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BACKGROUND: Plasmodium vivax (P. vivax) is the dominant Plasmodium spp. causing the disease malaria in low-transmission regions outside of Africa. These regions often feature high proportions of asymptomatic patients with sub-microscopic parasitaemia and relapses. Naturally acquired antibody responses are induced after Plasmodium infection, providing partial protection against high parasitaemia and clinical episodes. However, previous work has failed to address the presence and maintenance of such antibody responses to P. vivax particularly in low-transmission regions. METHODS: We followed 34 patients in western Thailand after symptomatic P. vivax infections to monitor antibody kinetics over 9 months, during which no recurrent infections occurred. We assessed total IgG, IgG subclass and IgM levels to up to 52 P. vivax proteins every 2-4 weeks using a multiplexed Luminex® assay and identified protein-specific variation in antibody longevity. Mathematical modelling was used to generate the estimated half-life of antibodies, long-, and short-lived antibody-secreting cells. RESULTS: Generally, an increase in antibody level was observed within 1-week post symptomatic infection, followed by an exponential decay of different rates. We observed mostly IgG1 dominance and IgG3 sub-dominance in this population. IgM responses followed similar kinetic patterns to IgG, with some proteins unexpectedly inducing long-lived IgM responses. We also monitored antibody responses against 27 IgG-immunogenic antigens in 30 asymptomatic individuals from a similar region. Our results demonstrate that most antigens induced robust and long-lived total IgG responses following asymptomatic infections in the absence of (detected) boosting infections. CONCLUSIONS: Our work provides new insights into the development and maintenance of naturally acquired immunity to P. vivax and will guide the potential use of serology to indicate immune status and/or identify populations at risk.
Assuntos
Malária Vivax , Malária , Anticorpos Antiprotozoários , Antígenos de Protozoários , Humanos , Cinética , Malária Vivax/epidemiologia , Plasmodium vivax , Proteínas de Protozoários , Tailândia/epidemiologiaRESUMO
Mammalian promoters can be separated into two classes, conserved TATA box-enriched promoters, which initiate at a well-defined site, and more plastic, broad and evolvable CpG-rich promoters. We have sequenced tags corresponding to several hundred thousand transcription start sites (TSSs) in the mouse and human genomes, allowing precise analysis of the sequence architecture and evolution of distinct promoter classes. Different tissues and families of genes differentially use distinct types of promoters. Our tagging methods allow quantitative analysis of promoter usage in different tissues and show that differentially regulated alternative TSSs are a common feature in protein-coding genes and commonly generate alternative N termini. Among the TSSs, we identified new start sites associated with the majority of exons and with 3' UTRs. These data permit genome-scale identification of tissue-specific promoters and analysis of the cis-acting elements associated with them.
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Evolução Molecular , Regiões Promotoras Genéticas , Regiões 3' não Traduzidas , Animais , Sequência de Bases , DNA , Genoma , Proteoma , TATA BoxRESUMO
BACKGROUND: DNA methylation in promoters is closely linked to downstream gene repression. However, whether DNA methylation is a cause or a consequence of gene repression remains an open question. If it is a cause, then DNA methylation may affect the affinity of transcription factors (TFs) for their binding sites (TFBSs). If it is a consequence, then gene repression caused by chromatin modification may be stabilized by DNA methylation. Until now, these two possibilities have been supported only by non-systematic evidence and they have not been tested on a wide range of TFs. An average promoter methylation is usually used in studies, whereas recent results suggested that methylation of individual cytosines can also be important. RESULTS: We found that the methylation profiles of 16.6% of cytosines and the expression profiles of neighboring transcriptional start sites (TSSs) were significantly negatively correlated. We called the CpGs corresponding to such cytosines "traffic lights". We observed a strong selection against CpG "traffic lights" within TFBSs. The negative selection was stronger for transcriptional repressors as compared with transcriptional activators or multifunctional TFs as well as for core TFBS positions as compared with flanking TFBS positions. CONCLUSIONS: Our results indicate that direct and selective methylation of certain TFBS that prevents TF binding is restricted to special cases and cannot be considered as a general regulatory mechanism of transcription.
