A simple interferometric method to measure the calibration factor and displacement amplification in piezoelectric flextensional actuators

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Probabilistic reasoning with a bayesian DNA device based on strand displacement

We present a computing model based on the DNA strand displacement technique which performs Bayesian inference. The model will take single stranded DNA as input data, representing the presence or absence of a specific molecular signal (evidence). The program logic encodes the prior probability of a disease and the conditional probability of a signal given the disease playing with a set of different DNA complexes and their ratios. When the input and program molecules interact, they release a different pair of single stranded DNA species whose relative proportion represents the application of Bayes? Law: the conditional probability of the disease given the signal. The models presented in this paper can empower the application of probabilistic reasoning in genetic diagnosis in vitro.

A coupled complex of T4 DNA replication helicase (gp41) and polymerase (gp43) can perform rapid and processive DNA strand-displacement synthesis

We have developed a coupled helicase–polymerase DNA unwinding assay and have used it to monitor the rate of double-stranded DNA unwinding catalyzed by the phage T4 DNA replication helicase (gp41). This procedure can be used to follow helicase activity in subpopulations in systems in which the unwinding-synthesis reaction is not synchronized on all the substrate-template molecules. We show that T4 replication helicase (gp41) and polymerase (gp43) can be assembled onto a loading site located near the end of a long double-stranded DNA template in the presence of a macromolecular crowding agent, and that this coupled “two-protein” system can carry out ATP-dependent strand displacement DNA synthesis at physiological rates (400 to 500 bp per sec) and with high processivity in the absence of other T4 DNA replication proteins. These results suggest that a direct helicase–polymerase interaction may be central to fast and processive double-stranded DNA replication, and lead us to reconsider the roles of the other replication proteins in processivity control.

Nucleic acid amplification testing for Neisseria gonorrhoeae: An ongoing challenge

Nucleic acid amplification tests (NAATs) for the detection of Neisseria gonorrhoeae became available in the early 1990s. Although offering several advantages over traditional detection methods, N. gonorrhoeae NAATs do have some limitations. These include cost, risk of carryover contamination, inhibition, and inability to provide antibiotic resistance data. In addition, there are sequence-related limitations that are unique to N. gonorrhoeae NAATs. In particular, false-positive results are a major consideration. These primarily stem from the frequent horizontal genetic exchange occurring within the Neisseria genus, leading to commensal Neisseria species acquiring N. gonorrhoeae genes. Furthermore, some N. gonorrhoeae subtypes may lack specific sequences targeted by a particular NAAT. Therefore, NAAT false-negative results because of sequence variation may occur in some gonococcal populations. Overall, the N. gonorrhoeae species continues to present a considerable challenge for molecular diagnostics. The need to evaluate N. gonorrhoeae NAATs before their use in any new patient population and to educate physicians on the limitations of these tests is emphasized in this review.

Vulvovaginal-swab or first-catch urine specimen to detect Chlamydia trachomatis in women in a community setting?

Screening for chlamydia in women is widely recommended. We evaluated the performance of two nucleic acid amplification tests for detecting Chlamydia trachomatis in self-collected vulvovaginal-swab and first-catch urine specimens from women in a community setting and a strategy for optimizing the sensitivity of an amplified enzyme immunoassay on vulvovaginal-swab specimens. We tested 2,745 paired vulvovaginal-swab and urine specimens by PCR (Roche Cobas) or strand displacement amplification (SDA; Becton Dickinson). There were 146 women infected with chlamydia. The assays detected 97.3% (95% confidence interval [CI], 93.1 to 99.2%) of infected patients with vulvovaginal-swab specimens and 91.8% (86.1 to 95.7%) with urine specimens. We tested 2,749 vulvovaginal-swab specimens with both a nucleic acid amplification test and a polymer conjugate-enhanced enzyme immunoassay with negative-gray-zone testing. The relative sensitivities obtained after retesting specimens in the negative gray zone were 74.3% (95% CI, 62.8 to 83.8%) with PCR and 58.3% (95% CI, 46.1 to 69.8%) with SDA. In community settings, both vulvovaginal-swab and first-catch urine specimens from women are suitable substrates for nucleic acid amplification tests, but enzyme immunoassays, even after negative-gray-zone testing, should not be used in screening programs.

