Evaluation of Contamination Inspection and Analysis Methods through Modeling System Performance

NASA Technical Reports Server (NTRS)

Seasly, Elaine; Dever, Jason; Stuban, Steven M. F.

2016-01-01

Contamination is usually identified as a risk on the risk register for sensitive space systems hardware. Despite detailed, time-consuming, and costly contamination control efforts during assembly, integration, and test of space systems, contaminants are still found during visual inspections of hardware. Improved methods are needed to gather information during systems integration to catch potential contamination issues earlier and manage contamination risks better. This research explores evaluation of contamination inspection and analysis methods to determine optical system sensitivity to minimum detectable molecular contamination levels based on IEST-STD-CC1246E non-volatile residue (NVR) cleanliness levels. Potential future degradation of the system is modeled given chosen modules representative of optical elements in an optical system, minimum detectable molecular contamination levels for a chosen inspection and analysis method, and determining the effect of contamination on the system. By modeling system performance based on when molecular contamination is detected during systems integration and at what cleanliness level, the decision maker can perform trades amongst different inspection and analysis methods and determine if a planned method is adequate to meet system requirements and manage contamination risk.

The agony of choice in dermatophyte diagnostics-performance of different molecular tests and culture in the detection of Trichophyton rubrum and Trichophyton interdigitale.

PubMed

Kupsch, C; Ohst, T; Pankewitz, F; Nenoff, P; Uhrlaß, S; Winter, I; Gräser, Y

2016-08-01

Dermatophytosis caused by dermatophytes of the genera Trichophyton and Microsporum belong to the most frequent mycoses worldwide. Molecular detection methods proved to be highly sensitive and enable rapid and accurate detection of dermatophyte species from clinical specimens. For the first time, we compare the performance of different molecular methods with each other and with conventional diagnostics in the detection of dermatophytoses caused by Trichophyton rubrum and Trichophyton interdigitale in clinical specimens (nail, skin and hair). The compared molecular methods comprise two already published PCR-ELISAs, a published quantitative RT-PCR as well as a newly developed PCR-ELISA targeting the internal transcribed spacer region. We investigated the sensitivity of the assays by analysing 375 clinical samples. In 148 specimens (39.5%) a positive result was gained in at least one of the four molecular tests or by culture, but the number of detected agents differed significantly between some of the assays. The most sensitive assay, a PCR-ELISA targeting a microsatellite region, detected 81 T. rubrum infections followed by an internal transcribed spacer PCR-ELISA (60), quantitative RT-PCR (52) and a topoisomerase II PCR-ELISA (51), whereas cultivation resulted in T. rubrum identification in 37 samples. The pros and cons of all four tests in routine diagnostics are discussed. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Nucleic Acid-Based Approaches for Detection of Viral Hepatitis

PubMed Central

Behzadi, Payam; Ranjbar, Reza; Alavian, Seyed Moayed

2014-01-01

Context: To determining suitable nucleic acid diagnostics for individual viral hepatitis agent, an extensive search using related keywords was done in major medical library and data were collected, categorized, and summarized in different sections. Results: Various types of molecular biology tools can be used to detect and quantify viral genomic elements and analyze the sequences. These molecular assays are proper technologies for rapidly detecting viral agents with high accuracy, high sensitivity, and high specificity. Nonetheless, the application of each diagnostic method is completely dependent on viral agent. Conclusions: Despite rapidity, automation, accuracy, cost-effectiveness, high sensitivity, and high specificity of molecular techniques, each type of molecular technology has its own advantages and disadvantages. PMID:25789132

Impact of transition from microscopy to molecular screening for detection of intestinal protozoa in Dutch patients.

PubMed

Svraka-Latifovic, S; Bouter, S; Naus, H; Bakker, L J; Timmerman, C P; Dorigo-Zetsma, J W

2014-11-01

Detection of intestinal protozoa by PCR methods has been described as being sensitive and specific, and as improving the diagnostic yield. Here we present the outcome of the transition from microscopy to molecular screening for detection of a select group of intestinal protozoa in faeces in our laboratory. Introduction of molecular screening for intestinal protozoa resulted in higher sensitivity, reduced hands-on-time, reduced time-to-results, leading to improved diagnostic efficiency. © 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.

Visual detection of glial cell line-derived neurotrophic factor based on a molecular translator and isothermal strand-displacement polymerization reaction

PubMed Central

Zhang, Li-Yong; Xing, Tao; Du, Li-Xin; Li, Qing-Min; Liu, Wei-Dong; Wang, Ji-Yue; Cai, Jing

2015-01-01

Background Glial cell line-derived neurotrophic factor (GDNF) is a small protein that potently promotes the survival of many types of neurons. Detection of GDNF is vital to monitoring the survival of sympathetic and sensory neurons. However, the specific method for GDNF detection is also un-discovered. The purpose of this study is to explore the method for protein detection of GDNF. Methods A novel visual detection method based on a molecular translator and isothermal strand-displacement polymerization reaction (ISDPR) has been proposed for the detection of GDNF. In this study, a molecular translator was employed to convert the input protein to output deoxyribonucleic acid signal, which was further amplified by ISDPR. The product of ISDPR was detected by a lateral flow biosensor within 30 minutes. Results This novel visual detection method based on a molecular translator and ISDPR has very high sensitivity and selectivity, with a dynamic response ranging from 1 pg/mL to 10 ng/mL, and the detection limit was 1 pg/mL of GDNF. Conclusion This novel visual detection method exhibits high sensitivity and selectivity, which is very simple and universal for GDNF detection to help disease therapy in clinical practice. PMID:25848224

Girsanov reweighting for path ensembles and Markov state models

NASA Astrophysics Data System (ADS)

Donati, L.; Hartmann, C.; Keller, B. G.

2017-06-01

The sensitivity of molecular dynamics on changes in the potential energy function plays an important role in understanding the dynamics and function of complex molecules. We present a method to obtain path ensemble averages of a perturbed dynamics from a set of paths generated by a reference dynamics. It is based on the concept of path probability measure and the Girsanov theorem, a result from stochastic analysis to estimate a change of measure of a path ensemble. Since Markov state models (MSMs) of the molecular dynamics can be formulated as a combined phase-space and path ensemble average, the method can be extended to reweight MSMs by combining it with a reweighting of the Boltzmann distribution. We demonstrate how to efficiently implement the Girsanov reweighting in a molecular dynamics simulation program by calculating parts of the reweighting factor "on the fly" during the simulation, and we benchmark the method on test systems ranging from a two-dimensional diffusion process and an artificial many-body system to alanine dipeptide and valine dipeptide in implicit and explicit water. The method can be used to study the sensitivity of molecular dynamics on external perturbations as well as to reweight trajectories generated by enhanced sampling schemes to the original dynamics.

Investigation of low-loss spectra and near-edge fine structure of polymers by PEELS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heckmann, W.

Transmission electron microscopy has changed from a purely imaging method to an analytical method. This has been facilitated particularly by equipping electron microscopes with energy filters and with parallel electron energy loss spectrometers (PEELS). Because of their relatively high energy resolution (1 to 2 eV) they provide information not only on the elements present but also on the type of bonds between the molecular groups. Polymers are radiation sensitive and the molecular bonds change as the spectrum is being recorded. This can be observed with PEEL spectrometers that are able to record spectra with high sensitivity and in rapid succession.

Molecular diagnosis of strongyloidiasis in a population of an endemic area through nested-PCR.

PubMed

Sharifdini, Meysam; Keyhani, Amir; Eshraghian, Mohammad Reza; Beigom Kia, Eshrat

2018-01-01

This study is aimed to diagnose and analyze strongyloidiasis in a population of an endemic area of Iran using nested-PCR, coupled with parasitological methods. Screening of strongyloidiasis infected people using reliable diagnostic techniques are essential to decrease the mortality and morbidity associated with this infection. Molecular methods have been proved to be highly sensitive and specific for detection of Strongyloides stercoralis in stool samples. A total of 155 fresh single stool samples were randomly collected from residents of north and northwest of Khouzestan Province, Iran. All samples were examined by parasitological methods including formalin-ether concentration and nutrient agar plate culture, and molecular method of nested-PCR. Infections with S. stercoralis were analyzed according to demographic criteria. Based on the results of nested-PCR method 15 cases (9.7%) were strongyloidiasis positive. Nested-PCR was more sensitive than parasitological techniques on single stool sampling. Elderly was the most important population index for higher infectivity with S. stercoralis . In endemic areas of S. stercoralis , old age should be considered as one of the most important risk factors of infection, especially among the immunosuppressed individuals.

Sensitivity for Diagnosing Group A Streptococcal Pharyngitis from Manufacturers is 10% Higher than Reported in Peer-Reviewed Publications.

