Porcine circovirus 3 (PCV3) is an emerging virus that was first identified in the United States in 2016. Since its first detection, PCV3 has already been found in America, Asia, and Europe. Although PCV3 has already been described in Brazil, knowledge of its detection and sequence variation before 2016 is limited, as well as its distribution in the main swine producing regions of Brazil. In this study, 67 porcine clinical samples collected from nine states in Brazil between 2006 and 2007 were analyzed for PCV3 infection by PCR. Results showed that 47.8% of the samples were PCV3 positive, across all nine states. Of the PCV3-positive samples, 37.5% were also positive for PCV2. Interestingly, no clinical signs were associated with samples that were detected singularly with PCV3 infection. Moreover, the positive PCV3 rate in healthy pigs was higher (29.8%) than that found in unhealthy pigs (17.9%), suggesting that most pigs could live with PCV3 infection without any clinical sign in the analyzed samples. Nucleotide sequence analysis showed that PCV3 strains obtained in this study shared 94.44% to 99.83% sequence identity at the open reading frame 2 (ORF2) gene level with available strains from different countries. PCV3 Brazilian sequences collected in 2006 and 2007 shared 97.94% to 99.62% identity with the strains obtained in 2016. The results of neutrality and selective pressure tests indicated that the PCV3 Cap protein seems unable to tolerate high levels of variation on its sequence. Phylogenetic analysis grouped the Brazilian strains in PCV3a and PCV3b genotypes clusters, both including strains collected in America, Asia, and Europe. Taking the results together, multiple events of introduction of PCV3 may have occurred in Brazil, and Brazilian PCV3 strains may show genetic stability over the past 10 years.

The thermotolerant yeast Kluyveromyces marxianus displays a potential to be used for ethanol production from both whey and lignocellulosic biomass at elevated temperatures, which is highly alluring to reduce the cost of the bioprocess. Nevertheless, contrary to Saccharomyces cerevisiae, K. marxianus cannot tolerate high ethanol concentrations. We report the transcriptional profile alterations in K. marxianus under ethanol stress in order to gain insights about mechanisms involved with ethanol response. Time-dependent changes have been characterized under the exposure of 6% ethanol and compared with the unstressed cells prior to the ethanol addition. Our results reveal that the metabolic flow through the central metabolic pathways is impaired under the applied ethanol stress. Consistent with these results, we also observe that genes involved with ribosome biogenesis are downregulated and gene-encoding heat shock proteins are upregulated. Remarkably, the expression of some gene-encoding enzymes related to unsaturated fatty acid and ergosterol biosynthesis decreases upon ethanol exposure, and free fatty acid and ergosterol measurements demonstrate that their content in K. marxianus does not change under this stress. These results are in contrast to the increase previously reported with S. cerevisiae subjected to ethanol stress and suggest that the restructuration of K. marxianus membrane composition differs in the two yeasts which gives important clues to understand the low ethanol tolerance of K. marxianus compared to S. cerevisiae.

This work discusses the biological and biochemical characterization of an extracellular protease produced by Pseudomonas fluorescens. The enzyme has a molecular weight of 49.486 kDa and hydrolyzes gelatin, casein, and azocasein, but not BSA. Its maximum activity is found at 37°C and pH 7.5, but it retained almost 70% activity at pH 10.0. It was shown to be a metalloprotease inhibited by Cu(2+), Ni(2+), Zn(2+), Hg(2+), Fe(2+), and Mg(2+), but induced by Mn(2+). After incubation at 100°C for 5min, the enzyme presented over 40% activity, but only 14 to 30% when submitted to milder heat treatments. This behavior may cause significant problems under conditions commonly used for the processing and storage of milk and dairy products, particularly UHT milk. A specific peptide sequenced by mass spectrometer analysis allowed the identification of gene that encodes this extracellular protease in the genome of Pseudomonas fluorescens 07A strain. The enzyme has 477 AA and highly conserved Ca(2+)- and Zn(2+)-binding domains, indicating that Ca(2+), the main ion in milk, is also a cofactor. This work contributes to the understanding of the biochemical aspects of enzyme activity and associates them with its sequence and structure. These findings are essential for the full understanding and control of these enzymes and the technological problems they cause in the dairy industry.

Coffee leaf rust caused by the fungus Hemileia vastatrix is one of the most important leaf diseases of coffee plantations worldwide. Current knowledge of the H . vastatrix genome is limited and only a small fraction of the total fungal secretome has been identified. In order to obtain a more comprehensive understanding of its secretome, we aimed to sequence and assemble the entire H . vastatrix genome using two next-generation sequencing platforms and a hybrid assembly strategy. This resulted in a 547 Mb genome of H . vastatrix race XXXIII (Hv33), with 13,364 predicted genes that encode 13,034 putative proteins with transcriptomic support. Based on this proteome, 615 proteins contain putative secretion peptides, and lack transmembrane domains. From this putative secretome, 111 proteins were identified as candidate effectors (EHv33) unique to H . vastatrix , and a subset consisting of 17 EHv33 genes was selected for a temporal gene expression analysis during infection. Five genes were significantly induced early during an incompatible interaction, indicating their potential role as pre-haustorial effectors possibly recognized by the resistant coffee genotype. Another nine genes were significantly induced after haustorium formation in the compatible interaction. Overall, we suggest that this fungus is able to selectively mount its survival strategy with effectors that depend on the host genotype involved in the infection process.

