Gene Expression Analysis in Bacteria by RT-qPCR.

Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) using fluorescent DNA-binding dyes is now a gold-standard methodology to study bacterial gene expression through relative quantitation of target mRNAs under specific experimental conditions, and recent developments in the technology allow for gene expression analysis in single cells. Nevertheless, several critical steps of the RT-qPCR protocol need to be carefully addressed in order to obtain reliable results, particularly regarding RNA sample quality and appropriate choice of reference genes. Besides, accurate reporting of study conditions is essential, as recommended by the MIQE guidelines. Herein, we provide a practical approach to quantitation of the transcript levels of bacterial genes using RT-qPCR, including a general protocol for obtaining good-quality bacterial RNA and a discussion on the selection and validation of candidate bacterial reference genes for data normalization.

Performance of 4T score and heparin-platelet factor 4 antibody in the diagnosis of heparin-induced thrombocytopenia (HIT) in cancer.

Cancer patients have characteristics which significantly influence the 4T score and heparin-platelet factor 4 antibody (H-PF4 ab). Our aim was to determine among cancer patients the correlation of the 4T score and H-PF4 ab with the serotonin release assay (SRA). We performed a retrospective analysis of records of cancer patients in whom H-PF4 polyclonal (IgG, IgM and IgA) enzyme-linked immunosorbent assay (ELISA) and SRA were evaluated. Cases were defined as heparin induced thrombocytopenia (HIT) when SRA confirmed the diagnosis. Logistic regression model and the receiver operating characteristic curves were conducted to identify the optimal cutting point for the optical density (OD) and 4T score to discriminate the SRA status. Among 246 patients, the optimal cutoff of 4T score for HIT diagnosis was 5 (sensitivity 90.0%, specificity 73.6%), and the optimal cutoff of H-PF4 polyclonal ELISA OD was 1.004 (sensitivity 81.8%, specificity 97.0%). Our findings suggest that cancer patients may need higher cutoff values for the 4T score. Conventional H-PF4 ab testing seem to perform similarly for the diagnosis of HIT when compared to published data from non-cancer cohorts. Additional studies are necessary to confirm our findings.

Limiting the Hydrolysis and Oxidation of Maleimide-Peptide Adducts Improves Detection of Protein Thiol Oxidation.

Oxidative stress, caused by reactive oxygen and nitrogen species (RONS), is important in the pathophysiology of many diseases. A key target of RONS is the thiol group of protein cysteine residues. Because thiol oxidation can affect protein function, mechanistic information about how oxidative stress affects tissue function can be ascertained by identifying oxidized proteins. The probes used must be specific and sensitive, such as maleimides for the alkylation of reduced cysteine thiols. However, we find that maleimide-alkylated peptides (MAPs) are oxidized and hydrolyzed under sample preparation conditions common for proteomic studies. This can result in up to 90% of the MAP signal being converted to oxidized or hydrolyzed MAPs, decreasing the sensitivity of the analysis. A substantial portion of these modifications were accounted for by Coomassie "blue silver" staining (∼14%) of gels and proteolytic digestion buffers (∼20%). More than 40% of the MAP signal can be retained with the use of thioglycolic acid during gel electrophoresis, trichloroethanol-UV protein visualization in gels, and proteolytic digestion buffer of pH 7.0 TRIS. This work demonstrates that it is possible to decrease modifications to MAPs through changes to the sample preparation workflow, enhancing the potential usefulness of maleimide in identifying oxidized peptides.

Development and Validation of Broad-Range Qualitative and Clade-Specific Quantitative Molecular Probes for Assessing Mercury Methylation in the Environment.

