Análise do transcriptoma das glândulas venenosas do escorpião Tityus stigmurus

In Brazil, accidents with scorpions are considered of medical importance, not only by the high incidence, but also for the potentiality of the venom from some species in determining severe clinical conditions. Tityus stigmurus is a widely distributed scorpion species in Northeastern Brazil and known to cause severe human envenomations, inducing pain, hyposthesia, edema, erythema, paresthesia, headaches and vomiting. The present study uses a transcriptomic approach to characterize the molecular repertoire from the non-stimulated venom gland of Tityus stigmurus scorpion. A cDNA library was constructed and 540 clones were sequenced and grouped into 37 clusters, with more than one EST (expressed sequence tag) and 116 singlets. Forty-one percent of ESTs belong to recognized toxin-coding sequences, with antimicrobial toxins (AMP-like) the most abundant transcripts, followed by alfa KTx- like, beta KTx-like, beta NaTx-like and alfa NaTx-like. Our analysis indicated that 34% include other possible venom molecules , whose transcripts correspond to anionic peptides, hypothetical secreted peptides, metalloproteinases, cystein-rich peptides and lectins. Fifteen percent of ESTs are similar to cellular transcripts. Sequences without good matches corresponded to 11%. This investigation provides the first global view of cDNAs from Tityus stigmurus. This approach enables characterization of a large number of venom gland component molecules, which belong either to known or atypical types of venom peptides and proteins from the Buthidae family

Estudo do genoma do vírus causador da mionecrose infecciosa em camarões e desenvolvimento de métodos para detecção de diferentes variantes

Shrimp farming is one of the activities that contribute most to the growth of global aquaculture. However, this business has undergone significant economic losses due to the onset of viral diseases such as Infectious Myonecrosis (IMN). The IMN is already widespread throughout Northeastern Brazil and affects other countries such as Indonesia, Thailand and China. The main symptom of disease is myonecrosis, which consists of necrosis of striated muscles of the abdomen and cephalothorax of shrimp. The IMN is caused by infectious myonecrosis virus (IMNV), a non-enveloped virus which has protrusions along its capsid. The viral genome consists of a single molecule of double-stranded RNA and has two Open Reading Frames (ORFs). The ORF1 encodes the major capsid protein (MCP) and a potential RNA binding protein (RBP). ORF2 encodes a probable RNA-dependent RNA polymerase (RdRp) and classifies IMNV in Totiviridae family. Thus, the objective of this research was study the IMNV complete genome and encoded proteins in order to develop a system differentiate virus isolates based on polymorphisms presence. The phylogenetic relationship among some totivirus was investigated and showed a new group to IMNV within Totiviridae family. Two new genomes were sequenced, analyzed and compared to two other genomes already deposited in GenBank. The new genomes were more similar to each other than those already described. Conserved and variable regions of the genome were identified through similarity graphs and alignments using the four IMNV sequences. This analyze allowed mapping of polymorphic sites and revealed that the most variable region of the genome is in the first half of ORF1, which coincides with the regions that possibly encode the viral protrusion, while the most stable regions of the genome were found in conserved domains of proteins that interact with RNA. Moreover, secondary structures were predicted for all proteins using various softwares and protein structural models were calculated using threading and ab initio modeling approaches. From these analyses was possible to observe that the IMNV proteins have motifs and shapes similar to proteins of other totiviruses and new possible protein functions have been proposed. The genome and proteins study was essential for development of a PCR-based detection system able to discriminate the four IMNV isolates based on the presence of polymorphic sites

Proteínas SAG1, SAG2, SAG3 de toxoplasma gondii como perspectivas para o desenvolvimento de protótipo vacinal contra a toxoplasmose

Toxoplasmosis is a zoonosis of worldwide distribution caused by the protozoan Toxoplasma gondii, triggering dangerous complications in immunocompromised patients and pregnant women, as well as having great economic impact for the livestock. So far the control of toxoplasmosis is made primarily by chemotherapy. However, most drugs used routinely have some limitations. In order to control this disease, several research groups, including ours, has been working to develop a medical-veterinary vaccine based on parasite antigens, vectors and protocols of immunization. In this study were implemented and standardized methodologies for amplification and cloning of recombinant immunogens in the system for the development of a prototype vaccine, based on the surface antigens of T. gondii and recombinant adenovirus encoding these antigens. Genes encoding BAG1, GRA2 and SAG1 proteins were amplified. We established a strategy for cloning SAG1, SAG2, SAG3 and TgAMA1- genes in recombinant system. The genes encoding SAG1 and SAG2 were cloned and their sequences showed high similarity with sequences from GenBank. The virtual translation of these proteins showed polymorphisms in the amino acid sequence, which can be correlated with levels of antigenicity. Simultaneously, the adenovirus encoding the SAGs (HAdSAGs) were expanded, purificated and characterizated. Immunization of C57bl/6 mice, using viral supernatant was not enought to elicit immune responses at high levels, being required HAdSAGs titration for future immunizations. Therefore, this study allowed the cloning of the two genes important for the development of a prototype vaccine. Besides, implementations methodologies that permit advancements in the development of a vaccine against toxoplasmosis using adenovirus to express proteins of the parasite

