Analysis of the dihydrofolate reductase-thymidylate synthase gene sequences in Plasmodium vivax field isolates that failed chloroquine treatment

<p>Abstract</p> <p>Background</p> <p>To use pyrimethamine as an alternative anti-malarial drug for chloroquine-resistant malaria parasites, it was necessary to determine the enzyme's genetic variation in dihydrofolate reductase-thymidylate syntase (DHFR-TS) among Korean strains.</p> <p>Methods</p> <p>Genetic variation of <it>dhfr-ts </it>genes of <it>Plasmodium vivax </it>clinical isolates from patients who did not respond to drug treatment (<it>n </it>= 11) in Korea were analysed. The genes were amplified using the polymerase chain reaction (PCR) with genomic DNA as a template.</p> <p>Results</p> <p>Sequence analysis showed that the open reading frame (ORF) of 1,857 nucleotides encoded a deduced protein of 618 amino acids (aa). Alignment with the DHFR-TS genes of other malaria parasites showed that a 231-residue DHFR domain and a 286-residue TS domain were seperated by a 101-aa linker region. This ORF shows 98.7% homology with the <it>P. vivax </it>Sal I strain (XM001615032) in the DHFR domain, 100% in the linker region and 99% in the TS domain. Comparison of the DHFR sequences from pyrimethamine-sensitive and pyrimethamine-resistant <it>P. vivax </it>isolates revealed that nine isolates belonged to the sensitive strain, whereas two isolates met the criteria for resistance. In these two isolates, the amino acid at position 117 is changed from serine to asparagine (S117N). Additionally, all Korean isolates showed a deletion mutant of THGGDN in short tandem repetitive sequences between 88 and 106 amino acid.</p> <p>Conclusions</p> <p>These results suggest that sequence variations in the DHFR-TS represent the prevalence of antifolate-resistant <it>P. vivax </it>in Korea. Two of 11 isolates have the Ser to Asn mutation in codon 117, which is the major determinant of pyrimethamine resistance in <it>P. vivax</it>. Therefore, the introduction of pyrimethamine for the treatment of chloroquine-resistant vivax malaria as alternative drug in Korea should be seriously considered.</p

Cloning and characterization of Plasmodium vivax serine hydroxymethyltransferase.

Serine hydroxymethyltransferase (SHMT), which catalyzes the reversible reaction of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate, is one of the three enzymes in dTMP synthesis pathway that is highly active during cell division and has been proposed as a potential chemotherapeutic target in infectious diseases and cancer. This is the first study to describe nucleotide and amino acid sequences of SHMT from the malaria parasite Plasmodium vivax. Sequencing of 12 P. vivax isolates revealed limited polymorphisms in 3 noncoding regions. Its biological function is also reported

Novel DNA Markers for Identification of Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, an important disease in the pig industry. Accurate and sensitive diagnostics such as DNA-based diagnostics are essential for preventing or responding to an outbreak. The specificity of DNA-based diagnostics depends on species-specific markers. Previously, an insertion element was found within an A. pleuropneumoniae-specific gene commonly used for A. pleuropneumoniae detection, prompting the need for additional species-specific markers. Herein, 12 marker candidates highly conserved (99 - 100% identity) among 34 A. pleuropneumoniae genomes (covering 13 serovars) were identified to be A. pleuropneumoniae-specific in silico, as these sequences are distinct from 30 genomes of 13 other Actinobacillus and problematic [Actinobacillus] species and more than 1700 genomes of other bacteria in the Pasteurellaceae family. Five marker candidates are within the apxIVA gene, a known A. pleuropneumoniae-specific gene, validating our in silico marker discovery method. Seven other A. pleuropneumoniae-specific marker candidates within the eamA, nusG, sppA, xerD, ybbN, ycfL, and ychJ genes were validated by polymerase chain reaction (PCR) to be specific to 129 isolates of A. pleuropneumoniae (covering all 19 serovars), but not to four closely related Actinobacillus species, four [Actinobacillus] species, or seven other bacterial species. This is the first study to identify A. pleuropneumoniae-specific markers through genome mining. Seven novel A. pleuropneumoniae-specific DNA markers were identified by a combination of in silico and molecular methods and can serve as additional or alternative targets for A. pleuropneumoniae diagnostics, potentially leading to better control of the disease. IMPORTANCE Species-specific markers are crucial for infectious disease diagnostics. Mutations within a marker sequence can lead to false-negative results, inappropriate treatment, and economic loss. The availability of several species-specific markers is therefore desirable. In this study, 12 DNA markers specific to A. pleuropneumoniae, a pig pathogen, were simultaneously identified. Five marker candidates are within a known A. pleuropneumoniae-specific gene. Seven novel markers can be used as additional targets in DNA-based diagnostics, which in turn can expedite disease diagnosis, assist farm management, and lead to better animal health and food security. The marker discovery strategy outlined herein requires less time, effort, and cost, and results in more markers compared with conventional methods. Identification of species-specific markers of other pathogens and corresponding infectious disease diagnostics are possible, conceivably improving health care and the economy