Two genes, hgcA and hgcB, are essential for microbial mercury (Hg) methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability, and biogeochemistry, are critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning known hgcAB genes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria, nine Firmicutes, and nine methanogenic Archaea genomes. A distinct PCR product at the expected size was confirmed for all hgcAB(+) strains tested via Sanger sequencing. Additionally, we developed clade-specific degenerate quantitative PCR (qPCR) primers that targeted hgcA for each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplified hgcA from 64%, 88%, and 86% of tested pure cultures of Deltaproteobacteria, Firmicutes, and Archaea, respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we tested hgcA and hgcAB recovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. The resulting data will be essential in developing accurate and robust predictive models of Hg methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics of hgcAB IMPORTANCE: The neurotoxin methylmercury (MeHg) poses a serious risk to human health. MeHg production in nature is associated with anaerobic microorganisms. The recent discovery of the Hg-methylating gene pair, hgcA and hgcB, has allowed us to design and optimize molecular probes against these genes within the genomic DNA for microorganisms known to methylate Hg. The protocols designed in this study allow for both qualitative and quantitative assessments of pure-culture or environmental samples. With these protocols in hand, we can begin to study the distribution of Hg-methylating organisms in nature via a cultivation-independent strategy.

3'-Biotin-tagged microRNA-27 does not associate with Argonaute proteins in cells.

Synthetic 3'-biotin-tagged microRNAs (miRNAs) have often been used to select interacting messenger RNA (mRNA) and noncoding RNA (ncRNA) targets. Here, we examined the extent of association of 3'-end biotinylated miR-27 with Argonaute (Ago) proteins in transfected human cells using a coimmunoprecipitation assay followed by Northern blot analysis. We report that biotinylated miR-27 does not efficiently associate with Ago compared to unmodified miR-27. These results suggest that 3'-end biotin-modified miRNAs are questionable monitors of miRNA function in cells.

A gold nanoparticles-modified aptamer beacon for urinary adenosine detection based on structure-switching/fluorescence-"turning on" mechanism.

A novel small molecule probe, aptamer beacon (AB), was introduced for adenosine (Ade) recognition and quantitative analysis. The Ade aptamer was engineered into an aptamer beacon by adding a gold nanoparticle-modified nucleotide sequence which is complementary to aptamer sequence (FDNA) at the 3'-end of FDNA. The fluorescence signal "turning on" was observed when AB was bound to Ade, which is attributed to a significant conformational change in AB from a FDNA/QDNA duplex to a FDNA-Ade complex. The Ade measurement was carried out in 20 mmol L(-1) Tris-HCl buffer solution of pH 7.4, ΔF signal linearly correlated with the concentration of Ade over the range of 2.0×10(-8) to 1.8×10(-6) mol L(-1). The limit of detection (LOD) for Ade is 6.0×10(-9) mol L(-1) with relative standard deviations (R.S.D) of 3.64-5.36%, and the recoveries were 98.6%, 100%, 102% (n=6), respectively. The present method has been successfully applied to determine Ade in human urine samples, and the obtained results were in good agreement with those obtained by the HPLC method. Our investigation shows that the unique properties of the AB could provide a promising potential for small molecules detection, and be benefit to extend the application of aptamer beacon technique.

Selective colorimetric sensing of cysteine in aqueous solutions using silver nanoparticles in the presence of Cr³+.

We here in report an extensive study on the development of a highly facile, selective and sensitive colorimetric probe for cysteine detection using silver nanoparticles (Ag NPs). The efficacy of the process relies upon the surface plasmon resonance properties of Ag NPs and the interaction of Ag-cysteine complex with chromium ions (Cr(3+)) in a ratio of 2:1. In the presence of Cr(3+), cysteine was able to induce the aggregation of Ag NPs thereby resulting in a change in yellow colour of the Ag colloid to purple. The reported probe has a limit of detection down to 1 nM which is to the best of our knowledge the lowest ever reported for the colorimetric detection of cysteine. Furthermore, a remarkable feature of this method is that it involves a simple technique exhibiting high selectivity to cysteine over other tested amino acids.

Real-time in situ calibration of an optically trapped probing system.

We present real-time in situ calibration of an optically trapped probing system. In the probing system, a micro/nanobead is stably trapped around the minimum of the field potential to serve as the measurement probe, whereas the random thermal force tends to destabilize it and causes Brownian motion around the equilibrium. The weighted recursive least-squares algorithm is applied to recursively update the system's parameters, such as the state transition coefficient, and to estimate specific system response and the unknown variance of the Gaussian white noise in real time according to the probe's motion. The real-time recursive algorithm was first applied to real-time calibration of measurement sensitivity and trapping stiffness for the case that the local temperature and the damping coefficient of the probe are known. It was then applied to estimate the probe's local temperature in real time. Two experiments were designed to illustrate the applicability of the real-time calibration method. The experimental results show that the recursive algorithm is able to real-time calibrate the trapping stiffness of the probing system and the measurement sensitivity of the back-focal-plane interferometry employed for position measurement. The experimental results also show that the method can estimate the probe's local temperature in real time.