Evolução do reparo por excisão de nucleotídeo

The phylogeny is one of the main activities of the modern taxonomists and a way to reconstruct the history of the life through comparative analysis of these sequences stored in their genomes aimed find any justification for the origin or evolution of them. Among the sequences with a high level of conservation are the genes of repair because it is important for the conservation and maintenance of genetic stability. Hence, variations in repair genes, as the genes of the nucleotide excision repair (NER), may indicate a possible gene transfer between species. This study aimed to examine the evolutionary history of the components of the NER. For this, sequences of UVRA, UVRB, UVRC and XPB were obtained from GenBank by Blast-p, considering 10-15 as cutoff to create a database. Phylogenetic studies were done using algorithms in PAUP programs, BAYES and PHYLIP package. Phylogenetic trees were build with protein sequences and with sequences of 16S ribosomal RNA for comparative analysis by the methods of parsimony, likelihood and Bayesian. The XPB tree shows that archaeal´s XPB helicases are similar to eukaryotic helicases. According to this data, we infer that the eukaryote nucleotide excision repair system had appeared in Archaea. At UVRA, UVRB and UVRC trees was found a monophyletic group formed by three species of epsilonproteobacterias class, three species of mollicutes class and archaeabacterias of Methanobacteria and Methanococci classes. This information is supported by a tree obtained with the proteins, UVRA, UVRB and UVRC concatenated. Thus, although there are arguments in the literature defending the horizontal transfer of the system uvrABC of bacteria to archaeabacterias, the analysis made in this study suggests that occurred a vertical transfer, from archaeabacteria, of both the NER genes: uvrABC and XPs. According the parsimony, this is the best way because of the occurrence of monophyletic groups, the time of divergence of classes and number of archaeabacterias species with uvrABC system

Expressão heteróloga de biossurfactantes identificados em bibliotecas metagenômicas

The microorganisms have a vast genetic diversity and they are present throughout the biosphere, however, only about 1% of the species can be cultivated by traditional cultivation techniques. Within this diversity there is a huge pool genetic and biological being explored. The metagenomics has enabled direct access to microbial genome derived from environmental samples using independent methods of cultivation. The methodology enables to obtain functional information about the proteins, as well as identify potential products with biotechnological interest and new industrially exploitable biological resources, such as new solutions to environmental impacts. Oil-contaminated areas are characterized by a large accumulation of hydrocarbons and surfactants may be used for bioremediation. Thus, the metagenomic approach was used in this study in order to select genes involved in the degradation and hydrocarbon emulsification. In a previous work, the environmental DNA (eDNA) was extracted from soil samples collected from two different areas (Caatinga and Saline River) of Rio Grande do Norte (Brazil), the metagenomic libraries were constructed and functionally analyzed. The clone able to degrade the oil was evaluated for the ability to synthesize biosurfactants. The sequence analysis revealed an ORF with 897 bp, 298 amino acids and a protein with around 34 kDa. The search for homology in GenBank revealed sequence similarity with a hypothetical protein of representatives Halobacteriaceae family, who were recently shown as strains producing biosurfactants. The presence of the inserted coding sequence and the acquired phenotype was confirmed. Primers were designed and the ORF amplified by PCR. The ORF was subcloned into pETDuet-1 expression vector for subsequent purification of the protein of interest containing a histidine tail. The tests performed to confirm the biosurfactant activity and the ability of hydrocarbon degradation showed positive results. The immunodetection test (western blot) using the monoclonal AntiHis® confirmed the presence of the environmental protein. This study was the first to report a possible protein with biosurfactant activity obtained from a metagenomic approach

Filogenia molecular das enzimas isocitrato liase e malato sintase e sua evolução em Viridiplanta