PubMed

Vachhani, Raj; Patel, Toral; Centor, Robert M; Estrada, Carlos A

2017-01-01

Meta-analyses based on peer-reviewed publications report a sensitivity of approximately 85% for rapid antigen streptococcus tests to diagnose group A streptococcal (GAS) pharyngitis. Because these meta-analyses excluded package inserts, we examined the test characteristics of rapid antigen streptococcal tests and molecular methods that manufacturers report in their package inserts. We included tests available in the US market (Food and Drug Administration, period searched 1993-2015) and used package insert data to calculate pooled sensitivity and specificity. To examine quality, we used the Quality Assessment of Diagnostic Accuracy Studies-2. We excluded 26 tests having different trade names but identical methods and data. The study design was prospective in 41.7% (10 of 24). The pooled sensitivity of the most commonly used method, lateral flow/immunochromatographic, was 95% (95% confidence interval [CI] 94-96) and the pooled specificity was 98% (96-98); 7108 patients. The pooled sensitivity of the polymerase chain reaction or molecular methods was 98% (95% CI 96-98) and the pooled specificity was 96% (95% CI 95-97); 5685 patients. Package inserts include sponsored studies that overestimate the sensitivity of rapid tests to diagnose GAS pharyngitis by approximately 10%. Physicians should understand that package inserts overestimate diagnostic test utility; a negative test cannot be used to exclude GAS pharyngitis.

Direct detection of microRNAs using isothermal amplification and molecular beacon with excellent sensitivity and specificity

NASA Astrophysics Data System (ADS)

Zhang, Wancun; Zhang, Qi; Qian, Zhiyu; Gu, Yueqing

2017-02-01

MicroRNAs (miRNAs) play important roles in a wide range of biological processes, including proliferation, development, metabolism, immunological response, tumorigenesis, and viral infection. The detection of miRNAs is imperative for gaining a better understanding of the functions of these biomolecules and has great potential for the early diagnosis of human disease as well as the discovery of new drugs through the use of miRNAs as targets. In this article, we develop a highly sensitive, and specific miRNA assay based on the two-stage isothermal amplification reactions and molecular beacon. The two-stage isothermal amplification reactions involves two templates and two-stage amplification reactions under isothermal conditions. The first template enables the amplification of miRNA, and the second template enables the conversion of miRNA to the reporter oligonucleotide(Y). Importantly, different miRNAs can be converted to the same Y seperately, which can hybridize with the same set of molecular beacon to generate fluorescent signals. This assay is highly sensitive and specific with a detection limit of 1 fM and can even discriminate single-nucleotide differences. Moreover, in combination with the specific templates, this method can be applied for multiplex miRNA assay by simply using the same molecular beacon. This method has potential to become a promising miRNA quantification method in biomedical research and clinical diagnosis.

Avian influenza virus RNA extraction

USDA-ARS?s Scientific Manuscript database

The efficient extraction and purification of viral RNA is critical for down-stream molecular applications whether it is the sensitive and specific detection of virus in clinical samples, virus gene cloning and expression, or quantification of avian influenza (AI) virus by molecular methods from expe...

The market trend analysis and prospects of cancer molecular diagnostics kits.

PubMed

Seo, Ju Hwan; Lee, Joon Woo; Cho, Daemyeong

2018-01-01

The molecular diagnostics market can be broadly divided into PCR (rt-PCR, d-PCR), NGS(Next Generation Sequencing), Microarray, FISH(Fluorescent in situ-hybridization) and other categories, based on the diagnostic technique. Also, depending on the disease being diagnosed, the market can also be divided into cancer, infectious diseases, HIV/STDs (herpes, syphilis), and women's health issues such as breast cancer, cervical cancer, ovarian cancer, HPV(human papillomavirus), and vaginitis.Chromosome analysis (including Fluorescent In-situ Hybridization) is one type of blood cancer diagnostic method, which involves the direct detection of individual cells with chromosomal translocation, but there have been problems of sensitivity when using this method. PCR targeting individual genes or the RT (reverse transcription)-PCR method offers outstanding sensitivity, but one drawback is the risk of false-positive reaction caused by contamination of samples, etc. Blood cancer molecular diagnostics kits allow us to overcome these shortcomings, and related products have been under development, with a focus on improving detection sensitivity, enabling multiple tests, and reducing the cost and diagnostic time. Blood cancer molecular diagnostics is usually performed based on platforms such as PCR. The global market for blood cancer molecular diagnostics kits is $ 335.9 million as of 2016 and is expected to reach $ 6980 million in 2026 with an average annual growth rate of 32.9%. The market in South Korea is anticipated to grow at an average annual rate of 28.9%, from $ 3.75 million as of 2016 to $ 60.89 million in 2026. The Market for blood cancer molecular diagnostics kits is judged to be higher in growth possibility due to the increase in the number of cancer patients.

Efficacy of the FilmArray blood culture identification panel for direct molecular diagnosis of infectious diseases from samples other than blood.

PubMed

Micó, Miquel; Navarro, Ferran; de Miniac, Daniela; González, Yésica; Brell, Albert; López, Cristina; Sánchez-Reus, Ferran; Mirelis, Beatriz; Coll, Pere

2015-12-01

Molecular-based techniques reduce the delay in diagnosing infectious diseases and therefore contribute to better patient outcomes. We assessed the FilmArray blood culture identification (BCID) panel (Biofire Diagnostics/bioMérieux) directly on clinical specimens other than blood: cerebrospinal, joint, pleural and ascitic fluids, bronchoscopy samples and abscesses. We compared the results from 88 samples obtained by culture-based techniques. The percentage of agreement between the two methods was 75 % with a Cohen κ value of 0.51. Global sensitivity and specificity using the FilmArray BCID panel were 71 and 97 %, respectively. Sensitivity was poorer in samples with a low bacterial load, such as ascitic and pleural fluids (25 %), whereas the sensitivity for abscess samples was high (89 %). These findings suggest that the FilmArray BCID panel could be useful to perform microbiological diagnosis directly from samples other than positive blood cultures, as it offers acceptable sensitivity and moderate agreement with conventional microbiological methods. Nevertheless, cost-benefit studies should be performed before introducing this method into algorithms for microbiological diagnostics.

Conventional and molecular diagnostic strategies for prosthetic joint infections.

PubMed

Esteban, Jaime; Sorlí, Luisa; Alentorn-Geli, Eduard; Puig, Lluís; Horcajada, Juan P

2014-01-01

An accurate diagnosis of prosthetic joint infection (PJI) is the mainstay for an optimized clinical management. This review analyzes different diagnostic strategies of PJI, with special emphasis on molecular diagnostic tools and their current and future applications. Until now, the culture of periprosthetic tissues has been considered the gold standard for the diagnosis of PJI. However, sonication of the implant increases the sensitivity of those cultures and is being increasingly adopted by many centers. Molecular diagnostic methods compared with intraoperative tissue culture, especially if combined with sonication, have a higher sensitivity, a faster turnaround time and are not influenced by previous antimicrobial therapy. However, they still lack a system for detection of antimicrobial susceptibility, which is crucial for an optimized and less toxic therapy of PJI. More studies are needed to assess the clinical value of these methods and their cost-effectiveness.

Ribozyme-mediated signal augmentation on a mass-sensitive biosensor.

PubMed

Knudsen, Scott M; Lee, Joonhyung; Ellington, Andrew D; Savran, Cagri A

2006-12-20

Mass-based detection methods such as the quartz crystal microbalance (QCM) offer an attractive option to label-based methods; however the sensitivity is generally lower by comparison. In particular, low-molecular-weight analytes can be difficult to detect based on mass addition alone. In this communication, we present the use of effector-dependent ribozymes (aptazymes) as reagents for augmenting small ligand detection on a mass-sensitive device. Two distinct aptazymes were chosen: an L1-ligase-based aptazyme (L1-Rev), which is activated by a small peptide (MW approximately 2.4 kDa) from the HIV-1 Rev protein, and a hammerhead cleavase-based aptazyme (HH-theo3) activated by theophylline (MW = 180 Da). Aptazyme activity was observed in real time, and low-molecular-weight analyte detection has been successfully demonstrated with both aptazymes.

Molecular Tools for Diagnosis of Visceral Leishmaniasis: Systematic Review and Meta-Analysis of Diagnostic Test Accuracy

PubMed Central

de Ruiter, C. M.; van der Veer, C.; Leeflang, M. M. G.; Deborggraeve, S.; Lucas, C.

2014-01-01

Molecular methods have been proposed as highly sensitive tools for the detection of Leishmania parasites in visceral leishmaniasis (VL) patients. Here, we evaluate the diagnostic accuracy of these tools in a meta-analysis of the published literature. The selection criteria were original studies that evaluate the sensitivities and specificities of molecular tests for diagnosis of VL, adequate classification of study participants, and the absolute numbers of true positives and negatives derivable from the data presented. Forty studies met the selection criteria, including PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA), and loop-mediated isothermal amplification (LAMP). The sensitivities of the individual studies ranged from 29 to 100%, and the specificities ranged from 25 to 100%. The pooled sensitivity of PCR in whole blood was 93.1% (95% confidence interval [CI], 90.0 to 95.2), and the specificity was 95.6% (95% CI, 87.0 to 98.6). The specificity was significantly lower in consecutive studies, at 63.3% (95% CI, 53.9 to 71.8), due either to true-positive patients not being identified by parasitological methods or to the number of asymptomatic carriers in areas of endemicity. PCR for patients with HIV-VL coinfection showed high diagnostic accuracy in buffy coat and bone marrow, ranging from 93.1 to 96.9%. Molecular tools are highly sensitive assays for Leishmania detection and may contribute as an additional test in the algorithm, together with a clear clinical case definition. We observed wide variety in reference standards and study designs and now recommend consecutively designed studies. PMID:24829226

A review on nanomechanical resonators and their applications in sensors and molecular transportation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arash, Behrouz; Rabczuk, Timon, E-mail: timon.rabczuk@uni-weimar.de; Jiang, Jin-Wu

2015-06-15

Nanotechnology has opened a new area in science and engineering, leading to the development of novel nano-electromechanical systems such as nanoresonators with ultra-high resonant frequencies. The ultra-high-frequency resonators facilitate wide-ranging applications such as ultra-high sensitive sensing, molecular transportation, molecular separation, high-frequency signal processing, and biological imaging. This paper reviews recent studies on dynamic characteristics of nanoresonators. A variety of theoretical approaches, i.e., continuum modeling, molecular simulations, and multiscale methods, in modeling of nanoresonators are reviewed. The potential application of nanoresonators in design of sensor devices and molecular transportation systems is introduced. The essence of nanoresonator sensors for detection of atomsmore » and molecules with vibration and wave propagation analyses is outlined. The sensitivity of the resonator sensors and their feasibility in detecting different atoms and molecules are particularly discussed. Furthermore, the applicability of molecular transportation using the propagation of mechanical waves in nanoresonators is presented. An extended application of the transportation methods for building nanofiltering systems with ultra-high selectivity is surveyed. The article aims to provide an up-to-date review on the mechanical properties and applications of nanoresonators, and inspire additional potential of the resonators.« less

Molecular diagnostics for human leptospirosis.