Assuntos
Citosina/metabolismo , Metilação de DNA , Fatores de Transcrição/metabolismo , Algoritmos , Sítios de Ligação , Biologia Computacional , Ilhas de CpG , Citosina/química , Humanos , Regiões Promotoras Genéticas , Fatores de Transcrição/química , Sítio de Iniciação de TranscriçãoRESUMO
BACKGROUND: Analyzing the RNA pool or transcription start sites requires effective means to convert RNA into cDNA libraries for digital expression counting. With current high-speed sequencers, it is necessary to flank the cDNAs with specific adapters. Adding template-switching oligonucleotides to reverse transcription reactions is the most commonly used approach when working with very small quantities of RNA even from single cells. RESULTS: Here we compared the performance of DNA-RNA, DNA-LNA and DNA oligonucleotides in template-switching during nanoCAGE library preparation. Test libraries from rat muscle and HeLa cell RNA were prepared in technical triplicates and sequenced for comparison of the gene coverage and distribution of the reads within transcripts. The DNA-RNA oligonucleotide showed the highest specificity for capped 5' ends of mRNA, whereas the DNA-LNA provided similar gene coverage with more reads falling within exons. CONCLUSIONS: While confirming the cap-specific preference of DNA-RNA oligonucleotides in template-switching reactions, our data indicate that DNA-LNA hybrid oligonucleotides could potentially find other applications in random RNA sequencing.
Assuntos
Biblioteca Gênica , Hibridização de Ácido Nucleico/genética , Oligonucleotídeos/metabolismo , RNA/metabolismo , Análise de Sequência de RNA/métodos , Moldes Genéticos , Animais , Genoma/genética , Células HeLa , Humanos , RatosRESUMO
Nucleic acid oligonucleotides are widely used in hybridization experiments for specific detection of complementary nucleic acid sequences. For design and application of oligonucleotides, an understanding of their thermodynamic properties is essential. Recently, exciton-controlled hybridization-sensitive fluorescent oligonucleotides (ECHOs) were developed as uniquely labeled DNA oligomers containing commonly one thymidine having two covalently linked thiazole orange dye moieties. The fluorescent signal of an ECHO is strictly hybridization-controlled, where the dye moieties have to intercalate into double-stranded DNA for signal generation. Here we analyzed the hybridization thermodynamics of ECHO/DNA duplexes, and thermodynamic parameters were obtained from melting curves of 64 ECHO/DNA duplexes measured by ultraviolet absorbance and fluorescence. Both methods demonstrated a substantial increase in duplex stability (ΔΔG°(37) ~ -2.6 ± 0.7 kcal mol(-1)) compared to that of DNA/DNA duplexes of the same sequence. With the exception of T·G mismatches, this increased stability was mostly unaffected by other mismatches in the position opposite the labeled nucleotide. A nearest neighbor model was constructed for predicting thermodynamic parameters for duplex stability. Evaluation of the nearest neighbor parameters by cross validation tests showed higher predictive reliability for the fluorescence-based than the absorbance-based parameters. Using our experimental data, a tool for predicting the thermodynamics of formation of ECHO/DNA duplexes was developed that is freely available at http://genome.gsc.riken.jp/echo/thermodynamics/. It provides reliable thermodynamic data for using the unique features of ECHOs in fluorescence-based experiments.