Epidemiological, social, diagnostic and economic evaluation of population screening for genital chlamydial infection

OBJECTIVES: To investigate epidemiological, social, diagnostic and economic aspects of chlamydia screening in non-genitourinary medicine settings. METHODS: Linked studies around a cross-sectional population-based survey of adult men and women invited to collect urine and (for women) vulvovaginal swab specimens at home and mail these to a laboratory for testing for Chlamydia trachomatis. Specimens were used in laboratory evaluations of an amplified enzyme immunoassay (PCE EIA) and two nucleic acid amplification tests [Cobas polymerase chain reaction (PCR), Becton Dickinson strand displacement amplification (SDA)]. Chlamydia-positive cases and two negative controls completed a risk factor questionnaire. Chlamydia-positive cases were invited into a randomised controlled trial of partner notification strategies. Samples of individuals testing negative completed psychological questionnaires before and after screening. In-depth interviews were conducted at all stages of screening. Chlamydia transmission and cost-effectiveness of screening were investigated in a transmission dynamic model. SETTING AND PARTICIPANTS: General population in the Bristol and Birmingham areas of England. In total, 19,773 women and men aged 16-39 years were randomly selected from 27 general practice lists. RESULTS: Screening invitations reached 73% (14,382/19,773). Uptake (4731 participants), weighted for sampling, was 39.5% (95% CI 37.7, 40.8%) in women and 29.5% (95% CI 28.0, 31.0%) in men aged 16-39 years. Chlamydia prevalence (219 positive results) in 16-24 year olds was 6.2% (95% CI 4.9, 7.8%) in women and 5.3% (95% CI 4.4, 6.3%) in men. The case-control study did not identify any additional factors that would help target screening. Screening did not adversely affect anxiety, depression or self-esteem. Participants welcomed the convenience and privacy of home-sampling. The relative sensitivity of PCR on male urine specimens was 100% (95% CI 89.1, 100%). The combined relative sensitivities of PCR and SDA using female urine and vulvovaginal swabs were 91.8% (86.1, 95.7, 134/146) and 97.3% (93.1, 99.2%, 142/146). A total of 140 people (74% of eligible) participated in the randomised trial. Compared with referral to a genitourinary medicine clinic, partner notification by practice nurses resulted in 12.4% (95% CI -3.7, 28.6%) more patients with at least one partner treated and 22.0% (95% CI 6.1, 37.8%) more patients with all partners treated. The health service and patients costs (2005 prices) of home-based postal chlamydia screening were 21.47 pounds (95% CI 19.91 pounds, 25.99) per screening invitation and 28.56 pounds (95% CI 22.10 pounds, 30.43) per accepted offer. Preliminary modelling found an incremental cost-effectiveness ratio (2003 prices) comparing screening men and women annually to no screening in the base case of 27,000 pounds/major outcome averted at 8 years. If estimated screening uptake and pelvic inflammatory disease incidence were increased, the cost-effectiveness ratio fell to 3700 pounds/major outcome averted. CONCLUSIONS: Proactive screening for chlamydia in women and men using home-collected specimens was feasible and acceptable. Chlamydia prevalence rates in men and women in the general population are similar. Nucleic acid amplification tests can be used on first-catch urine specimens and vulvovaginal swabs. The administrative costs of proactive screening were similar to those for opportunistic screening. Using empirical estimates of screening uptake and incidence of complications, screening was not cost-effective.

Comparison of amplification methods for transcriptomic analyses of low abundance prokaryotic RNA sources

Microarrays have established as instrumental for bacterial detection, identification, and genotyping as well as for transcriptomic studies. For gene expression analyses using limited numbers of bacteria (derived from in vivo or ex vivo origin, for example), RNA amplification is often required prior to labeling and hybridization onto microarrays. Evaluation of the fidelity of the amplification methods is crucial for the robustness and reproducibility of microarray results. We report here the first utilization of random primers and the highly processive Phi29 phage polymerase to amplify material for transcription profiling analyses. We compared two commercial amplification methods (GenomiPhi and MessageAmp kits) with direct reverse-transcription as the reference method, focusing on the robustness of mRNA quantification using either microarrays or quantitative RT-PCR. Both amplification methods using either poly-A tailing followed by in vitro transcription, or direct strand displacement polymerase, showed appreciable linearity. Strand displacement technique was particularly affordable compared to in vitro transcription-based (IVT) amplification methods and consisted in a single tube reaction leading to high amplification yields. Real-time measurements using low-, medium-, and highly expressed genes revealed that this simple method provided linear amplification with equivalent results in terms of relative messenger abundance as those obtained by conventional direct reverse-transcription.