A crowdsourcing evaluation of the NIH chemical probes.

Between 2004 and 2008, the US National Institutes of Health Molecular Libraries and Imaging initiative pilot phase funded 10 high-throughput screening centers, resulting in the deposition of 691 assays into PubChem and the nomination of 64 chemical probes. We crowdsourced the Molecular Libraries and Imaging initiative output to 11 experts, who expressed medium or high levels of confidence in 48 of these 64 probes.

Gene probes versus classical methods in the identification of mycobacteria.

OBJECTIVE: The emergence of tuberculosis/HIV co-infection and the increase in the number of cases of infection with nontuberculous mycobacteria (NTM) require rapid laboratory test results in the isolation and identification of mycobacteria. The objective of this study was to evaluate the identification of mycobacteria by means of gene probes in comparison with that obtained using classical biochemical methods. METHODS: Between 2002 and 2004, 178 mycobacterial cultures, all testing positive for acid-fast bacilli, were analyzed. Samples were obtained from clinical specimens of patients with respiratory symptoms or with clinical suspicion of pulmonary tuberculosis/mycobacteriosis who were treated in the greater metropolitan area of Santos. RESULTS: The gene probe identified 137 samples (77%) as Mycobacterium tuberculosis complex and 41 (23%) as NTM. Discordant results between the methods (3%) were obtained only in the year of implementation (2002). When comparing the methods, the sensitivity, specificity, positive predictive value and negative predictive value of the gene probe method were 98%, 93%, 98% and 93%, respectively. CONCLUSIONS: Despite the cost, the identification of mycobacteria using the molecular technique is faster: maximum 3 h vs. 28-30 days for classical methods. The use of gene probes is a validated molecular technique. It is fast, easy to use and readily available on the market. It has high specificity and sensitivity, which justifies its implementation and routine use in referral laboratories, since it facilitates the diagnosis providing agile clinical interventions.

Estudo comparativo entre um sistema de sonda genética e métodos clássicos na identificação das micobactérias / Gene probes versus classical methods in the identification of mycobacteria

OBJETIVO: O aparecimento da co-infecção tuberculose/HIV e o aumento de casos de doenças provocadas por micobactérias não-tuberculosas (MNT) exigem repostas laboratoriais rápidas tanto no isolamento como na identificação das micobactérias. O objetivo deste trabalho foi avaliar a identificação das micobactérias através de sonda genética em comparação com os métodos bioquímicos clássicos. MÉTODOS: Entre 2002 e 2004, foram analisadas 178 culturas de micobactérias, confirmadas como bacilos álcool-ácido resistentes e obtidas de isolados clínicos de pacientes sintomáticos respiratórios ou com suspeita clínica de tuberculose pulmonar e/ou micobacterioses, atendidos nas Unidades de Saúde da Baixada Santista. RESULTADOS: A sonda genética identificou 137 amostras (77%) como complexo Mycobacterium tuberculosis e 41 (23%) como MNT. A discordância observada de 3% entre os métodos ocorreu apenas no ano de implantação (2002). Ao comparar os métodos, a sensibilidade, especificidade, valor preditivo positivo e valor preditivo negativo da sonda genética foram 98%, 93%, 98% e 93%, respectivamente. CONCLUSÕES: Apesar do custo elevado, a identificação de micobactérias pela técnica molecular é mais rápida: máximo de 3 h vs. 28-30 dias para os métodos clássicos. A utilização de sondas genéticas é uma técnica molecular validada, simples e disponível no mercado, com elevada especificidade, sensibilidade e rapidez, o que justifica sua implantação e uso rotineiro em laboratórios de referência, facilitando o diagnóstico e permitindo uma intervenção clínica ágil.