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Benzotiazóis/química , DNA/química , Quinolinas/química , Timidina/química , Pareamento Incorreto de Bases , Sequência de Bases , DNA/genética , Desenho de Fármacos , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Termodinâmica , Temperatura de TransiçãoRESUMO
Next-generation sequencing is excellently suited to evaluate the abundance of mRNAs to study gene expression. Here we compare two alternative technologies, cap analysis of gene expression (CAGE) and serial analysis of gene expression (SAGE), for the same RNA samples. Along with quantifying gene expression levels, CAGE can be used to identify tissue-specific transcription start sites, while SAGE monitors 3'-end usage. We used both methods to get more insight into the transcriptional control of myogenesis, studying differential gene expression in differentiated and proliferating C2C12 myoblast cells with statistical evaluation of reproducibility and differential gene expression. Both CAGE and SAGE provided highly reproducible data (Pearson's correlations >0.92 among biological triplicates). With both methods we found around 10,000 genes expressed at levels >2 transcripts per million (approximately 0.3 copies per cell), with an overlap of 86%. We identified 4304 and 3846 genes differentially expressed between proliferating and differentiated C2C12 cells by CAGE and SAGE, respectively, with an overlap of 2144. We identified 196 novel regulatory regions with preferential use in proliferating or differentiated cells. Next-generation sequencing of CAGE and SAGE libraries provides consistent expression levels and can enrich current genome annotations with tissue-specific promoters and alternative 3'-UTR usage.
Assuntos
Perfilação da Expressão Gênica/métodos , Mioblastos/metabolismo , Análise de Sequência de RNA , Regiões 3' não Traduzidas , Animais , Linhagem Celular , Camundongos , Modelos Biológicos , Desenvolvimento Muscular/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reprodutibilidade dos Testes , Alinhamento de Sequência , Sítio de Iniciação de TranscriçãoRESUMO
A more sensitive surveillance tool is needed to identify Plasmodium vivax infections for treatment and to accelerate malaria elimination efforts. To address this challenge, our laboratory has developed an eight-antigen panel that detects total IgG as serological markers of P. vivax exposure within the prior 9 months. The value of these markers has been established for use in areas with low transmission. In moderate-high transmission areas, there is evidence that total IgG is more long-lived than in areas with low transmission, resulting in poorer performance of these markers in these settings. Antibodies that are shorter-lived may be better markers of recent infection for use in moderate-high transmission areas. Using a multiplex assay, the antibody temporal kinetics of total IgG, IgG1, IgG3, and IgM against 29 P. vivax antigens were measured over 36 weeks following asymptomatic P. vivax infection in Papua New Guinean children (n = 31), from an area with moderate-high transmission intensity. IgG3 declined faster to background than total IgG, IgG1, and IgM. Based on these kinetics, IgG3 performance was then assessed for classifying recent exposure in a cohort of Peruvian individuals (n = 590; age 3-85 years) from an area of moderate transmission intensity. Using antibody responses against individual antigens, the highest performance of IgG3 in classifying recent P. vivax infections in the prior 9 months was to one of the Pv-fam-a proteins assessed (PVX_125728) (AUC = 0.764). Surprisingly, total IgG was overall a better marker of recent P. vivax infection, with the highest individual classification performance to RBP2b1986-2653 (PVX_094255) (AUC = 0.838). To understand the acquisition of IgG3 in this Peruvian cohort, relevant epidemiological factors were explored using a regression model. IgG3 levels were positively associated with increasing age, living in an area with (relatively) higher transmission intensity, and having three or more PCR-detected blood-stage P. vivax infections within the prior 13 months. Overall, we found that IgG3 did not have high accuracy for detecting recent exposure to P. vivax in the Peruvian cohort, with our data suggesting that this is due to the high levels of prior exposure required to acquire high IgG3 antibody levels.