RecA tests homology at both pairing and strand exchange

RecA is a 38-kDa protein from Escherichia coli that polymerizes on single-stranded DNA, forming a nucleoprotein filament that pairs with homologous duplex DNA and carries out strand exchange in vitro. To observe the effects of mismatches on the kinetics of the RecA-catalyzed recombination reaction, we used assays based upon fluorescence energy transfer that can differentiate between the pairing and strand displacement phases. Oligonucleotide sequences that produced 2–14% mismatches in the heteroduplex product of strand exchange were tested, as well as completely homologous and heterologous sequences. The equilibrium constant for pairing decreased as the number of mismatches increased, which appeared to result from both a decrease in the rate of formation and an increase in the rate of dissociation of the intermediates. In addition, the rate of strand displacement decreased with increasing numbers of mismatches, roughly in proportion to the number of mismatches. The equilibrium constant for pairing and the rate constant for strand displacement both decreased 6-fold as the heterology increased to 14%. These results suggest that discrimination of homology from heterology occurs during both pairing and strand exchange.

Programming Molecular Devices using Nucleic Acid Hairpins

Nucleic Acid hairpins have been a subject of study for the last four decades. They are composed of single strand that is

hybridized to itself, and the central section forming an unhybridized loop. In nature, they stabilize single stranded RNA, serve as nucleation

sites for RNA folding, protein recognition signals, mRNA localization and regulation of mRNA degradation. On the other hand,

DNA hairpins in biological contexts have been studied with respect to forming cruciform structures that can regulate gene expression.

The use of DNA hairpins as fuel for synthetic molecular devices, including locomotion, was proposed and experimental demonstrated in 2003. They

were interesting because they bring to the table an on-demand energy/information supply mechanism.

The energy/information is hidden (from hybridization) in the hairpin’s loop, until required.

The energy/information is harnessed by opening the stem region, and exposing the single stranded loop section.

The loop region is now free for possible hybridization and help move the system into a thermodynamically favourable state.

The hidden energy and information coupled with

programmability provides another functionality, of selectively choosing what reactions to hide and

what reactions to allow to proceed, that helps develop a topological sequence of events.

Hairpins have been utilized as a source of fuel for many different DNA devices. In this thesis, we program four different

molecular devices using DNA hairpins, and experimentally validate them in the

laboratory. 1) The first device: A

novel enzyme-free autocatalytic self-replicating system composed entirely of DNA that operates isothermally. 2) The second

device: Time-Responsive Circuits using DNA have two properties: a) asynchronous: the final output is always correct

regardless of differences in the arrival time of different inputs.

b) renewable circuits which can be used multiple times without major degradation of the gate motifs

(so if the inputs change over time, the DNA-based circuit can re-compute the output correctly based on the new inputs).

3) The third device: Activatable tiles are a theoretical extension to the Tile assembly model that enhances

its robustness by protecting the sticky sides of tiles until a tile is partially incorporated into a growing assembly.

4) The fourth device: Controlled Amplification of DNA catalytic system: a device such that the amplification

of the system does not run uncontrollably until the system runs out of fuel, but instead achieves a finite

amount of gain.

Nucleic acid circuits with the ability

to perform complex logic operations have many potential practical applications, for example the ability to achieve point of care diagnostics.

We discuss the designs of our DNA Hairpin molecular devices, the results we have obtained, and the challenges we have overcome

to make these truly functional.

Unique genome replication mechanism of the archaeal virus AFV1.

The exceptional genomic content and genome organization of the Acidianus filamentous virus 1 (AFV1) that infects the hyperthermophilic archaeon Acidianus hospitalis suggest that this virus might exploit an unusual mechanism of genome replication. An analysis of replicative intermediates of the viral genome by two-dimensional (2D) agarose gel electrophoresis revealed that viral genome replication starts by the formation of a D-loop and proceeds via strand displacement replication. Characterization of replicative intermediates using dark-field electron microscopy, in combination with the 2D agarose gel electrophoresis data, suggests that recombination plays a key role in the termination of AFV1 genome replication through the formation of terminal loops. A terminal protein was found to be attached to the ends of the viral genome. The results allow us to postulate a model of genome replication that relies on recombination events for initiation and termination.