PubMed

Waggoner, Jesse J; Pinsky, Benjamin A

2016-10-01

The definitive diagnosis of leptospirosis, which results from infection with spirochetes of the genus Leptospira, currently relies on the use of culture, serological testing (microscopic agglutination testing), and molecular detection. The purpose of this review is to describe new molecular diagnostics for Leptospira and discuss advancements in the use of available methods. Efforts have been focused on improving the clinical sensitivity of Leptospira detection using molecular methods. In this review, we describe a reoptimized pathogenic species-specific real-time PCR (targeting lipL32) that has demonstrated improved sensitivity, findings by two groups that real-time reverse-transcription PCR assays targeting the 16S rrs gene can improve detection, and two new loop-mediated amplification techniques. Quantitation of leptospiremia, detection in different specimen types, and the complementary roles played by molecular detection and microscopic agglutination testing will be discussed. Finally, a protocol for Leptospira strain subtyping using variable number tandem repeat targets and high-resolution melting will be described. Molecular diagnostics have an established role for the diagnosis of leptospirosis and provide an actionable diagnosis in the acute setting. The use of real-time reverse-transcription PCR for testing serum/plasma and cerebrospinal fluid, when available, may improve the detection of Leptospira without decreasing clinical specificity.

Quantification of the fluorine containing drug 5-fluorouracil in cancer cells by GaF molecular absorption via high-resolution continuum source molecular absorption spectrometry

NASA Astrophysics Data System (ADS)

Krüger, Magnus; Huang, Mao-Dong; Becker-Roß, Helmut; Florek, Stefan; Ott, Ingo; Gust, Ronald

The development of high-resolution continuum source molecular absorption spectrometry made the quantification of fluorine feasible by measuring the molecular absorption as gallium monofluoride (GaF). Using this new technique, we developed on the example of 5-fluorouracil (5-FU) a graphite furnace method to quantify fluorine in organic molecules. The effect of 5-FU on the generation of the diatomic GaF molecule was investigated. The experimental conditions such as gallium nitrate amount, temperature program, interfering anions (represented as corresponding acids) and calibration for the determination of 5-FU in standard solution and in cellular matrix samples were investigated and optimized. The sample matrix showed no effect on the sensitivity of GaF molecular absorption. A simple calibration curve using an inorganic sodium fluoride solution can conveniently be used for the calibration. The described method is sensitive and the achievable limit of detection is 0.23 ng of 5-FU. In order to establish the concept of "fluorine as a probe in medicinal chemistry" an exemplary application was selected, in which the developed method was successfully demonstrated by performing cellular uptake studies of the 5-FU in human colon carcinoma cells.

Resonance Rayleigh scattering method for highly sensitive detection of chitosan using aniline blue as probe

NASA Astrophysics Data System (ADS)

Zhang, Weiai; Ma, Caijuan; Su, Zhengquan; Bai, Yan

2016-11-01

This paper describes a highly sensitive and accurate approach using aniline blue (AB) (water soluble) as a probe to determine chitosan (CTS) through Resonance Rayleigh scattering (RRS). Under optimum experimental conditions, the intensities of RRS were linearly proportional to the concentration of CTS in the range from 0.01 to 3.5 μg/mL, and the limit of detection (LOD) was 6.94 ng/mL. Therefore, a new and highly sensitive method based on RRS for the determination of CTS has been developed. Furthermore, the effect of molecular weight of CTS and the effect of the degree of deacetylation of CTS on the accurate quantification of CTS was studied. The experimental data was analyzed by linear regression analysis, which indicated that the molecular weight and the degree of deacetylation of CTS had no statistical significance and this method could be used to determine CTS accurately. Meanwhile, this assay was applied for CTS determination in health products with satisfactory results.

Resonance ionization for analytical spectroscopy

DOEpatents

Hurst, George S.; Payne, Marvin G.; Wagner, Edward B.

1976-01-01

This invention relates to a method for the sensitive and selective analysis of an atomic or molecular component of a gas. According to this method, the desired neutral component is ionized by one or more resonance photon absorptions, and the resultant ions are measured in a sensitive counter. Numerous energy pathways are described for accomplishing the ionization including the use of one or two tunable pulsed dye lasers.

Serological and molecular tools to diagnose visceral leishmaniasis: 2-years’ experience of a single center in Northern Italy

PubMed Central

Ortalli, Margherita; Attard, Luciano; Vanino, Elisa; Gaibani, Paolo; Vocale, Caterina; Rossini, Giada; Cagarelli, Roberto; Pierro, Anna; Billi, Patrizia; Mastroianni, Antonio; Di Cesare, Simona; Codeluppi, Mauro; Franceschini, Erica; Melchionda, Fraia; Gramiccia, Marina; Scalone, Aldo; Gentilomi, Giovanna A.; Landini, Maria P.

2017-01-01

The diagnosis of visceral leishmaniasis (VL) remains challenging, due to the limited sensitivity of microscopy, the poor performance of serological methods in immunocompromised patients and the lack of standardization of molecular tests. The aim of this study was to implement a combined diagnostic workflow by integrating serological and molecular tests with standardized clinical criteria. Between July 2013 and June 2015, the proposed workflow was applied to specimens obtained from 94 in-patients with clinical suspicion of VL in the Emilia-Romagna region, Northern Italy. Serological tests and molecular techniques were employed. Twenty-one adult patients (22%) had a confirmed diagnosis of VL by clinical criteria, serology and/or real-time polymerase chain reaction; 4 of these patients were HIV-positive. Molecular tests exhibited higher sensitivity than serological tests for the diagnosis of VL. In our experience, the rK39 immunochromatographic test was insufficiently sensitive for use as a screening test for the diagnosis of VL caused by L. infantum in Italy. However, as molecular tests are yet not standardized, further studies are required to identify an optimal screening test for Mediterranean VL. PMID:28832646

Towards a high sensitivity small animal PET system based on CZT detectors (Conference Presentation)

NASA Astrophysics Data System (ADS)

Abbaszadeh, Shiva; Levin, Craig

2017-03-01

Small animal positron emission tomography (PET) is a biological imaging technology that allows non-invasive interrogation of internal molecular and cellular processes and mechanisms of disease. New PET molecular probes with high specificity are under development to target, detect, visualize, and quantify subtle molecular and cellular processes associated with cancer, heart disease, and neurological disorders. However, the limited uptake of these targeted probes leads to significant reduction in signal. There is a need to advance the performance of small animal PET system technology to reach its full potential for molecular imaging. Our goal is to assemble a small animal PET system based on CZT detectors and to explore methods to enhance its photon sensitivity. In this work, we reconstruct an image from a phantom using a two-panel subsystem consisting of six CZT crystals in each panel. For image reconstruction, coincidence events with energy between 450 and 570 keV were included. We are developing an algorithm to improve sensitivity of the system by including multiple interaction events.

Method for fabrication and verification of conjugated nanoparticle-antibody tuning elements for multiplexed electrochemical biosensors.

PubMed

La Belle, Jeffrey T; Fairchild, Aaron; Demirok, Ugur K; Verma, Aman

2013-05-15

There is a critical need for more accurate, highly sensitive and specific assay for disease diagnosis and management. A novel, multiplexed, single sensor using rapid and label free electrochemical impedance spectroscopy tuning method has been developed. The key challenges while monitoring multiple targets is frequency overlap. Here we describe the methods to circumvent the overlap, tune by use of nanoparticle (NP) and discuss the various fabrication and characterization methods to develop this technique. First sensors were fabricated using printed circuit board (PCB) technology and nickel and gold layers were electrodeposited onto the PCB sensors. An off-chip conjugation of gold NP's to molecular recognition elements (with verification technique) is described as well. A standard covalent immobilization of the molecular recognition elements is also discussed with quality control techniques. Finally use and verification of sensitivity and specificity is also presented. By use of gold NP's of various sizes, we have demonstrated the possibility and shown little loss of sensitivity and specificity in the molecular recognition of inflammatory markers as "model" targets for our tuning system. By selection of other sized NP's or NP's of various materials, the tuning effect can be further exploited. The novel platform technology developed could be utilized in critical care, clinical management and at home health and disease management. Copyright © 2013 Elsevier Inc. All rights reserved.