OBJECTIVE: The emergence of tuberculosis/HIV co-infection and the increase in the number of cases of infection with nontuberculous mycobacteria (NTM) require rapid laboratory test results in the isolation and identification of mycobacteria. The objective of this study was to evaluate the identification of mycobacteria by means of gene probes in comparison with that obtained using classical biochemical methods. METHODS: Between 2002 and 2004, 178 mycobacterial cultures, all testing positive for acid-fast bacilli, were analyzed. Samples were obtained from clinical specimens of patients with respiratory symptoms or with clinical suspicion of pulmonary tuberculosis/mycobacteriosis who were treated in the greater metropolitan area of Santos. RESULTS: The gene probe identified 137 samples (77%) as Mycobacterium tuberculosis complex and 41 (23%) as NTM. Discordant results between the methods (3%) were obtained only in the year of implementation (2002). When comparing the methods, the sensitivity, specificity, positive predictive value and negative predictive value of the gene probe method were 98%, 93%, 98% and 93%, respectively. CONCLUSIONS: Despite the cost, the identification of mycobacteria using the molecular technique is faster: maximum 3 h vs. 28-30 days for classical methods. The use of gene probes is a validated molecular technique. It is fast, easy to use and readily available on the market. It has high specificity and sensitivity, which justifies its implementation and routine use in referral laboratories, since it facilitates the diagnosis providing agile clinical interventions.

Single nucleotide specific detection of DNA by native chemical ligation of fluorescence labeled PNA-probes.

DNA-directed chemical ligations provide the opportunity to diagnose DNA sequences with very high sequence specificity. Fluorescent labels have been attached to reactive probes to enable the homogeneous detection of DNA and RNA. However, it has frequently been found that the attachment of fluorescent labels results in decreases of ligation fidelity. Herein we describe the development of a fluorogenic ligation reaction that provides for 10(2)-fold to perfect sequence selectivity. The reaction is based on the isocysteine-mediated native chemical PNA ligation. It is shown that DNA-induced rate accelerations of approximately 43.000-fold can be obtained through subtle variations of the ligation conditions. PNA-thioesters and isocysteine-PNA conjugates were labeled with FAM and TMR fluorophores, respectively. For gaining rapid synthetic access, a convenient on-resin labeling approach was developed. A new PNA monomer featuring an Alloc-protected lysine side chain was synthesized and coupled in solid-phase PNA synthesis. In the event of a ligation reaction the two fluorophores are brought into proximity. It is shown that fluorescence resonance energy transfer provides a positive fluorescence signal which is specific for product formation rather than for loss of starting materials. Single base mutations can be detected within minutes and with very high sequence selectivity at optimized conditions.

Calibration of probe volume in fluorescence correlation spectroscopy.

In fluorescence correlation spectroscopy (FCS), an accurate evaluation of the probe volume is the basis of correct interpretation of experimental data and solution of an appropriate diffusion model. Poor fitting convergence has been a problem in the determination of the dimensional parameters, the beam radius, omega, and the distance along the optical axis of the probe volume, l. In this work, the instability of fitting during the calibration process is investigated by examining the chi(2) surfaces. We demonstrate that the minimum of chi(2) in the omega dimension is well defined for both converging and diverging data. The difficulty of fitting comes from the l dimension. The uncertainty in l could be significantly larger than that in omega, as determined by F-statistics. A modified calibration process is recommended based on examining two data treatment methods, combining several short data sets into a single long run and averaging the correlation functions of several short data sets. It is found that by using the mean of several converging correlation functions from short data sets instead of a long time correlation, more stable and consistent dimensional parameters are extracted to define the probe volume.

[Standardization of conditions for PCR detection of Leishmania spp. DNA in sand flies (Diptera, Psychodidae)]. / Padronização de condições para detecção de DNA de Leishmania spp. em flebotomíneos (Diptera, Psychodidae) pela reação em cadeia da polimerase.

The correct identification of etiological agents in vector insects is crucial for epidemiological studies. Identification of flagellates in such vectors, usually by dissection of the digestive tract and microscopic observation of the contents as well as attempts at parasite isolation from insects in culture media, have proven operationally inadequate and with poor diagnostic specificity, since female sand flies are also hosts for other flagellates like Trypanosoma and Endotrypanum. Due to the efficiency and specificity of DNA target sequence amplification by polymerase chain reaction (PCR), the latter could be used to investigate the presence of Leishmania in sand flies, although the insects need to be properly stored and the Leishmania DNA extracted using appropriate methodology. This paper describes methodologies to standardize sand fly storage and Leishmania DNA extraction in such specimens as a more practical method in field studies.