L'élastographie ultrasonore dynamique vasculaire : une nouvelle modalité d'imagerie non-invasive pour la caractérisation mécanique de la thrombose veineuse

L’accident thromboembolique veineux, tel que la thrombose veineuse profonde (TVP) ou thrombophlébite des membres inférieurs, est une pathologie vasculaire caractérisée par la formation d’un caillot sanguin causant une obstruction partielle ou totale de la lumière sanguine. Les embolies pulmonaires sont une complication mortelle des TVP qui surviennent lorsque le caillot se détache, circule dans le sang et produit une obstruction de la ramification artérielle irriguant les poumons. La combinaison d’outils et de techniques d’imagerie cliniques tels que les règles de prédiction cliniques (signes et symptômes) et les tests sanguins (D-dimères) complémentés par un examen ultrasonographique veineux (test de compression, écho-Doppler), permet de diagnostiquer les premiers épisodes de TVP. Cependant, la performance de ces outils diagnostiques reste très faible pour la détection de TVP récurrentes. Afin de diriger le patient vers une thérapie optimale, la problématique n’est plus basée sur la détection de la thrombose mais plutôt sur l’évaluation de la maturité et de l’âge du thrombus, paramètres qui sont directement corrélées à ses propriétés mécaniques (e.g. élasticité, viscosité). L’élastographie dynamique (ED) a récemment été proposée comme une nouvelle modalité d’imagerie non-invasive capable de caractériser quantitativement les propriétés mécaniques de tissus. L’ED est basée sur l’analyse des paramètres acoustiques (i.e. vitesse, atténuation, pattern de distribution) d’ondes de cisaillement basses fréquences (10-7000 Hz) se propageant dans le milieu sondé. Ces ondes de cisaillement générées par vibration externe, ou par source interne à l’aide de la focalisation de faisceaux ultrasonores (force de radiation), sont mesurées par imagerie ultrasonore ultra-rapide ou par résonance magnétique. Une méthode basée sur l’ED adaptée à la caractérisation mécanique de thromboses veineuses permettrait de quantifier la sévérité de cette pathologie à des fins d’amélioration diagnostique. Cette thèse présente un ensemble de travaux reliés au développement et à la validation complète et rigoureuse d’une nouvelle technique d’imagerie non-invasive élastographique pour la mesure quantitative des propriétés mécaniques de thromboses veineuses. L’atteinte de cet objectif principal nécessite une première étape visant à améliorer les connaissances sur le comportement mécanique du caillot sanguin (sang coagulé) soumis à une sollicitation dynamique telle qu’en ED. Les modules de conservation (comportement élastique, G’) et de perte (comportement visqueux, G’’) en cisaillement de caillots sanguins porcins sont mesurés par ED lors de la cascade de coagulation (à 70 Hz), et après coagulation complète (entre 50 Hz et 160 Hz). Ces résultats constituent les toutes premières mesures du comportement dynamique de caillots sanguins dans une gamme fréquentielle aussi étendue. L’étape subséquente consiste à mettre en place un instrument innovant de référence (« gold standard »), appelé RheoSpectris, dédié à la mesure de la viscoélasticité hyper-fréquence (entre 10 Hz et 1000 Hz) des matériaux et biomatériaux. Cet outil est indispensable pour valider et calibrer toute nouvelle technique d’élastographie dynamique. Une étude comparative entre RheoSpectris et la rhéométrie classique est réalisée afin de valider des mesures faites sur différents matériaux (silicone, thermoplastique, biomatériaux, gel). L’excellente concordance entre les deux technologies permet de conclure que RheoSpectris est un instrument fiable pour la mesure mécanique à des fréquences difficilement accessibles par les outils actuels. Les bases théoriques d’une nouvelle modalité d’imagerie élastographique, nommée SWIRE (« shear wave induced resonance dynamic elastography »), sont présentées et validées sur des fantômes vasculaires. Cette approche permet de caractériser les propriétés mécaniques d’une inclusion confinée (e.g. caillot sanguin) à partir de sa résonance (amplification du déplacement) produite par la propagation d’ondes de cisaillement judicieusement orientées. SWIRE a également l’avantage d’amplifier l’amplitude de vibration à l’intérieur de l’hétérogénéité afin de faciliter sa détection et sa segmentation. Finalement, la méthode DVT-SWIRE (« Deep venous thrombosis – SWIRE ») est adaptée à la caractérisation de l’élasticité quantitative de thromboses veineuses pour une utilisation en clinique. Cette méthode exploite la première fréquence de résonance mesurée dans la thrombose lors de la propagation d’ondes de cisaillement planes (vibration d’une plaque externe) ou cylindriques (simulation de la force de radiation par génération supersonique). DVT-SWIRE est appliquée sur des fantômes simulant une TVP et les résultats sont comparés à ceux donnés par l’instrument de référence RheoSpectris. Cette méthode est également utilisée avec succès dans une étude ex vivo pour l’évaluation de l’élasticité de thromboses porcines explantées après avoir été induites in vivo par chirurgie.