Porcine circovirus 3 (PCV3) is an emerging virus that was identified in the United States in 2016. Since its first identification, PCV3 has been identified in Brazil, China, United States, Poland, and Republic of Korea. In this study, we used molecular phylogenetic analysis of available sequences to address questions surrounding the emergence of PCV3 in porcine world industry. Our data indicate that PCV3 did not emerge through recombination events among currently known circoviruses and that its speciation is not a recent evolutionary event. The most common recent ancestor analysis suggests that PCV3 lineages have emerged over the past 50 years. PCV3 is not genetically closely related with other Porcine circovirus and it has been evolving undetected for some time in swine and probably in bovine population. We also found groups of genetically related isolates of PCV3 originated from different countries that may be associated with dispersal routes, suggesting that PCV3 has already been circulating in pig-producing countries for some time before its first detection.

Bacteria of the genus Desulfovibrio belong to the group of Sulphate Reducing Bacteria (SRB). SRB generate significant liabilities in the petroleum industry, mainly due to their ability to microbiologically induce corrosion, biofilm formation and H2S production. Bacteriophages are an alternative control method for SRB, whose information for this group of bacteria however, is scarce. The present study developed a workflow for the identification of complete prophages in Desulfovibrio. Poly-lysogenesis was shown to be common in Desulfovibrio. In the 47 genomes analyzed 53 complete prophages were identified. These were classified within the order Caudovirales, with 69.82% belonging to the Myoviridade family. More than half the prophages identified have genes coding for lysozyme or holin. Four of the analyzed bacterial genomes present prophages with identity above 50% in the same strain, whose comparative analysis demonstrated the existence of colinearity between the sequences. Of the 17 closed bacterial genomes analyzed, 6 have the CRISPR-Cas system classified as inactive. The identification of bacterial poly-lysogeny, the proximity between the complete prophages and the possible inactivity of the CRISPR-Cas in closed bacterial genomes analyzed allowed the choice of poly-lysogenic strains with prophages belonging to the Myoviridae family for the isolation of prophages and testing of related strains for subsequent studies.

Porcine circovirus-2 (PCV-2) is an emerging virus associated with a number of different syndromes in pigs known as Porcine Circovirus Associated Diseases (PCVAD). Since its identification and characterization in the early 1990s, PCV-2 has achieved a worldwide distribution, becoming endemic in most pig-producing countries, and is currently considered as the main cause of losses on pig farms. In this study, we analyzed the main routes of the spread of PCV-2 between pig-producing countries using phylogenetic and phylogeographical approaches. A search for PCV-2 genome sequences in GenBank was performed, and the 420 PCV-2 sequences obtained were grouped into haplotypes (group of sequences that showed 100% identity), based on the infinite sites model of genome evolution. A phylogenetic hypothesis was inferred by Bayesian Inference for the classification of viral strains and a haplotype network was constructed by Median Joining to predict the geographical distribution of and genealogical relationships between haplotypes. In order to establish an epidemiological and economic context in these analyses, we considered all information about PCV-2 sequences available in GenBank, including papers published on viral isolation, and live pig trading statistics available on the UN Comtrade database (http://comtrade.un.org/). In these analyses, we identified a strong correlation between the means of PCV-2 dispersal predicted by the haplotype network and the statistics on the international trading of live pigs. This correlation provides a new perspective on the epidemiology of PCV-2, highlighting the importance of the movement of animals around the world in the emergence of new pathogens, and showing the need for effective sanitary barriers when trading live animals.

Ki-1/57 (HABP4) and CGI-55 (SERBP1) are regulatory proteins and paralogs with 40.7% amino acid sequence identity and 67.4% similarity. Functionally, they have been implicated in the regulation of gene expression on both the transcriptional and mRNA metabolism levels. A link with tumorigenesis is suggested, since both paralogs show altered expression levels in tumor cells and the Ki-1/57 gene is found in a region of chromosome 9q that represents a haplotype for familiar colon cancer. However, the target genes regulated by Ki-1/57 and CGI-55 are unknown. Here, we analyzed the alterations of the global transcriptome profile after Ki-1/57 or CGI-55 overexpression in HEK293T cells by DNA microchip technology. We were able to identify 363 or 190 down-regulated and 50 or 27 up-regulated genes for Ki-1/57 and CGI-55, respectively, of which 20 were shared between both proteins. Expression levels of selected genes were confirmed by qRT-PCR both after protein overexpression and siRNA knockdown. The majority of the genes with altered expression were associated to proliferation, apoptosis and cell cycle control processes, prompting us to further explore these contexts experimentally. We observed that overexpression of Ki-1/57 or CGI-55 results in reduced cell proliferation, mainly due to a G1 phase arrest, whereas siRNA knockdown of CGI-55 caused an increase in proliferation. In the case of Ki-1/57 overexpression, we found protection from apoptosis after treatment with the ER-stress inducer thapsigargin. Together, our data give important new insights that may help to explain these proteins putative involvement in tumorigenic events.