechnological tools.

Isolation of Madre de Dios Virus (Orthobunyavirus; Bunyaviridae), an Oropouche Virus Species Reassortant, from a Monkey in Venezuela.

Oropouche virus (OROV), genus Orthobunyavirus, family Bunyaviridae, is an important cause of human illness in tropical South America. Herein, we report the isolation, complete genome sequence, genetic characterization, and phylogenetic analysis of an OROV species reassortant, Madre de Dios virus (MDDV), obtained from a sick monkey (Cebus olivaceus Schomburgk) collected in a forest near Atapirire, a small rural village located in Anzoategui State, Venezuela. MDDV is one of a growing number of naturally occurring OROV species reassortants isolated in South America and was known previously only from southern Peru.

Zika virus complete genome from Salvador, Bahia, Brazil.

In May 2015 the first autochthonous Zika virus infection was reported in Brazil. Rapid and urgent measures are needed to contain the ongoing outbreak. Here we report the full-length ZIKV coding sequence from Bahia. Genetic analysis of outbreak sequences will be essential for characterizing the diversity of circulating strains, identifying hotspots of virus transmission and guiding public health control. Rapid and urgent measures are needed to contain the ongoing outbreak.

Comparative modeling and molecular dynamics suggest high carboxylase activity of the Cyanobium sp. CACIAM14 RbcL protein.

Rubisco catalyzes the first step reaction in the carbon fixation pathway, bonding atmospheric CO2/O2 to ribulose 1,5-bisphosphate; it is therefore considered one of the most important enzymes in the biosphere. Genetic modifications to increase the carboxylase activity of rubisco are a subject of great interest to agronomy and biotechnology, since this could increase the productivity of biomass in plants, algae and cyanobacteria and give better yields in crops and biofuel production. Thus, the aim of this study was to characterize in silico the catalytic domain of the rubisco large subunit (rbcL gene) of Cyanobium sp. CACIAM14, and identify target sites to improve enzyme affinity for ribulose 1,5-bisphosphate. A three-dimensional model was built using MODELLER 9.14, molecular dynamics was used to generate a 100 ns trajectory by AMBER12, and the binding free energy was calculated using MM-PBSA, MM-GBSA and SIE methods with alanine scanning. The model obtained showed characteristics of form-I rubisco, with 15 beta sheets and 19 alpha helices, and maintained the highly conserved catalytic site encompassing residues Lys175, Lys177, Lys201, Asp203, and Glu204. The binding free energy of the enzyme-substrate complexation of Cyanobium sp. CACIAM14 showed values around -10 kcal mol(-1) using the SIE method. The most important residues for the interaction with ribulose 1,5-bisphosphate were Arg295 followed by Lys334. The generated model was successfully validated, remaining stable during the whole simulation, and demonstrated characteristics of enzymes with high carboxylase activity. The binding analysis revealed candidates for directed mutagenesis sites to improve rubisco's affinity.

Analysis of a Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) for yellow fever diagnostic.

Yellow Fever virus (YFV) is an important human pathogen in tropical areas of Africa and South America. Although an efficient vaccine is available and has been used since the early 1940s, sylvatic YFV transmission still occurs in forested areas where anthropogenic actions are present, such as mineral extraction, rearing livestock and agriculture, and ecological tourism. In this context, two distinct techniques based on the RT-PCR derived method have been previously developed, however both methods are expensive due to the use of thermo cyclers and labeled probes. We developed isothermal genome amplification, which is a rapid, sensitive, specific and low cost molecular approach for YFV genome detection. This assay used a set of degenerate primers designed for the NS1 gene and was able to amplify, within 30 min in isothermal conditions, the YFV 17D vaccine strain derived from an African wild prototype strain (Asibi), as well as field strains from Brazil, other endemic countries from South and Central America, and the Caribbean. The generic RT-LAMP assay could be helpful for YFV surveillance in field and rapid response during outbreaks in endemic areas.

Emergence and potential for spread of Chikungunya virus in Brazil.

BACKGROUND: In December 2013, an outbreak of Chikungunya virus (CHIKV) caused by the Asian genotype was notified in the Caribbean. The outbreak has since spread to 38 regions in the Americas. By September 2014, the first autochthonous CHIKV infections were confirmed in Oiapoque, North Brazil, and in Feira de Santana, Northeast Brazil. METHODS: We compiled epidemiological and clinical data on suspected CHIKV cases in Brazil and polymerase-chain-reaction-based diagnostic was conducted on 68 serum samples from patients with symptom onset between April and September 2014. Two imported and four autochthonous cases were selected for virus propagation, RNA isolation, full-length genome sequencing, and phylogenetic analysis. We then followed CDC/PAHO guidelines to estimate the risk of establishment of CHIKV in Brazilian municipalities. RESULTS: We detected 41 CHIKV importations and 27 autochthonous cases in Brazil. Epidemiological and phylogenetic analyses indicated local transmission of the Asian CHIKV genotype in Oiapoque. Unexpectedly, we also discovered that the ECSA genotype is circulating in Feira de Santana. The presumed index case of the ECSA genotype was an individual who had recently returned from Angola and developed symptoms in Feira de Santana. We estimate that, if CHIKV becomes established in Brazil, transmission could occur in 94% of municipalities in the country and provide maps of the risk of importation of each strain of CHIKV in Brazil. CONCLUSIONS: The etiological strains associated with the early-phase CHIKV outbreaks in Brazil belong to the Asian and ECSA genotypes. Continued surveillance and vector mitigation strategies are needed to reduce the future public health impact of CHIKV in the Americas.