Fast microwave-assisted pyrolysis (fMAP) in the presence of a microwave absorbent (SiC) and catalyst (HZSM-5) was tested on a Chlorella sp. strain and on a Nannochloropsis strain. The liquid products were characterized, and the effects of temperature and catalyst:biomass ratio were analyzed. For Chlorella sp., a temperature of 550 °C, with no catalyst were the optimal conditions, resulting in a maximum bio-oil yield of 57 wt.%. For Nannochloropsis, a temperature of 500 °C, with 0.5 of catalyst ratio were shown to be the optimal condition, resulting in a maximum bio-oil yield of 59 wt.%. These results show that the use of microwave absorbents in fMAP increased bio-oil yields and quality, and it is a promising technology to improve the commercial application and economic outlook of microwave pyrolysis technology. Additionally, the use of a different catalyst needs to be considered to improve the bio-oil characteristics.
Desulfovibrio gigas is a model organism of sulfate-reducing bacteria of which energy metabolism and stress response have been extensively studied. The complete genomic context of this organism was however, not yet available. The sequencing of the D. gigas genome provides insights into the integrated network of energy conserving complexes and structures present in this bacterium. Comparison with genomes of other Desulfovibrio spp. reveals the presence of two different CRISPR/Cas systems in D. gigas. Phylogenetic analysis using conserved protein sequences (encoded by rpoB and gyrB) indicates two main groups of Desulfovibrio spp, being D. gigas more closely related to D. vulgaris and D. desulfuricans strains. Gene duplications were found such as those encoding fumarate reductase, formate dehydrogenase, and superoxide dismutase. Complexes not yet described within Desulfovibrio genus were identified: Mnh complex, a v-type ATP-synthase as well as genes encoding the MinCDE system that could be responsible for the larger size of D. gigas when compared to other members of the genus. A low number of hydrogenases and the absence of the codh/acs and pfl genes, both present in D. vulgaris strains, indicate that intermediate cycling mechanisms may contribute substantially less to the energy gain in D. gigas compared to other Desulfovibrio spp. This might be compensated by the presence of other unique genomic arrangements of complexes such as the Rnf and the Hdr/Flox, or by the presence of NAD(P)H related complexes, like the Nuo, NfnAB or Mnh.
The Binary (Bin) toxin from the entomopathogenic bacterium Lysinibacillus sphaericus acts on larvae of the culicid Culex quinquefasciatus through its binding to Cqm1, a midgut-bound ?-glucosidase. Specific binding by the BinB subunit to the Cqm1 receptor is essential for toxicity however the toxin is unable to bind to the Cqm1 ortholog from the refractory species Aedes aegypti (Aam1). Here, to investigate the molecular basis for the interaction between Cqm1 and BinB, recombinant Cqm1 and Aam1 were first expressed as soluble forms in Sf9 cells. The two proteins were found to display the same glycosilation patterns and BinB binding properties as the native ?-glucosidases. Chimeric constructs were then generated through the exchange of reciprocal fragments between the corresponding cqm1 and aam1 cDNAs. Subsequent expression and binding experiments defined a Cqm1 segment encompassing residues S129 and A312 as critical for the interaction with BinB. Through site directed mutagenesis experiments, replacing specific sets of residues from Cqm1 with those of Aam1, the 159GG160 doublet was required for this interaction. Molecular modeling mapped these residues to an exposed loop within the Cqm1's structure, compatible with a target site for BinB and providing a possible explanation for its lack of binding to Aam1.
The potential use of CRISPR loci genotyping to elucidate population dynamics and microevolution of 146 Yersinia pestis strains from different biovars and locations was investigated in this work. The majority of strains from the Orientalis biovar presented specific spacer arrays, allowing for the establishment of a CRISPR signature for their respective isolates. Twenty-one new spacers were found in the Y. pestis strains from plague foci in Brazil. Ninety-three (64%) strains were grouped in the G1 genotype, whereas the others were distributed in 35 genotypes. This study allowed observing a microevolutionary process in a group of Y. pestis isolated from Brazil. We also identified specific genotypes of Y. pestis that were important for the establishment of the bacteria in plague foci in Brazil. The data have provided supporting evidence for the diversity and dynamics of CRISPR loci present in the genome of Y. pestis strains from plague foci in Brazil.
NorR protein was shown to be responsible for the transcriptional regulation of flavorubredoxin and its associated oxidoreductase in Escherichia coli. Since Desulfovibrio gigas has a rubredoxin:oxygen oxidoreductase (ROO) that is involved in both oxidative and nitrosative stress response, a NorR-like protein was searched in D. gigas genome. We have found two putative norR coding units in its genome. To study the role of the protein designated as NorR1-like (NorR1L) in the presence of nitrosative stress, a norR1L null mutant of D. gigas was created and a phenotypic analysis was performed under the nitrosating agent GSNO. We show that under these conditions, the growth of both D. gigas mutants ?roo and ?norR1-like is impaired. In order to confirm that D. gigas NorR1-like may play identical function as the NorR of E. coli, we have complemented the E. coli ?norR mutant strain with the norR1-like gene and have evaluated growth when nitrosative stress was imposed. The growth phenotype of E. coli ?norR mutant strain was recovered under these conditions. We also found that induction of roo gene expression is completely abolished in the norR1L mutant strain of D. gigas subjected to nitrosative stress. It is identified in ?-proteobacteria, for the first time a transcription factor that is involved in nitrosative stress response and regulates the rd-roo gene expression.
Campylobacter fetus subsp. venerealis is the etiologic agent of bovine genital campylobacteriosis, a sexually transmitted disease of cattle that is of worldwide importance. The complete sequencing and annotation of the genome of the type strain C. fetus subsp. venerealis NCTC 10354(T) are reported.