Direct Analysis of Low-Volatile Molecular Marker Extract from Airborne Particulate Matter Using Sensitivity Correction Method

PubMed Central

Irei, Satoshi

2016-01-01

Molecular marker analysis of environmental samples often requires time consuming preseparation steps. Here, analysis of low-volatile nonpolar molecular markers (5-6 ring polycyclic aromatic hydrocarbons or PAHs, hopanoids, and n-alkanes) without the preseparation procedure is presented. Analysis of artificial sample extracts was directly conducted by gas chromatography-mass spectrometry (GC-MS). After every sample injection, a standard mixture was also analyzed to make a correction on the variation of instrumental sensitivity caused by the unfavorable matrix contained in the extract. The method was further validated for the PAHs using the NIST standard reference materials (SRMs) and then applied to airborne particulate matter samples. Tests with the SRMs showed that overall our methodology was validated with the uncertainty of ~30%. The measurement results of airborne particulate matter (PM) filter samples showed a strong correlation between the PAHs, implying the contributions from the same emission source. Analysis of size-segregated PM filter samples showed that their size distributions were found to be in the PM smaller than 0.4 μm aerodynamic diameter. The observations were consistent with our expectation of their possible sources. Thus, the method was found to be useful for molecular marker studies. PMID:27127511

Molecular diagnosis of toxoplasmosis in immunocompromised patients.

PubMed

Robert-Gangneux, Florence; Belaz, Sorya

2016-08-01

Toxoplasmosis in immunocompromised patients is associated with a high mortality rate. Molecular techniques are important tools to diagnose acute disease in immunocompromised patients, but there are various methods with variable efficiency. Some of them have been validated for the diagnosis of congenital toxoplasmosis, but the impact of their use has not been evaluated in immunocompromised patients. Toxoplasmosis is of increasing importance in non-HIV immunocompromised patients. In addition, the picture of disease shows greater severity in South America, both in immunocompetent study participants and in congenitally infected infants. These epidemiological differences could influence the sensitivity of diagnostic methods. This review analyzes recent data on molecular diagnosis and compares them with older ones, in light of progress gained in molecular techniques and of recent epidemiological findings. Most recent studies were conducted in South America and used PCR targeting the B1 gene. PCR on blood could allow diagnosing a significant proportion of patients with ocular toxoplasmosis in Brazil. Quantitative PCR methods with specific probes should be used to improve sensitivity and warrant specificity. Performance of quantitative PCR targeting the repeated 529 bp sequence for the diagnosis of toxoplasmosis in immunocompromised patients needs evaluation in field studies in South America and in western countries.

Parasite detection in patients with post kala-azar dermal leishmaniasis in India: a comparison between molecular and immunological methods

PubMed Central

Salotra, P; Sreenivas, G; Beena, K R; Mukherjee, A; Ramesh, V

2003-01-01

Aims: To evaluate the sensitivity and specificity of serological, immunohistochemical, and molecular methods in the diagnosis of post kala-azar dermal leishmaniasis (PKDL). Methods: Twenty five patients with confirmed PKDL and 25 controls were included in the study. G2D10, a monoclonal antibody against Leishmania, was used for the immunohistochemical (IHC) staining of lesion sections to visualise anti-Leishmania donovani antibodies. The diagnostic usefulness of IHC was compared with enzyme linked immunosorbent assay (ELISA) with a recombinant (rk39) antigen, and a species specific polymerase chain reaction (PCR) assay, amplifying a kinetoplast minicircle DNA sequence. Results: IHC detected 22 of 25 PKDL cases, giving a sensitivity of 88%. The diagnostic sensitivity of both the ELISA and PCR tests was higher (96%). All of the 25 controls examined were negative in PCR, indicating 100% specificity of the test, whereas ELISA showed 96% specificity. Conclusions: IHC with G2D10 significantly enhances the sensitivity of detection of PKDL over routine haematoxylin and eosin staining. ELISA with a recombinant antigen is an economical and practical assay. PCR is the most sensitive and specific diagnostic method for PKDL. The tests described would facilitate the recognition of patients with PKDL, enabling timely treatment, which would contribute greatly to the control of kala-azar. PMID:14600129

Excited state X-ray absorption spectroscopy: Probing both electronic and structural dynamics

NASA Astrophysics Data System (ADS)

Neville, Simon P.; Averbukh, Vitali; Ruberti, Marco; Yun, Renjie; Patchkovskii, Serguei; Chergui, Majed; Stolow, Albert; Schuurman, Michael S.

2016-10-01

We investigate the sensitivity of X-ray absorption spectra, simulated using a general method, to properties of molecular excited states. Recently, Averbukh and co-workers [M. Ruberti et al., J. Chem. Phys. 140, 184107 (2014)] introduced an efficient and accurate L 2 method for the calculation of excited state valence photoionization cross-sections based on the application of Stieltjes imaging to the Lanczos pseudo-spectrum of the algebraic diagrammatic construction (ADC) representation of the electronic Hamiltonian. In this paper, we report an extension of this method to the calculation of excited state core photoionization cross-sections. We demonstrate that, at the ADC(2)x level of theory, ground state X-ray absorption spectra may be accurately reproduced, validating the method. Significantly, the calculated X-ray absorption spectra of the excited states are found to be sensitive to both geometric distortions (structural dynamics) and the electronic character (electronic dynamics) of the initial state, suggesting that core excitation spectroscopies will be useful probes of excited state non-adiabatic dynamics. We anticipate that the method presented here can be combined with ab initio molecular dynamics calculations to simulate the time-resolved X-ray spectroscopy of excited state molecular wavepacket dynamics.

Apparatus for molecular weight separation

DOEpatents

Smith, Richard D.; Liu, Chuanliang

2001-01-01

The present invention relates generally to an apparatus and method for separating high molecular weight molecules from low molecular weight molecules. More specifically, the invention relates to the use of microdialysis for removal of the salt (low molecular weight molecules) from a nucleotide sample (high molecular weight molecules) for ESI-MS analysis. The dialysis or separation performance of the present invention is improved by (1) increasing dialysis temperature thereby increasing desalting efficiency and improving spectrum quality; (2) adding piperidine and imidazole to the dialysis buffer solution and reducing charge states and further increasing detection sensitivity for DNA; (3) using low concentrations (0-2.5 mM NH4OAc) of dialysis buffer and shifting the DNA negative ions to higher charge states, producing a nearly 10-fold increase in detection sensitivity and a slightly decreased desalting efficiency, (4) conducting a two-stage separation or (5) any combination of (1), (2), (3) and (4).

Microdialysis unit for molecular weight separation

DOEpatents

Smith, Richard D.; Liu, Chuanliang

1999-01-01

The present invention relates generally to an apparatus and method for separating high molecular weight molecules from low molecular weight molecules. More specifically, the invention relates to the use of microdialysis for removal of the salt (low molecular weight molecules) from a nucleotide sample (high molecular weight molecules) for ESI-MS analysis. The dialysis or separation performance of the present invention is improved by (1) increasing dialysis temperature thereby increasing desalting efficiency and improving spectrum quality; (2) adding piperidine and imidazole to the dialysis buffer solution and reducing charge states and further increasing detection sensitivity for DNA; (3) using low concentrations (0-2.5 mM NH4OAc) of dialysis buffer and shifting the DNA negative ions to higher charge states, producing a nearly 10-fold increase in detection sensitivity and a slightly decreased desalting efficiency, or (4) any combination of (1), (2), and (3).

Molecular tools for diagnosis of visceral leishmaniasis: systematic review and meta-analysis of diagnostic test accuracy.

PubMed

de Ruiter, C M; van der Veer, C; Leeflang, M M G; Deborggraeve, S; Lucas, C; Adams, E R

2014-09-01

Molecular methods have been proposed as highly sensitive tools for the detection of Leishmania parasites in visceral leishmaniasis (VL) patients. Here, we evaluate the diagnostic accuracy of these tools in a meta-analysis of the published literature. The selection criteria were original studies that evaluate the sensitivities and specificities of molecular tests for diagnosis of VL, adequate classification of study participants, and the absolute numbers of true positives and negatives derivable from the data presented. Forty studies met the selection criteria, including PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA), and loop-mediated isothermal amplification (LAMP). The sensitivities of the individual studies ranged from 29 to 100%, and the specificities ranged from 25 to 100%. The pooled sensitivity of PCR in whole blood was 93.1% (95% confidence interval [CI], 90.0 to 95.2), and the specificity was 95.6% (95% CI, 87.0 to 98.6). The specificity was significantly lower in consecutive studies, at 63.3% (95% CI, 53.9 to 71.8), due either to true-positive patients not being identified by parasitological methods or to the number of asymptomatic carriers in areas of endemicity. PCR for patients with HIV-VL coinfection showed high diagnostic accuracy in buffy coat and bone marrow, ranging from 93.1 to 96.9%. Molecular tools are highly sensitive assays for Leishmania detection and may contribute as an additional test in the algorithm, together with a clear clinical case definition. We observed wide variety in reference standards and study designs and now recommend consecutively designed studies. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Molecular imaging in neuroendocrine tumors: molecular uptake mechanisms and clinical results.