Assuntos
Malária Falciparum , Malária Vivax , Malária , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antiprotozoários , Infecções Assintomáticas , Biomarcadores , Criança , Pré-Escolar , Humanos , Imunoglobulina G , Imunoglobulina M , Malária Vivax/diagnóstico , Pessoa de Meia-Idade , Plasmodium falciparum , Plasmodium vivax , Adulto JovemRESUMO
Serological markers are a promising tool for surveillance and targeted interventions for Plasmodium vivax malaria. P. vivax is closely related to the zoonotic parasite P. knowlesi, which also infects humans. P. vivax and P. knowlesi are co-endemic across much of South East Asia, making it important to design serological markers that minimize cross-reactivity in this region. To determine the degree of IgG cross-reactivity against a panel of P. vivax serological markers, we assayed samples from human patients with P. knowlesi malaria. IgG antibody reactivity is high against P. vivax proteins with high sequence identity with their P. knowlesi ortholog. IgG reactivity peaks at 7 days post-P. knowlesi infection and is short-lived, with minimal responses 1 year post-infection. We designed a panel of eight P. vivax proteins with low levels of cross-reactivity with P. knowlesi. This panel can accurately classify recent P. vivax infections while reducing misclassification of recent P. knowlesi infections.
Assuntos
Malária Vivax , Malária , Plasmodium knowlesi , Humanos , Imunoglobulina G , Malária/diagnóstico , Malária Vivax/diagnóstico , Plasmodium vivaxRESUMO
Cell-free protein synthesis (CFPS) systems are gaining more importance as universal tools for basic research, applied sciences, and product development with new technologies emerging for their application. Huge progress was made in the field of synthetic biology using CFPS to develop new proteins for technical applications and therapy. Out of the available CFPS systems, wheat germ cell-free protein synthesis (WG-CFPS) merges the highest yields with the use of a eukaryotic ribosome, making it an excellent approach for the synthesis of complex eukaryotic proteins including, for example, protein complexes and membrane proteins. Separating the translation reaction from other cellular processes, CFPS offers a flexible means to adapt translation reactions to protein needs. There is a large demand for such potent, easy-to-use, rapid protein expression systems, which are optimally serving protein requirements to drive biochemical and structural biology research. We summarize here a general workflow for a wheat germ system providing examples from the literature, as well as applications used for our own studies in structural biology. With this review, we want to highlight the tremendous potential of the rapidly evolving and highly versatile CFPS systems, making them more widely used as common tools to recombinantly prepare particularly challenging recombinant eukaryotic proteins.
RESUMO
To achieve malaria elimination, new tools are required to explicitly target Plasmodium vivax. Recently, a novel panel of P. vivax proteins were identified and validated as serological markers for detecting recent exposure to P. vivax within the last 9 months. In order to improve the sensitivity and specificity of these markers, immunoglobulin M (IgM) in addition to immunoglobulin G (IgG) antibody responses were compared with a down-selected panel of 20 P. vivax proteins. IgM was tested using archival plasma samples from observational cohort studies conducted in malaria-endemic regions of Thailand and Brazil. IgM responses to these proteins generally had poorer classification performance than IgG.
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Malaria transmission-blocking vaccines (TBVs) prevent the completion of the developmental lifecycle of malarial parasites within the mosquito vector, effectively blocking subsequent infections. The mosquito midgut protein Anopheline alanyl aminopeptidase N (AnAPN1) is the leading, mosquito-based TBV antigen. Structure-function studies identified two Class II epitopes that can induce potent transmission-blocking (T-B) antibodies, informing the design of the next-generation AnAPN1. Here, we functionally screened new immunogens and down-selected to the UF6b construct that has two glycine-linked copies of the T-B epitopes. We then established a process for manufacturing UF6b and evaluated in outbred female CD1 mice the immunogenicity of the preclinical product with the human-safe adjuvant Glucopyranosyl Lipid Adjuvant in a liposomal formulation with saponin QS21 (GLA-LSQ). UF6b:GLA-LSQ effectively immunofocused the humoral response to one of the key T-B epitopes resulting in potent T-B activity, underscoring UF6b as a prime TBV candidate to aid in malaria elimination and eradication efforts.