The plant metabolism consists of a complex network of physical and chemical events resulting in photosynthesis, respiration, synthesis and degradation of organic compounds. This is only possible due to the different kinds of responses to many environmental variations that a plant could be subject through evolution, leading also to conquering new surroundings. The glyoxylate cycle is a metabolic pathway found in glyoxysomes plant, which has unique role in the seedling establishment. Considered as a variation of the citric acid cycle, it uses an acetyl coenzyme A molecule, derived from lipids beta-oxidation to synthesize compounds which are used in carbohydrate synthesis. The Malate synthase (MLS) and Isocitrate lyase (ICL) enzyme of this cycle are unique and essential in regulating the biosynthesis of carbohydrates. Because of the absence of decarboxylation steps as rate-limiting steps, detailed studies of molecular phylogeny and evolution of these proteins enables the elucidation of the effects of this route presence in the evolutionary processes involved in their distribution across the genome from different plant species. Therefore, the aim of this study was to establish a relationship between the molecular evolution of the characteristics of enzymes from the glyoxylate cycle (isocitrate lyase and malate synthase) and their molecular phylogeny, among green plants (Viridiplantae). For this, amino acid and nucleotide sequences were used, from online repositories as UniProt and Genbank. Sequences were aligned and then subjected to an analysis of the best-fit substitution models. The phylogeny was rebuilt by distance methods (neighbor-joining) and discrete methods (maximum likelihood, maximum parsimony and Bayesian analysis). The identification of structural patterns in the evolution of the enzymes was made through homology modeling and structure prediction from protein sequences. Based on comparative analyzes of in silico models and from the results of phylogenetic inferences, both enzymes show significant structure conservation and their topologies in agreement with two processes of selection and specialization of the genes. Thus, confirming the relevance of new studies to elucidate the plant metabolism from an evolutionary perspective

Análise do transcriptoma das glândulas venenosas do escorpião Tityus stigmurus

In Brazil, accidents with scorpions are considered of medical importance, not only by the high incidence, but also for the potentiality of the venom from some species in determining severe clinical conditions. Tityus stigmurus is a widely distributed scorpion species in Northeastern Brazil and known to cause severe human envenomations, inducing pain, hyposthesia, edema, erythema, paresthesia, headaches and vomiting. The present study uses a transcriptomic approach to characterize the molecular repertoire from the non-stimulated venom gland of Tityus stigmurus scorpion. A cDNA library was constructed and 540 clones were sequenced and grouped into 37 clusters, with more than one EST (expressed sequence tag) and 116 singlets. Forty-one percent of ESTs belong to recognized toxin-coding sequences, with antimicrobial toxins (AMP-like) the most abundant transcripts, followed by alfa KTx- like, beta KTx-like, beta NaTx-like and alfa NaTx-like. Our analysis indicated that 34% include other possible venom molecules , whose transcripts correspond to anionic peptides, hypothetical secreted peptides, metalloproteinases, cystein-rich peptides and lectins. Fifteen percent of ESTs are similar to cellular transcripts. Sequences without good matches corresponded to 11%. This investigation provides the first global view of cDNAs from Tityus stigmurus. This approach enables characterization of a large number of venom gland component molecules, which belong either to known or atypical types of venom peptides and proteins from the Buthidae family

Estudo do genoma do vírus causador da mionecrose infecciosa em camarões e desenvolvimento de métodos para detecção de polimorfismos

Shrimp farming is one of the activities that contribute most to the growth of global aquaculture. However, this business has undergone significant economic losses due to the onset of viral diseases such as Infectious Myonecrosis (IMN). The IMN is already widespread throughout Northeastern Brazil and affects other countries such as Indonesia, Thailand and China. The main symptom of disease is myonecrosis, which consists of necrosis of striated muscles of the abdomen and cephalothorax of shrimp. The IMN is caused by infectious myonecrosis virus (IMNV), a non-enveloped virus which has protrusions along its capsid. The viral genome consists of a single molecule of double-stranded RNA and has two Open Reading Frames (ORFs). The ORF1 encodes the major capsid protein (MCP) and a potential RNA binding protein (RBP). ORF2 encodes a probable RNA-dependent RNA polymerase (RdRp) and classifies IMNV in Totiviridae family. Thus, the objective of this research was study the IMNV complete genome and encoded proteins in order to develop a system differentiate virus isolates based on polymorphisms presence. The phylogenetic relationship among some totivirus was investigated and showed a new group to IMNV within Totiviridae family. Two new genomes were sequenced, analyzed and compared to two other genomes already deposited in GenBank. The new genomes were more similar to each other than those already described. Conserved and variable regions of the genome were identified through similarity graphs and alignments using the four IMNV sequences. This analyze allowed mapping of polymorphic sites and revealed that the most variable region of the genome is in the first half of ORF1, which coincides with the regions that possibly encode the viral protrusion, while the most stable regions of the genome were found in conserved domains of proteins that interact with RNA. Moreover, secondary structures were predicted for all proteins using various softwares and protein structural models were calculated using threading and ab initio modeling approaches. From these analyses was possible to observe that the IMNV proteins have motifs and shapes similar to proteins of other totiviruses and new possible protein functions have been proposed. The genome and proteins study was essential for development of a PCR-based detection system able to discriminate the four IMNV isolates based on the presence of polymorphic sites