PubMed

Koopmans, Klaas P; Neels, Oliver N; Kema, Ido P; Elsinga, Philip H; Links, Thera P; de Vries, Elisabeth G E; Jager, Pieter L

2009-09-01

Neuroendocrine tumors can originate almost everywhere in the body and consist of a great variety of subtypes. This paper focuses on molecular imaging methods using nuclear medicine techniques in neuroendocrine tumors, coupling molecular uptake mechanisms of radiotracers with clinical results. A non-systematic review is presented on receptor based and metabolic imaging methods. Receptor-based imaging covers the molecular backgrounds of somatostatin, vaso-intestinal peptide (VIP), bombesin and cholecystokinin (CCK) receptors and their link with nuclear imaging. Imaging methods based on specific metabolic properties include meta-iodo-benzylguanide (MIBG) and dimercapto-sulphuric acid (DMSA-V) scintigraphy as well as more modern positron emission tomography (PET)-based methods using radio-labeled analogues of amino acids, glucose, dihydroxyphenylalanine (DOPA), dopamine and tryptophan. Diagnostic sensitivities are presented for each imaging method and for each neuroendocrine tumor subtype. Finally, a Forest plot analysis of diagnostic performance is presented for each tumor type in order to provide a comprehensive overview for clinical use.

Perspectives on Non-Animal Alternatives for Assessing Sensitization Potential in Allergic Contact Dermatitis

PubMed Central

Sharma, Nripen S.; Jindal, Rohit; Mitra, Bhaskar; Lee, Serom; Li, Lulu; Maguire, Tim J.; Schloss, Rene; Yarmush, Martin L.

2014-01-01

Skin sensitization remains a major environmental and occupational health hazard. Animal models have been used as the gold standard method of choice for estimating chemical sensitization potential. However, a growing international drive and consensus for minimizing animal usage have prompted the development of in vitro methods to assess chemical sensitivity. In this paper, we examine existing approaches including in silico models, cell and tissue based assays for distinguishing between sensitizers and irritants. The in silico approaches that have been discussed include Quantitative Structure Activity Relationships (QSAR) and QSAR based expert models that correlate chemical molecular structure with biological activity and mechanism based read-across models that incorporate compound electrophilicity. The cell and tissue based assays rely on an assortment of mono and co-culture cell systems in conjunction with 3D skin models. Given the complexity of allergen induced immune responses, and the limited ability of existing systems to capture the entire gamut of cellular and molecular events associated with these responses, we also introduce a microfabricated platform that can capture all the key steps involved in allergic contact sensitivity. Finally, we describe the development of an integrated testing strategy comprised of two or three tier systems for evaluating sensitization potential of chemicals. PMID:24741377

Sensitivity Analysis and Uncertainty Quantification for the LAMMPS Molecular Dynamics Simulation Code

DOE Office of Scientific and Technical Information (OSTI.GOV)

Picard, Richard Roy; Bhat, Kabekode Ghanasham

2017-07-18

We examine sensitivity analysis and uncertainty quantification for molecular dynamics simulation. Extreme (large or small) output values for the LAMMPS code often occur at the boundaries of input regions, and uncertainties in those boundary values are overlooked by common SA methods. Similarly, input values for which code outputs are consistent with calibration data can also occur near boundaries. Upon applying approaches in the literature for imprecise probabilities (IPs), much more realistic results are obtained than for the complacent application of standard SA and code calibration.

Diagnosis of amphimeriasis by LAMPhimerus assay in human stool samples long-term storage onto filter paper

PubMed Central

Calvopiña, Manuel; Buendía-Sánchez, María; López-Abán, Julio; Vicente, Belén; Muro, Antonio

2018-01-01

Amphimeriasis, a fish-borne zoonotic disease caused by the liver fluke Amphimerus spp., has recently been reported as an emerging disease affecting an indigenous Ameridian group, the Chachi, living in Ecuador. The only method for diagnosing amphimeriasis was the microscopic detection of eggs from the parasite in patients' stool samples with very low sensitivity. Our group developed an ELISA technique for detection of anti-Amphimerus IgG in human sera and a molecular method based on LAMP technology (named LAMPhimerus) for specific and sensitive parasite DNA detection. The LAMPhimerus method showed to be much more sensitive than classical parasitological methods for amphimeriasis diagnosis using human stool samples for analysis. The objective of this work is to demonstrate the feasibility of using dried stool samples on filter paper as source of DNA in combination with the effectiveness of our previously designed LAMPhimerus assay for successfully Amphimerus sp. detection in clinical stool samples. A total of 102 untreated and undiluted stool samples collected from Chachi population were spread as thin layer onto common filter paper for easily transportation to our laboratory and stored at room temperature for one year until DNA extraction. When LAMPhimerus method was applied for Amphimerus sp. DNA detection, a higher number of positive results was detected (61/102; 59.80%) in comparison to parasitological methods (38/102; 37.25%), including 28/61 (45.90%) microscopy-confirmed Amphimerus sp. infections. The diagnostic parameters for the sensitivity and specificity werecalculated for our LAMPhimerus assay, which were 79.17% and 65.98%, respectively. We demonstrate, for the first time, that common filter paper is useful for easy collection and long-term storage of human stool samples for later DNA extraction and molecular analysis of human-parasitic trematode eggs. This simple, economic and easily handling method combined with the specific and sensible LAMPhimerus assay has the potential to beused as an effective molecular large-scale screening test for amphimeriasis-endemic areas. PMID:29444135

A method for fast safety screening of explosives in terms of crystal packing and molecular stability.

PubMed

Hu, Xiaohua; Chen, Nana; Li, Weichen

2016-07-01

Safety prediction is crucial to the molecular design or the material design of explosives, and the predictions based on any single factor alone will cause much inaccuracy, leading to a desire for a method on multi-bases. The presented proposes an improved method for fast screening explosive safety by combining a crystal packing factor and a molecular one, that is, steric hindrance against shear slide in crystal and molecular stability, denoted by intermolecular friction symbol (IFS) and bond dissociation energy (BDE) of trigger linkage respectively. Employing this BDE-IFS combined method, we understand the impact sensitivities of 24 existing explosives, and predict those of two energetic-energetic cocrystals of the observed CL-20/BTF and the supposed HMX/TATB. As a result, a better understanding is implemented by the combined method relative to molecular stability alone, verifying its improvement of more accurate predictions and the feasibility of IFS to graphically reflect molecular stacking in crystals. Also, this work verifies that the explosive safety is strongly related with its crystal stacking, which determines steric hindrance and influences shear slide.

Real-time fluorescence loop mediated isothermal amplification for the diagnosis of malaria.

PubMed

Lucchi, Naomi W; Demas, Allison; Narayanan, Jothikumar; Sumari, Deborah; Kabanywanyi, Abdunoor; Kachur, S Patrick; Barnwell, John W; Udhayakumar, Venkatachalam

2010-10-29

Molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. The recently developed loop-mediated isothermal amplification (LAMP) method is relatively simple and can be improved for better use in endemic countries. In this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform. We refer to this as the RealAmp method. Published genus-specific primers were used to test the utility of this method. DNA derived from different species of malaria parasites was used for the initial characterization. Clinical samples of P. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested PCR method. Additionally, directly boiled parasite preparations were compared with a conventional DNA isolation method. The RealAmp method was found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was generally less than 60 minutes. All human-infecting Plasmodium species were detected. The sensitivity and specificity of RealAmp in detecting P. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested PCR method. In addition, this method consistently detected P. falciparum from directly boiled blood samples. This RealAmp method has great potential as a field usable molecular tool for diagnosis of malaria. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and operational programs.

SYMPOSIUM ON MULTIMODALITY CARDIOVASCULAR MOLECULAR IMAGING IMAGING TECHNOLOGY - PART 2

PubMed Central

de Kemp, Robert A.; Epstein, Frederick H.; Catana, Ciprian; Tsui, Benjamin M.W.; Ritman, Erik L.