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Thailand is aiming for malaria elimination by the year 2030. However, the high proportion of asymptomatic infections and the presence of the hidden hypnozoite stage of Plasmodium vivax are impeding these efforts. We hypothesized that a validated surveillance tool utilizing serological markers of recent exposure to P. vivax infection could help to identify areas of ongoing transmission. The objective of this exploratory study was to assess the ability of P. vivax serological exposure markers to detect residual transmission "hot-spots" in Western Thailand. Total IgG levels were measured against a panel of 23 candidate P. vivax serological exposure markers using a multiplexed bead-based assay. A total of 4,255 plasma samples from a cross-sectional survey conducted in 2012 of endemic areas in the Kanchanaburi and Ratchaburi provinces were assayed. We compared IgG levels with multiple epidemiological factors that are associated with an increased risk of P. vivax infection in Thailand, including age, gender, and spatial location, as well as Plasmodium infection status itself. IgG levels to all proteins were significantly higher in the presence of a P. vivax infection (n = 144) (T-test, p < 0.0001). Overall seropositivity rates varied from 2.5% (PVX_097625, merozoite surface protein 8) to 16.8% (PVX_082670, merozoite surface protein 7), with 43% of individuals seropositive to at least 1 protein. Higher IgG levels were associated with older age (>18 years, p < 0.05) and males (17/23 proteins, p < 0.05), supporting the paradigm that men have a higher risk of infection than females in this setting. We used a Random Forests algorithm to predict which individuals had exposure to P. vivax parasites in the last 9-months, based on their IgG antibody levels to a panel of eight previously validated P. vivax proteins. Spatial clustering was observed at the village and regional level, with a moderate correlation between PCR prevalence and sero-prevalence as predicted by the algorithm. Our data provides proof-of-concept for application of such surrogate markers as evidence of recent exposure in low transmission areas. These data can be used to better identify geographical areas with asymptomatic infection burdens that can be targeted in elimination campaigns.
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Most commonly used intercalating fluorescent dyes in DNA detection are lacking any sequence specificity, whereas so-called Exciton Primers can overcome this limitation by functioning as "sequence-specific dyes." After hybridization to complementary sequences, the fluorescence of Exciton Primers provides sequence-specific signals for real-time monitoring of amplification reactions. Applied to the SmartAmp2 mutation detection process, Exciton Primers show high signal strength with low background leading to a superior specificity and sensitivity compared to SYBR Green I. Signal strength can be further enhanced using multiple dyes within one Exciton Primer or use of multiple Exciton Primers in the same amplification reaction. Here we demonstrate the use of Exciton Primers for genotyping a single nucleotide polymorphism (SNP) in the VKORC1 locus (-1639G>A) relevant for Warfarin dosing as an example for Exciton Primers mediated genotyping by SmartAmp2. The genotyping assay can use only one labeled Exciton Primer for endpoint detection, or simultaneously by real-time monitoring detect wild-type and mutant alleles in a one-tube reaction using two Exciton Primers having different dyes. Working directly from blood samples, Exciton Primer mediated genotyping by SmartAmp2 offers superior solutions for rapid point-of-care testing.