Financiamento de rodovias por meio da cobrança de pedágio : uma avaliação da Empresa Gaúcha de Rodovias

O presente trabalho discorre a respeito dos métodos de financiamento empregados em rodovias, com maior enfoque na cobrança de pedágio, e faz uma análise da eficiência econômica e operacional da Empresa Gaúcha de Rodovias (EGR), uma empresa estatal que atua na cobrança de pedágio em rodovias do estado do Rio Grande do Sul. Após uma revisão da literatura sobre os métodos de financiamento de rodovias e de um breve panorama da situação atual das rodovias no Brasil, foram elencadas empresas privadas concessionárias de rodovias para servirem de base para comparações e futura análise do desempenho da EGR. Foram estudados, além da EGR, os grupos Triunfo e EcoRodovias, representados pelos seus setores de concessão de rodovias, nos anos de 2013 e 2014. As comparações e análises foram baseadas em indicadores de desempenho desenvolvidos ao longo do trabalho, os quais abrangeram elementos como número de acidentes, número de atendimentos, extensão dos trechos administrados, volume de tráfego, custo dos serviços prestados, despesas operacionais das empresas e volume total arrecadado. A definição dos indicadores foi limitada pela disponibilidade de dados, os quais foram coletados a partir de pesquisa documental realizada junto às empresas e à Agência Nacional de Transportes Terrestres (ANTT). Os indicadores apontaram que as rodovias administradas pela EGR, quando comparadas com as das demais empresas estudadas, apresentam bom desempenho no quesito segurança. Entretanto, todos os demais indicadores foram desfavoráveis à empresa, concluindo-se, por fim, que a Empresa Gaúcha de Rodovias apresenta, de um modo geral, desempenhos econômico e operacional inferiores quando comparada com empresas privadas concessionárias de rodovias de porte semelhante

Molecular characterization of dihydrofolate reductase in relation to antifolate resistance in Plasmodium vivax.

The genes encoding the wild-type and six (five single and one double) mutant dihydrofolate reductase (DHFR) domains of the human malaria parasite, Plasmodium vivax (Pv), were cloned and expressed in Escherichia coli. The catalytic activities and the kinetic parameters of the purified recombinant wild-type and the mutant PvDHFRs were determined. Generally, all the PvDHFR mutants yielded enzymes with poorer catalytic activities when compared to the wild type enzyme. The widely used antifolates, pyrimethamine and cycloguanil, were effective inhibitors of the wild-type PvDHFR, but were approximately 60 to &gt;4000 times less active against the mutant enzymes. In contrast to the analogous S108N mutation of Plasmodium falciparum DHFR (PfDHFR), the single S117N mutation in PvDHFR conferred approximately 4000- and approximately 1600-fold increased resistance to pyrimethamine and cycloguanil, respectively, compared to the wild-type PvDHFR. The S58R+S117N double mutant PvDHFR was 10- to 25-fold less resistant than the S117N mutant to the inhibitors, but also exhibited higher kcat/Km value than the single mutant. The antifolate WR99210 was equally effective against both the wild-type and SP21 (S58R+S117N) mutant DHFRs, but was much less effective against some of the single mutants. Data on kinetic parameters and inhibitory constant suggest that the wild-type P. vivax is susceptible to antimalarial antifolates and that point mutations in the DHFR domain of P. vivax are responsible for antifolate resistance

Crystal structure of dihydrofolate reductase from Plasmodium vivax: Pyrimethamine displacement linked with mutation-induced resistance

Pyrimethamine (Pyr) targets dihydrofolate reductase of Plasmodium vivax (PvDHFR) as well as other malarial parasites, but its use as antimalarial is hampered by the widespread high resistance. Comparison of the crystal structures of PvDHFR from wild-type and the Pyr-resistant (SP21, Ser-58 → Arg + Ser-117 → Asn) strain as complexes with NADPH and Pyr or its analog lacking p-Cl (Pyr20) clearly shows that the steric conflict arising from the side chain of Asn-117 in the mutant enzyme, accompanied by the loss of binding to Ser-120, is mainly responsible for the reduction in binding of Pyr. Pyr20 still effectively inhibits both the wild-type and SP21 proteins, and the x-ray structures of these complexes show how Pyr20 fits into both active sites without steric strain. These structural insights suggest a general approach for developing new generations of antimalarial DHFR inhibitors that, by only occupying substrate space of the active site, would retain binding affinity with the mutant enzymes