Reliable molecular markers are essential for a better understanding of the molecular epidemiology of Plasmodium vivax, which is a neglected human malaria parasite. The aim of this study was to analyze the genetic diversity of P. vivax isolates from the Brazilian Amazon using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the highly polymorphic merozoite surface protein-3alpha (PvMSP-3?) gene. To accomplish this, 60 isolates of P. vivax from different endemic areas in the Brazilian Amazon were collected. The PvMSP-3? gene was amplified by nested-PCR. Three major types of the PvMSP-3? locus were detected at different frequencies: type A (68%), B (15%) and C (17%). A single sample showed two PCR fragments, which corresponded to infection with types A and C. PCR-RFLP analysis using the HhaI restriction enzyme for 52 isolates clearly identified 11 haplotypes, eight of which were from type A, two from type B and only one from type C. Seven other isolates did not show a clear pattern using PCR-RFLP. This result might be due to multiple clone infections. This study showed a high diversity of the PvMSP-3? gene among P. vivax isolates from the Brazilian Amazon, but also indicated that the detection performance of PCR-RFLP of the PvMSP-3? gene may not be sufficient to detect multiple clone infections.
Few genetic markers have been described to analyze populations of Plasmodium vivax. The genetic variability of P. vivax has been analyzed mainly among isolates taken from areas ranging from hyper- to holoendemic areas. These studies of genetic variability have neglected many areas with different epidemiologic profiles. The purpose of this study was to analyze the genetic variability of P. vivax isolates from four different Brazilian Amazon areas. We chose to study the five most polymorphic tandem repeats (TRs) identified so far. All TRs studied were polymorphic in at least one studied population, with a modal allele at nearly all loci. Expected heterozygosity ranged from 0.462 to 0.666 and did not correlate with the repeat array length. The genetic distances among the populations varied from 0.027 to 0.241, and did not correlate with their geographic separation. Tandem repeats identified in P. vivax isolates failed to allow geographic clustering.
The Trypanosomatids parasites Leishmania braziliensis, Leishmania major and Leishmania infantum are important human pathogens. Despite of years of study and genome availability, effective vaccine has not been developed yet, and the chemotherapy is highly toxic. Therefore, it is clear just interdisciplinary integrated studies will have success in trying to search new targets for developing of vaccines and drugs. An essential part of this rationale is related to protein-protein interaction network (PPI) study which can provide a better understanding of complex protein interactions in biological system. Thus, we modeled PPIs for Trypanosomatids through computational methods using sequence comparison against public database of protein or domain interaction for interaction prediction (Interolog Mapping) and developed a dedicated combined system score to address the predictions robustness. The confidence evaluation of network prediction approach was addressed using gold standard positive and negative datasets and the AUC value obtained was 0.94. As result, 39,420, 43,531 and 45,235 interactions were predicted for L. braziliensis, L. major and L. infantum respectively. For each predicted network the top 20 proteins were ranked by MCC topological index. In addition, information related with immunological potential, degree of protein sequence conservation among orthologs and degree of identity compared to proteins of potential parasite hosts was integrated. This information integration provides a better understanding and usefulness of the predicted networks that can be valuable to select new potential biological targets for drug and vaccine development. Network modularity which is a key when one is interested in destabilizing the PPIs for drug or vaccine purposes along with multiple alignments of the predicted PPIs were performed revealing patterns associated with protein turnover. In addition, around 50% of hypothetical protein present in the networks received some degree of functional annotation which represents an important contribution since approximately 60% of Leishmania predicted proteomes has no predicted function.
Plasmodium vivax infection is characterized by a dormant hepatic stage, the hypnozoite that is activated at varying periods of time after clearance of the primary acute blood-stage, resulting in relapse. Differentiation between treatment failure and new infections requires characterization of initial infections, relapses, and clone multiplicity in vivax malaria infections.
Epitope prediction using computational methods represents one of the most promising approaches to vaccine development. Reduction of time, cost, and the availability of completely sequenced genomes are key points and highly motivating regarding the use of reverse vaccinology. Parasites of genus Leishmania are widely spread and they are the etiologic agents of leishmaniasis. Currently, there is no efficient vaccine against this pathogen and the drug treatment is highly toxic. The lack of sufficiently large datasets of experimentally validated parasites epitopes represents a serious limitation, especially for trypanomatids genomes. In this work we highlight the predictive performances of several algorithms that were evaluated through the development of a MySQL database built with the purpose of: a) evaluating individual algorithms prediction performances and their combination for CD8+ T cell epitopes, B-cell epitopes and subcellular localization by means of AUC (Area Under Curve) performance and a threshold dependent method that employs a confusion matrix; b) integrating data from experimentally validated and in silico predicted epitopes; and c) integrating the subcellular localization predictions and experimental data. NetCTL, NetMHC, BepiPred, BCPred12, and AAP12 algorithms were used for in silico epitope prediction and WoLF PSORT, Sigcleave and TargetP for in silico subcellular localization prediction against trypanosomatid genomes.
Signal peptide is one of the most important motifs involved in protein trafficking and it ultimately influences protein function. Considering the expected functional conservation among orthologs it was hypothesized that divergence in signal peptides within orthologous groups is mainly due to N-terminal protein sequence misannotation. Thus, discrepancies in signal peptide prediction of orthologous proteins were used to identify misannotated proteins in five Plasmodium species.
Hexose transporters (HT) are membrane proteins involved in the uptake of energy-supplying glucose and other hexoses into the cell. Previous studies employing the Differential Display technique have shown that the transcription level of the HT gene from T. cruzi (TcrHT) is higher in an in vitro-induced benznidazole (BZ)-resistant population of the parasite (17 LER) than in its susceptible counterpart (17 WTS).
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