Assuntos
Primers do DNA/metabolismo , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único/genética , Benzotiazóis , Diaminas , Corantes Fluorescentes/química , Genótipo , Humanos , Oxigenases de Função Mista/genética , Compostos Orgânicos/metabolismo , Quinolinas , Vitamina K Epóxido RedutasesRESUMO
Multiplexed bead-based assays that use Luminex® xMAP® technology have become popular for measuring antibodies against proteins of interest in many fields, including malaria and more recently SARS-CoV-2/COVID-19. There are currently two formats that are widely used: non-magnetic beads or magnetic beads. Data are lacking regarding the comparability of results obtained using these two types of beads, and for assays run on different instruments. Whilst non-magnetic beads can only be run on flow-based instruments (such as the Luminex® 100/200™ or Bio-Plex® 200), magnetic beads can be run on both these and the newer MAGPIX® instruments. In this study we utilized a panel of purified recombinant Plasmodium vivax proteins and samples from malaria-endemic areas to measure P. vivax-specific IgG responses using different combinations of beads and instruments. We directly compared: i) non-magnetic versus magnetic beads run on a Bio-Plex® 200, ii) magnetic beads run on the Bio-Plex® 200 versus MAGPIX® and iii) non-magnetic beads run on a Bio-Plex® 200 versus magnetic beads run on the MAGPIX®. We also performed an external comparison of our optimized assay. We observed that IgG antibody responses, measured against our panel of P. vivax proteins, were moderately-strongly correlated in all three of our comparisons (pearson r>0.5 for 18/19 proteins), however higher amounts of protein were required for coupling to magnetic beads. Our external comparison indicated that results generated in different laboratories using the same coupled beads are also highly comparable (pearson r>0.7), particularly if a reference standard curve is used.
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Separação Celular/métodos , Imunoglobulina G/imunologia , Separação Imunomagnética/métodos , Antígenos de Protozoários/imunologia , Criança , Pré-Escolar , Feminino , Humanos , Fenômenos Magnéticos , Malária/imunologia , Malária Vivax/imunologia , Masculino , Microesferas , Papua Nova Guiné/epidemiologia , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , TecnologiaRESUMO
A major gap in the Plasmodium vivax elimination toolkit is the identification of individuals carrying clinically silent and undetectable liver-stage parasites, called hypnozoites. This study developed a panel of serological exposure markers capable of classifying individuals with recent P. vivax infections who have a high likelihood of harboring hypnozoites. We measured IgG antibody responses to 342 P. vivax proteins in longitudinal clinical cohorts conducted in Thailand and Brazil and identified candidate serological markers of exposure. Candidate markers were validated using samples from year-long observational cohorts conducted in Thailand, Brazil and the Solomon Islands and antibody responses to eight P. vivax proteins classified P. vivax infections in the previous 9 months with 80% sensitivity and specificity. Mathematical models demonstrate that a serological testing and treatment strategy could reduce P. vivax prevalence by 59-69%. These eight antibody responses can serve as a biomarker, identifying individuals who should be targeted with anti-hypnozoite therapy.
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Biomarcadores/sangue , Malária Vivax/diagnóstico , Testes Sorológicos/métodos , Adulto , Brasil/epidemiologia , Criança , Estudos de Coortes , Diagnóstico Precoce , Humanos , Imunoglobulina G/análise , Imunoglobulina G/sangue , Controle de Infecções/métodos , Estudos Longitudinais , Malária Vivax/sangue , Malária Vivax/epidemiologia , Melanesia/epidemiologia , Plasmodium vivax/fisiologia , Prevalência , Sensibilidade e Especificidade , Testes Sorológicos/normas , Tailândia/epidemiologia , Fatores de TempoRESUMO
The cloning of cDNAs, copies of cellular RNA, is one of the classical technologies in molecular biology. Over the past 30 years cDNA cloning technologies have been improved to enable the cloning of large cDNA collections, which are fundamental to today's understanding of the utilization of genetic information. With the discovery of noncoding RNAs, additional new approaches to the cloning of short RNAs have been developed. However, with the realization that much larger portions of genomes are transcribed than anticipated from genome annotations, cDNA cloning faces new challenges to uncover rare transcripts and to make the corresponding cDNAs available for functional studies. This review provides an overview on the current status of cDNA cloning and possibilities for the discovery and characterization of new RNA families.