Repair of abasic sites in DNA

Repair of both normal and reduced AP sites is activated by AP endonuclease, which recognizes and cleaves a phosphodiester bond 5' to the AP site. For a short period of time an incised AP site is occupied by poly(ADP-ribose) polymerase and then DNA polymerase beta adds one nucleotide into the repair gap and simultaneously removes the 5'-sugar phosphate. Finally, the DNA ligase III/XRCC1 complex accomplishes repair by sealing disrupted DNA ends. However, long-patch BER pathway, which is involved in the removal of reduced abasic sites, requires further DNA synthesis resulting in strand displacement and the generation of a damage-containing flap that is later removed by the flap endonuclease. Strand-displacement DNA synthesis is accomplished by DNA polymerase delta/epsilon and DNA ligase I restores DNA integrity. DNA synthesis by DNA polymerase delta/epsilon is dependent on proliferating cell nuclear antigen, which also stimulates the DNA ligase I and flap endonuclease. These repair events are supported by multiple protein-protein interactions. (C) 2003 Elsevier B.V. All rights reserved.

Evolutionary dynamics of the immunodominant repeats of the Plasmodium vivax malaria-vaccine candidate circumsporozoite protein (CSP)

The circumsporozoite protein (CSP) of Plasmodium vivax, a major target for malaria vaccine development, has immunodominant B-cell epitopes mapped to central nonapeptide repeat arrays. To determine whether rearrangements of repeat motifs during mitotic DNA replication of parasites create significant CSP diversity under conditions of low effective meiotic recombination rates, we examined csp alleles from sympatric P. vivax isolates systematically sampled from an area of low malaria endemicity in Brazil over a period of 14 months. Nine unique csp types, comprising six different nona peptide repeats, were observed in 45 isolates analyzed. Identical or nearly identical repeats predominated in most arrays, consistent with their recent expansion. We found strong linkage disequilibrium at sites across the chromosome 8 segment flanking the csp locus, consistent with rare meiotic recombination in this region. We conclude that CSP repeat diversity may not be severely constrained by rare meiotic recombination in areas of low malaria endemicity. New repeat variants may be readily created by nonhomologous recombination even when meiotic recombination is rare, with potential implications for CSP-based vaccine development. (C) 2010 Elsevier B.V. All rights reserved.

Plasmodium vivax: Microsatellite analysis of multiple-clone infections

We used mixtures of genomic DNA from two genetically distinct isolates from Brazil, 42M and 312M, to investigate how accurately 12-locus microsatellite typing describes the overall genetic diversity and characterizes multilocus haplotypes in multiple-clone Plasmodium vivax infections. We found varying PCR amplification efficiencies of microsatellite alleles; for example, from the same 1:1 mixture of 42M and 312M DNA we amplified predominantly 312M-type alleles at 10 loci and 42M-type alleles at 2 loci. All microsatellite alleles were accurately scored in 1:0.5 and 1:0.25 312M:42M DNA mixtures, even when minor peak heights did not meet previously suggested criteria for minor allele detection in multiple-clone infections. Relative proportions of major and minor alleles were unaffected by multiple displacement amplification of template DNA prior to PCR-based microsatellite typing. Although microsatellite typing may detect minor alleles in clone mixtures, amplification biases may lead to inaccurate assignment of predominant haplotypes in multiple-clone P. vivax infections. (C) 2008 Elsevier Inc. All rights reserved.

Método de detecção interferométrica de fase, com baixa profundidade de modulação, aplicado à medição de deslocamentos nanométricos em atuadores e mini-manipuladores piezoelétricos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)