Interpretive criteria for use of AccuProbe for identification of Mycobacterium avium complex directly from 7H9 broth cultures.

Rapid identification of Mycobacterium avium complex (MAC) is possible by use of AccuProbe (Gen-Probe, San Diego, Calif.). To evaluate the reliability of the MAC AccuProbe for testing 7H9 cultures inoculated with broth from MGIT cultures positive for acid-fast bacilli or growth on a solid medium, we compared probe results to results obtained by sequencing a portion of the 16S rRNA gene. Isolates were sequenced if the MAC probe result was <100,000 relative light units (RLU) or if it was > or =100,000 RLU and the colony morphology was not classic or there were two colony types. For the 1,389 cultures tested in phase 1, conducted to evaluate cutoff values for the MAC probe in testing of 7H9 cultures inoculated with broth from MGIT cultures, the sensitivity and specificity of the MAC AccuProbe were 97.7% and 88.8%, respectively, according to the manufacturer's interpretive criteria (> or =30,000 RLU is positive). If the cutoff for a positive result were 80,000 RLU, the specificity would be 100% and the sensitivity 92.3%. Of the 344 isolates in phase 2, which was conducted to confirm the 80,000-RLU cutoff for a positive result and therefore included only isolates with a MAC probe result of < or =100,000 RLU, 13 of 16 with results of > or =30,000 but <80,000 RLU were identified as mycobacteria other than MAC, including five Mycobacterium tuberculosis complex isolates. These data support the use of 80,000 RLU as the cutoff for a positive result in testing of 7H9 broth cultures with the MAC AccuProbe.

Highly multiplexed molecular inversion probe genotyping: over 10,000 targeted SNPs genotyped in a single tube assay.

Large-scale genetic studies are highly dependent on efficient and scalable multiplex SNP assays. In this study, we report the development of Molecular Inversion Probe technology with four-color, single array detection, applied to large-scale genotyping of up to 12,000 SNPs per reaction. While generating 38,429 SNP assays using this technology in a population of 30 trios from the Centre d'Etude Polymorphisme Humain family panel as part of the International HapMap project, we established SNP conversion rates of approximately 90% with concordance rates >99.6% and completeness levels >98% for assays multiplexed up to 12,000plex levels. Furthermore, these individual metrics can be "traded off" and, by sacrificing a small fraction of the conversion rate, the accuracy can be increased to very high levels. No loss of performance is seen when scaling from 6,000plex to 12,000plex assays, strongly validating the ability of the technology to suppress cross-reactivity at high multiplex levels. The results of this study demonstrate the suitability of this technology for comprehensive association studies that use targeted SNPs in indirect linkage disequilibrium studies or that directly screen for causative mutations.

Semi-automated, single-band peak-fitting analysis of hydroxyl radical nucleic acid footprint autoradiograms for the quantitative analysis of transitions.

Hydroxyl radical footprinting can probe the solvent accessibility of the ribose moiety of the individual nucleotides of DNA and RNA. Semi-automated analytical tools are presented for the quantitative analyses of nucleic acid footprint transitions in which processes such as folding or ligand binding are followed as a function of time or ligand concentration. Efficient quantitation of the intensities of the electrophoretic bands comprising the footprinting reaction products is achieved by fitting a series of Lorentzian curves to line profiles obtained from gels utilizing sequentially relaxed constraints consistent with electrophoretic mobility. An automated process of data 'standardization' has been developed that corrects for differences in the loading amounts in the electrophoresis. This process enhances the accuracy of the derived transitions and makes generating them easier. Together with visualization of the processed footprinting in false-color two-dimensional maps, DNA and RNA footprinting data can be accurately, precisely and efficiently processed allowing transitions to be objectively and comprehensively analyzed. The utility of this new analysis approach is illustrated by its application to the ion-meditated folding of a large RNA molecule.