Assuntos
Clonagem Molecular/métodos , DNA Complementar/genética , Animais , Bases de Dados de Ácidos Nucleicos , Biblioteca Gênica , Técnicas Genéticas/tendências , Vetores Genéticos , Humanos , RNA/genética , Splicing de RNARESUMO
Transcripts in all eukaryotes are characterized by the 5'-end specific cap structure in mRNAs. Cap Analysis Gene Expression or CAGE makes use of these caps to specifically obtain cDNA fragments from the 5'-end of RNA and sequences those at high throughput for transcript identification and genome-wide mapping of transcription start sites for coding and noncoding genes. Here, we provide an improved version of our nanoCAGE protocol that has been developed for preparing CAGE libraries from as little as 50 ng of total RNA within three standard working days. Key steps in library preparation have been improved over our previously published protocol to obtain libraries having a good 5'-end selection and a more equal size distribution for higher sequencing efficiency on Illumina MiSeq and HiSeq sequencers. We recommend nanoCAGE as the method of choice for transcriptome profiling projects even from limited amounts of RNA, and as the best approach for genome-wide mapping of transcription start sites within promoter regions.
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Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Capuzes de RNA , RNA Mensageiro/genética , RNA não Traduzido/genética , Transcriptoma , Biblioteca Gênica , Regiões Promotoras Genéticas , RNA Mensageiro/química , RNA não Traduzido/química , Software , Sítio de Iniciação de TranscriçãoRESUMO
The global prevalence of malaria has decreased over the past fifteen years, but similar gains have not been realized against Plasmodium vivax because this species is less responsive to conventional malaria control interventions aimed principally at P. falciparum. Approximately half of all malaria cases outside of Africa are caused by P. vivax. This species places dormant forms in human liver that cause repeated clinical attacks without involving another mosquito bite. The diagnosis of acute patent P. vivax malaria relies primarily on light microscopy. Specific rapid diagnostic tests exist but typically perform relatively poorly compared to those for P. falciparum. Better diagnostic tests are needed for P. vivax. To guide their development, FIND, in collaboration with P. vivax experts, identified the specific diagnostic needs associated with this species and defined a series of three distinct target product profiles, each aimed at a particular diagnostic application: (i) point-of-care of acutely ill patients for clinical care purposes; (ii) point-of-care asymptomatic and otherwise sub-patent residents for public health purposes, e.g., mass screen and treat campaigns; and (iii) ultra-sensitive not point-of-care diagnosis for epidemiological research/surveillance purposes. This report presents and discusses the rationale for these P. vivax-specific diagnostic target product profiles. These contribute to the rational development of fit-for-purpose diagnostic tests suitable for the clinical management, control and elimination of P. vivax malaria.
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Malária Falciparum/diagnóstico , Malária Vivax/diagnóstico , Plasmodium falciparum/isolamento & purificação , Plasmodium vivax/isolamento & purificação , Sistemas Automatizados de Assistência Junto ao Leito , Testes Diagnósticos de Rotina , Humanos , Malária Falciparum/parasitologia , Malária Falciparum/prevenção & controle , Malária Vivax/parasitologia , Malária Vivax/prevenção & controle , Especificidade da EspécieRESUMO
Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download.
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Primers do DNA/genética , Técnicas de Genotipagem/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Pareamento Incorreto de Bases , Sondas de Oligonucleotídeos/genética , Taq Polimerase/metabolismoRESUMO
The electrophoretic mobility shift assay (EMSA) is the most frequently used experiment for studying protein-DNA interactions and to identify DNA-binding proteins. Protein-DNA complexes formed during EMSA experiments can be further analyzed by shift-western blotting, where the protein and DNA components contained in a polyacrylamide gel are transferred to stacked membranes: First a nitrocellulose membrane retains the proteins while double-stranded DNA passes through the nitrocellulose membrane and binds only to a charged membrane placed below. Immobilized proteins can then be stained with specific antibodies while the DNA can be detected by a radioactive label or a nonradioactive detection system. Shift-western blotting can overcome many limitations of supershift experiments and allows for the analysis of complex protein-DNA complexes containing multiple protein factors. Moreover, proteins and/or DNA may be recovered from membranes after the blotting step for further analysis by other means.