2013-01-01

Rationale The ability to trace or identify specific molecules within a specific anatomic location provides insight into metabolic pathways, tissue components and tracing of solute transport mechanisms. With the increasing use of small animals for research such imaging must have sufficiently high spatial resolution to allow anatomic localization as well as sufficient specificity and sensitivity to provide an accurate description of the molecular distribution and concentration. Methods Imaging methods based on electromagnetic radiation, such as PET, SPECT, MRI and CT, are increasingly applicable due to recent advances in novel scanner hardware, image reconstruction software and availability of novel molecules which have enhanced sensitivity in these methodologies. Results Micro-PET has been advanced by development of detector arrays that provide higher resolution and positron emitting elements that allow new molecular tracers to be labeled. Micro-MRI has been improved in terms of spatial resolution and sensitivity by increased magnet field strength and development of special purpose coils and associated scan protocols. Of particular interest is the associated ability to image local mechanical function and solute transport processes which can be directly related to the molecular information. This is further strengthened by the synergistic integration of the PET with MRI. Micro-SPECT has been improved by use of coded aperture imaging approaches as well as image reconstruction algorithms which can better deal with the photon limited scan data. The limited spatial resolution can be partially overcome by integrating the SPECT with CT. Micro-CT by itself provides exquisite spatial resolution of anatomy, but recent developments of high spatial resolution photon counting and spectrally-sensitive imaging arrays, combined with x-ray optical devices, have promise for actual molecular identification by virtue of the chemical bond lengths of molecules, especially of bio-polymers. Conclusion With the increasing use of small animals for evaluating new clinical imaging techniques as well as providing increased insights into patho-physiological phenomena, the availability of improved detection systems, scanning protocols and associated software, the repertoire of molecular imaging is greatly increased in sensitivity and specificity. PMID:20457793

Comparison of Nested Polymerase Chain Reaction and Real-Time Polymerase Chain Reaction with Parasitological Methods for Detection of Strongyloides stercoralis in Human Fecal Samples

PubMed Central

Sharifdini, Meysam; Mirhendi, Hossein; Ashrafi, Keyhan; Hosseini, Mostafa; Mohebali, Mehdi; Khodadadi, Hossein; Kia, Eshrat Beigom

2015-01-01

This study was performed to evaluate nested polymerase chain reaction (PCR) and real-time PCR methods for detection of Strongyloides stercoralis in fecal samples compared with parasitological methods. A total of 466 stool samples were examined by conventional parasitological methods (formalin ether concentration [FEC] and agar plate culture [APC]). DNA was extracted using an in-house method, and mitochondrial cytochrome c oxidase subunit 1 and 18S ribosomal genes were amplified by nested PCR and real-time PCR, respectively. Among 466 samples, 12.7% and 18.2% were found infected with S. stercoralis by FEC and APC, respectively. DNA of S. stercoralis was detected in 18.9% and 25.1% of samples by real-time PCR and nested PCR, respectively. Considering parasitological methods as the diagnostic gold standard, the sensitivity and specificity of nested PCR were 100% and 91.6%, respectively, and that of real-time PCR were 84.7% and 95.8%, respectively. However, considering sequence analyzes of the selected nested PCR products, the specificity of nested PCR is increased. In general, molecular methods were superior to parasitological methods. They were more sensitive and more reliable in detection of S. stercoralis in comparison with parasitological methods. Between the two molecular methods, the sensitivity of nested PCR was higher than real-time PCR. PMID:26350449

Determination of phenformin hydrochloride using molecular imprinting technology coupled with flow-injection chemiluminescence.

PubMed

Liu, Zhenbo; Jia, Fengyan; Wang, Wenwen; Wang, Cuixia; Liu, Yongming

2012-01-01

A novel method was developed using molecular imprinting technology (MIT) coupled with flow-injection chemiluminescence (FI-CL) for highly sensitive detection of phenformin hydrochloride (PH). The phenformin imprinted polymer was synthesized with methacrylic acid (MAA) as a functional monomer and ethylene glycol dimethacrylate (EGDMA) as a cross-linker. Newly synthesized molecularly imprinted polymer (MIP) particles were packed into a column as a selective recognition element for determination of PH. A CL method for the determination of PH was developed based on the CL reaction of PH with N-bromosuccinimide sensitized by eosin Y in basic media. The optimization of detection conditions was investigated. The CL intensity responded linearly to the concentration of PH in the range 0.09-2.0 µg/mL, with a correlation coefficient of 0.9920. The detection limit was 0.031 µg/mL. The relative standard deviation for the determination of 1.0 µg/mL PH solution was 1.0% (n = 11). The method was applied to the determination of PH in urine samples, with satisfactory results. Copyright © 2011 John Wiley & Sons, Ltd.

Stimulated Raman scattering (SRS) spectroscopic OCT (Conference Presentation)

NASA Astrophysics Data System (ADS)

Robles, Francisco E.; Zhou, Kevin C.; Fischer, Martin C.; Warren, Warren S.

2017-02-01

Optical coherence tomography (OCT) enables non-invasive, high-resolution, tomographic imaging of biological tissues by leveraging principles of low coherence interferometry; however, OCT lacks molecular specificity. Spectroscopic OCT (SOCT) overcomes this limitation by providing depth-resolved spectroscopic signatures of chromophores, but SOCT has been limited to a couple of endogenous molecules, namely hemoglobin and melanin. Stimulated Raman scattering, on the other hand, can provide highly specific molecular information of many endogenous species, but lacks the spatial and spectral multiplexing capabilities of SOCT. In this work we integrate the two methods, SRS and SOCT, to enable simultaneously multiplexed spatial and spectral imaging with sensitivity to many endogenous biochemical species that play an important role in biology and medicine. The method, termed SRS-SOCT, has the potential to achieve fast, volumetric, and highly sensitive label-free molecular imaging, which would be valuable for many applications. We demonstrate the approach by imaging excised human adipose tissue and detecting the lipids' Raman signatures in the high-wavenumber region. Details of this method along with validations and results will be presented.

A combination of immunohistochemistry and molecular approaches improves highly sensitive detection of BRAF mutations in papillary thyroid cancer.

PubMed

Martinuzzi, Claudia; Pastorino, Lorenza; Andreotti, Virginia; Garuti, Anna; Minuto, Michele; Fiocca, Roberto; Bianchi-Scarrà, Giovanna; Ghiorzo, Paola; Grillo, Federica; Mastracci, Luca

2016-09-01

The optimal method for BRAF mutation detection remains to be determined despite advances in molecular detection techniques. The aim of this study was to compare, against classical Sanger sequencing, the diagnostic performance of two of the most recently developed, highly sensitive methods: BRAF V600E immunohistochemistry (IHC) and peptide nucleic-acid (PNA)-clamp qPCR. BRAF exon 15 mutations were searched in formalin-fixed paraffin-embedded tissues from 86 papillary thyroid carcinoma using the three methods. The limits of detection of Sanger sequencing in borderline or discordant cases were quantified by next generation sequencing. BRAF mutations were found in 74.4 % of cases by PNA, in 71 % of cases by IHC, and in 64 % of cases by Sanger sequencing. Complete concordance for the three methods was observed in 80 % of samples. Better concordance was observed with the combination of two methods, particularly PNA and IHC (59/64) (92 %), while the combination of PNA and Sanger was concordant in 55 cases (86 %). Sensitivity of the three methods was 99 % for PNA, 94.2 % for IHC, and 89.5 % for Sanger. Our data show that IHC could be used as a cost-effective, first-line method for BRAF V600E detection in daily practice, followed by PNA analysis in negative or uninterpretable cases, as the most efficient method. PNA-clamp quantitative PCR is highly sensitive and complementary to IHC as it also recognizes other mutations besides V600E and it is suitable for diagnostic purposes.

Development of a fast and efficient method for hepatitis A virus concentration from green onion.

PubMed

Zheng, Yan; Hu, Yuan

2017-11-01

Hepatitis A virus (HAV) can cause serious liver disease and even death. HAV outbreaks are associated with the consumption of raw or minimally processed produce, making it a major public health concern. Infections have occurred despite the fact that effective HAV vaccine has been available. Development of a rapid and sensitive HAV detection method is necessary for an investigation of an HAV outbreak. Detection of HAV is complicated by the lack of a reliable culture method. In addition, due to the low infectious dose of HAV, these methods must be very sensitive. Current methods rely on efficient sample preparation and concentration steps followed by sensitive molecular detection techniques. Using green onions which was involved in most recent HAV outbreaks as a representative produce, a method of capturing virus particles was developed using carboxyl-derivatized magnetic beads in this study. Carboxyl beads, like antibody-coated beads or cationic beads, detect HAV at a level as low as 100 pfu/25g of green onions. RNA from virus concentrated in this manner can be released by heat-shock (98°C 5min) for molecular detection without sacrificing sensitivity. Bypassing the RNA extraction procedure saves time and removes multiple manipulation steps, which makes large scale HAV screening possible. In addition, the inclusion of beef extract and pectinase rather than NP40 in the elution buffer improved the HAV liberation from the food matrix over current methods by nearly 10 fold. The method proposed in this study provides a promising tool to improve food risk assessment and protect public health. Published by Elsevier B.V.

Applied Genomics: Data Mining Reveals Species-Specific Malaria Diagnostic Targets More Sensitive than 18S rRNA▿†‡

PubMed Central

Demas, Allison; Oberstaller, Jenna; DeBarry, Jeremy; Lucchi, Naomi W.; Srinivasamoorthy, Ganesh; Sumari, Deborah; Kabanywanyi, Abdunoor M.; Villegas, Leopoldo; Escalante, Ananias A.; Kachur, S. Patrick; Barnwell, John W.; Peterson, David S.; Udhayakumar, Venkatachalam; Kissinger, Jessica C.

2011-01-01

Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treatment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting Plasmodium, differentiating between Plasmodium species, and detecting subclinical infections. Despite available whole-genome sequence data for Plasmodium falciparum and P. vivax, the majority of PCR-based methods still rely on the 18S rRNA gene targets. Historically, this gene has served as the best target for diagnostic assays. However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of nested PCR. New diagnostic targets are needed. Ideal targets will be species specific, highly sensitive, and amenable to both single-step and multiplex PCRs. We have mined the genomes of P. falciparum and P. vivax to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We show that these targets (Pvr47 and Pfr364) exist in 14 to 41 copies and are more sensitive than 18S rRNA when utilized in a single-step PCR. Parasites are routinely detected at levels of 1 to 10 parasites/μl. The reaction can be multiplexed to detect both species in a single reaction. We have examined 7 P. falciparum strains and 91 P. falciparum clinical isolates from Tanzania and 10 P. vivax strains and 96 P. vivax clinical isolates from Venezuela, and we have verified a sensitivity and specificity of ∼100% for both targets compared with a nested 18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a powerful alternative molecular diagnostic method for the detection of P. falciparum and P. vivax in conventional or multiplex PCR platforms. PMID:21525225

Predicting drug-induced liver injury using ensemble learning methods and molecular fingerprints.

PubMed

Ai, Haixin; Chen, Wen; Zhang, Li; Huang, Liangchao; Yin, Zimo; Hu, Huan; Zhao, Qi; Zhao, Jian; Liu, Hongsheng

2018-05-21

Drug-induced liver injury (DILI) is a major safety concern in the drug-development process, and various methods have been proposed to predict the hepatotoxicity of compounds during the early stages of drug trials. In this study, we developed an ensemble model using three machine learning algorithms and 12 molecular fingerprints from a dataset containing 1,241 diverse compounds. The ensemble model achieved an average accuracy of 71.1±2.6%, sensitivity of 79.9±3.6%, specificity of 60.3±4.8%, and area under the receiver operating characteristic curve (AUC) of 0.764±0.026 in five-fold cross-validation and an accuracy of 84.3%, sensitivity of 86.9%, specificity of 75.4%, and AUC of 0.904 in an external validation dataset of 286 compounds collected from the Liver Toxicity Knowledge Base (LTKB). Compared with previous methods, the ensemble model achieved relatively high accuracy and sensitivity. We also identified several substructures related to DILI. In addition, we provide a web server offering access to our models (http://ccsipb.lnu.edu.cn/toxicity/HepatoPred-EL/).

Inkjet-printed gold nanoparticle surfaces for the detection of low molecular weight biomolecules by laser desorption/ionization mass spectrometry.

PubMed

Marsico, Alyssa L M; Creran, Brian; Duncan, Bradley; Elci, S Gokhan; Jiang, Ying; Onasch, Timothy B; Wormhoudt, Joda; Rotello, Vincent M; Vachet, Richard W

2015-11-01

Effective detection of low molecular weight compounds in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is often hindered by matrix interferences in the low m/z region of the mass spectrum. Here, we show that monolayer-protected gold nanoparticles (AuNPs) can serve as alternate matrices for the very sensitive detection of low molecular weight compounds such as amino acids. Amino acids can be detected at low fmol levels with minimal interferences by properly choosing the AuNP deposition method, density, size, and monolayer surface chemistry. By inkjet-printing AuNPs at various densities, we find that AuNP clusters are essential for obtaining the greatest sensitivity. Graphical Abstract ᅟ.

Quantum Dot-Fullerene Based Molecular Beacon Nanosensors for Rapid, Highly Sensitive Nucleic Acid Detection.

PubMed

Liu, Ye; Kannegulla, Akash; Wu, Bo; Cheng, Li-Jing

2018-05-15

Spherical fullerene (C 60 ) can quench the fluorescence of a quantum dot (QD) through energy transfer and charge transfer processes, with the quenching efficiency regulated by the number of proximate C 60 on each QD. With the quenching property and its small size compared with other nanoparticle-based quenchers, it is advantageous to group a QD reporter and multiple C 60 -labeled oligonucleotide probes to construct a molecular beacon (MB) probe for sensitive, robust nucleic acid detection. We demonstrated a rapid, high-sensitivity DNA detection method using the nanosensors composed of QD-C 60 based MBs carried by magnetic nanoparticles (MNPs). The assay was accelerated by first dispersing the nanosensors in analytes for highly efficient DNA capture resulting from short-distance 3-dimensional diffusion of targets to the sensor surface and then concentrating the nanosensors to a substrate by magnetic force to amplify the fluorescence signal for target quantification. The enhanced mass transport enabled a rapid detection (< 10 min) with a small sample volume (1-10 µl). The high signal-to-noise ratio produced by the QD-C 60 pairs and magnetic concentration yielded a detection limit of 100 fM (~106 target DNA copies for a 10 µl analyte). The rapid, sensitive, label-free detection method will benefit the applications in point-of-care molecular diagnostic technologies.

Evaluation by latent class analysis of a magnetic capture based DNA extraction followed by real-time qPCR as a new diagnostic method for detection of Echinococcus multilocularis in definitive hosts.

PubMed

Maas, Miriam; van Roon, Annika; Dam-Deisz, Cecile; Opsteegh, Marieke; Massolo, Alessandro; Deksne, Gunita; Teunis, Peter; van der Giessen, Joke

2016-10-30

A new method, based on a magnetic capture based DNA extraction followed by qPCR, was developed for the detection of the zoonotic parasite Echinococcus multilocularis in definitive hosts. Latent class analysis was used to compare this new method with the currently used phenol-chloroform DNA extraction followed by single tube nested PCR. In total, 60 red foxes and coyotes from three different locations were tested with both molecular methods and the sedimentation and counting technique (SCT) or intestinal scraping technique (IST). Though based on a limited number of samples, it could be established that the magnetic capture based DNA extraction followed by qPCR showed similar sensitivity and specificity as the currently used phenol-chloroform DNA extraction followed by single tube nested PCR. All methods have a high specificity as shown by Bayesian latent class analysis. Both molecular assays have higher sensitivities than the combined SCT and IST, though the uncertainties in sensitivity estimates were wide for all assays tested. The magnetic capture based DNA extraction followed by qPCR has the advantage of not requiring hazardous chemicals like the phenol-chloroform DNA extraction followed by single tube nested PCR. This supports the replacement of the phenol-chloroform DNA extraction followed by single tube nested PCR by the magnetic capture based DNA extraction followed by qPCR for molecular detection of E. multilocularis in definitive hosts. Copyright © 2016 Elsevier B.V. All rights reserved.

KRAS and BRAF Mutation Detection: Is Immunohistochemistry a Possible Alternative to Molecular Biology in Colorectal Cancer?

PubMed Central

Borrini, Francesco; Bolognese, Antonio; Lamy, Aude; Sabourin, Jean-Christophe

2015-01-01

KRAS genotyping is mandatory in metastatic colorectal cancer treatment prior to undertaking antiepidermal growth factor receptor (EGFR) monoclonal antibody therapy. BRAF V600E mutation is often present in colorectal carcinoma with CpG island methylator phenotype and microsatellite instability. Currently, KRAS and BRAF evaluation is based on molecular biology techniques such as SNaPshot or Sanger sequencing. As molecular testing is performed on formalin-fixed paraffin-embedded (FFPE) samples, immunodetection would appear to be an attractive alternative for detecting mutations. Thus, our objective was to assess the validity of KRAS and BRAF immunodetection of mutations compared with the genotyping reference method in colorectal adenocarcinoma. KRAS and BRAF genotyping was assessed by SNaPshot. A rabbit anti-human KRAS polyclonal antibody was tested on 33 FFPE colorectal tumor samples with known KRAS status. Additionally, a mouse anti-human BRAF monoclonal antibody was tested on 30 FFPE tumor samples with known BRAF status. KRAS immunostaining demonstrated both poor sensitivity (27%) and specificity (64%) in detecting KRAS mutation. Conversely, BRAF immunohistochemistry showed perfect sensitivity (100%) and specificity (100%) in detecting V600E mutation. Although molecular biology remains the reference method for detecting KRAS mutation, immunohistochemistry could be an attractive method for detecting BRAF V600E mutation in colorectal cancer. PMID:25983749

Microdisc gel electrophoresis in sodium dodecyl sulfate of organic material from rat otoconial complexes

NASA Technical Reports Server (NTRS)

Ross, M. D.; Pote, K. G.; Rarey, K. E.; Verma, L. M.

1981-01-01

The gravity receptors of all vertebrates utilize a 'test mass' consisting of a complex arrangement of mineral and organic substance that lies over the sensory receptor areas. In most vertebrates, the mineral is a polymorph of calcium carbonate in the form of minute, single crystals called otoconia. An investigation is conducted to determine the number of proteins in otoconial complexes and their molecular weights. The investigation makes use of a microdisk gel electrophoresis method reported by Gainer (1971). The most important finding of the reported research is that analysis of the proteins of the organic material of the otoconial complexes is possible when sensitive microanalytical methods are employed. Further modification of the basic technique employed and the inclusion of other sensitive staining methods should mean that, in the future, protein separation by molecular weight will be possible in sample pools containing only two otoconial masses.

[Histopathological Diagnosis of Invasive Fungal Infections in Formalin-Fixed and Paraffin-Embedded Tissues in Conjunction with Molecular Methods].

PubMed

Shinozaki, Minoru; Tochigi, Naobumi; Sadamoto, Sota; Yamagata Murayama, Somay; Wakayama, Megumi; Nemoto, Tetsuo

2018-01-01

The main objective of this study was to evaluate the relationship between histopathology, polymerase chain reaction (PCR), and in situ hybridization (ISH) for the identification of causative fungi in formalin-fixed and paraffin-embedded (FFPE) tissue specimens. Since pathogenic fungi in tissue specimens can be difficult to identify morphologically, PCR and ISH have been usually employed as auxiliary procedures. However, little comparison has been made on the sensitivity and specificity of PCR and ISH using FFPE specimens. Therefore, to compare and clarify the reproducibility and usefulness of PCR and ISH as auxiliary procedures for histological identification, we performed histopathological review, PCR assays, and ISH to identify pathogenic fungi in 59 FFPE tissue specimens obtained from 49 autopsies. The following are the main findings for this retrospective review: i) even for cases classified as "mold not otherwise specified" (MNOS), two cases could be identified as Aspergillus species by molecular methods; ii) all cases classified as non-zygomycetes mold (NZM) were Aspergillus species and were not identified by molecular methods as other fungi; iii) all 3 cases classified as zygomycetes mold (ZM) could be identified by molecular methods as Mucorales; iv) except for 1 case identified by molecular methods as Trichosporon spp., 5 cases were originally identified as dimorphic yeast (DY). As a measure of nucleic acid integrity, PCR and ISH successfully detected human and fungal nucleic acids in approximately 60% of the specimens. Detection of Aspergillus DNA by nested PCR assay and by ISH against the A. fumigatus ALP gene were similarly sensitive and significant (p<0.01). Thus, our findings demonstrated the potential risk of error in the classification of fungi based on pathological diagnosis. Combining molecular methods such as ISH and PCR on FFPE specimens with pathological diagnosis should improve diagnostic accuracy of fungal infection.

[Epidemiologic diagnostic of nosocomial suppurative-septic infections of Pseudomonas etiology based on intraspecies typing of causative agent].

PubMed

Fel'dblium, I V; Zakharova, Iu A; Nikolaeva, A M; Fedotova, O S

2013-01-01

Scientific justification of optimization of epidemiologic diagnostic of suppurative-septic infection (SSI) caused by Pseudomonas aeruginosa based on comparability of antibiotic sensitivity and beta-lactamase production. Intraspecies typing of 37 P. aeruginosa strains isolated during microbiological monitoring of 106 patients and 131 objects of clinical environment of surgical and obstetrician hospitals by using a complex ofphenotypic and molecular-biological methods including determination of sensitivity to antibiotics by serial dilutions method and PCR-diagnostics with determination of TEM, SHV, CTX, OXA, MBL, VIM genes was performed. P. aeruginosa strains combined into groups by isolation location during studies turned out to be heterogeneous by sensitivity to antibiotics and beta-lactamase production that allowed to form subgroups of strains by focality attribute. Isolates recovered from different SSI foci had significant differences in minimal inhibitory concentration (MIC) reaching 1024 times. MIC parameter within subgroups did not exceed 8 - 16 consequent dilutions. Use of a complex of phenotypic and molecular-biologic methods of causative agent typing including determination of sensitivity to antibiotics by serial dilutions method and evaluation of beta-lactamase production allowed to establish a mechanism of development of SSI epidemic process caused by P. aeruginosa, detect origins and reservoirs of infection in hospital, modes and factors of transmission and reach maximum justification of epidemiologic control and prophylaxis measures of localization of foci of nosocomial infections of pseudomonas etiology.

A Novel Molecular Test to Diagnose Canine Visceral Leishmaniasis at the Point of Care

PubMed Central

Castellanos-Gonzalez, Alejandro; Saldarriaga, Omar A.; Tartaglino, Lilian; Gacek, Rosana; Temple, Elissa; Sparks, Hayley; Melby, Peter C.; Travi, Bruno L.

2015-01-01

Dogs are the principal reservoir hosts of zoonotic visceral leishmaniasis (VL) but current serological methods are not sensitive enough to detect all subclinically infected animals, which is crucial to VL control programs. Polymerase chain reaction (PCR) methods have greater sensitivity but require expensive equipment and trained personnel, impairing its implementation in endemic areas. We developed a diagnostic test that uses isothermal recombinase polymerase amplification (RPA) to detect Leishmania infantum. This method was coupled with lateral flow (LF) reading with the naked eye to be adapted as a point-of-care test. The L. infantum RPA-LF had an analytical sensitivity similar to real time-PCR, detecting DNA of 0.1 parasites spiked in dog blood, which was equivalent to 40 parasites/mL. There was no cross amplification with dog or human DNA or with Leishmania braziliensis, Leishmania amazonensis, or Trypanosoma cruzi. The test also amplified Leishmania donovani strains (N = 7). In a group of clinically normal dogs (N = 30), RPA-LF detected more subclinical infections than rK39 strip test, a standard serological method (50% versus 13.3% positivity, respectively; P = 0.005). Also, RPA-LF detected L. infantum in noninvasive mucosal samples of dogs with a sensitivity comparable to blood samples. This novel molecular test may have a positive impact in leishmaniasis control programs. PMID:26240156

Measurement issues associated with quantitative molecular biology analysis of complex food matrices for the detection of food fraud.

PubMed

Burns, Malcolm; Wiseman, Gordon; Knight, Angus; Bramley, Peter; Foster, Lucy; Rollinson, Sophie; Damant, Andrew; Primrose, Sandy

2016-01-07

Following a report on a significant amount of horse DNA being detected in a beef burger product on sale to the public at a UK supermarket in early 2013, the Elliott report was published in 2014 and contained a list of recommendations for helping ensure food integrity. One of the recommendations included improving laboratory testing capacity and capability to ensure a harmonised approach for testing for food authenticity. Molecular biologists have developed exquisitely sensitive methods based on the polymerase chain reaction (PCR) or mass spectrometry for detecting the presence of particular nucleic acid or peptide/protein sequences. These methods have been shown to be specific and sensitive in terms of lower limits of applicability, but they are largely qualitative in nature. Historically, the conversion of these qualitative techniques into reliable quantitative methods has been beset with problems even when used on relatively simple sample matrices. When the methods are applied to complex sample matrices, as found in many foods, the problems are magnified resulting in a high measurement uncertainty associated with the result which may mean that the assay is not fit for purpose. However, recent advances in the technology and the understanding of molecular biology approaches have further given rise to the re-assessment of these methods for their quantitative potential. This review focuses on important issues for consideration when validating a molecular biology assay and the various factors that can impact on the measurement uncertainty of a result associated with molecular biology approaches used in detection of food fraud, with a particular focus on quantitative PCR-based and proteomics assays.

Metal oxide-encapsulated dye-sensitized photoanodes for dye-sensitized solar cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hupp, Joseph T.; Son, Ho-Jin

2016-01-12

Dye-sensitized semiconducting metal oxide films for photoanodes, photoanodes incorporating the films and DSCs incorporating the photoanodes are provided. Also provided are methods for making the dye sensitized semiconducting metal oxide films. The methods of making the films are based on the deposition of an encapsulating layer of a semiconducting metal oxide around the molecular anchoring groups of photosensitizing dye molecules adsorbed to a porous film of the semiconducting metal oxide. The encapsulating layer of semiconducting metal oxide is formed in such a way that it is not coated over the chromophores of the adsorbed dye molecules and, therefore, allows themore » dye molecules to remain electrochemically addressable.« less

A Sensitive TLRH Targeted Imaging Technique for Ultrasonic Molecular Imaging

PubMed Central

Hu, Xiaowen; Zheng, Hairong; Kruse, Dustin E.; Sutcliffe, Patrick; Stephens, Douglas N.; Ferrara, Katherine W.

2010-01-01

The primary goals of ultrasound molecular imaging are the detection and imaging of ultrasound contrast agents (microbubbles), which are bound to specific vascular surface receptors. Imaging methods that can sensitively and selectively detect and distinguish bound microbubbles from freely circulating microbubbles (free microbubbles) and surrounding tissue are critically important for the practical application of ultrasound contrast molecular imaging. Microbubbles excited by low frequency acoustic pulses emit wide-band echoes with a bandwidth extending beyond 20 MHz; we refer to this technique as TLRH (transmission at a low frequency and reception at a high frequency). Using this wideband, transient echo, we have developed and implemented a targeted imaging technique incorporating a multi-frequency co-linear array and the Siemens Antares® imaging system. The multi-frequency co-linear array integrates a center 5.4 MHz array, used to receive echoes and produce radiation force, and two outer 1.5 MHz arrays used to transmit low frequency incident pulses. The targeted imaging technique makes use of an acoustic radiation force sub-sequence to enhance accumulation and a TLRH imaging sub-sequence to detect bound microbubbles. The radiofrequency (RF) data obtained from the TLRH imaging sub-sequence are processsed to separate echo signatures between tissue, free microbubbles, and bound microbubbles. By imaging biotin-coated microbubbles targeted to avidin-coated cellulose tubes, we demonstrate that the proposed method has a high contrast-to-tissue ratio (up to 34 dB) and a high sensitivity to bound microbubbles (with the ratio of echoes from bound microbubbles versus free microbubbles extending up to 23 dB). The effects of the imaging pulse acoustic pressure, the radiation force sub-sequence and the use of various slow-time filters on the targeted imaging quality are studied. The TLRH targeted imaging method is demonstrated in this study to provide sensitive and selective detection of bound microbubbles for ultrasound molecularly-targeted imaging. PMID:20178897

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salvador Palau, A.; Eder, S. D., E-mail: sabrina.eder@uib.no; Kaltenbacher, T.

Time-of-flight (TOF) is a standard experimental technique for determining, among others, the speed ratio S (velocity spread) of a molecular beam. The speed ratio is a measure for the monochromaticity of the beam and an accurate determination of S is crucial for various applications, for example, for characterising chromatic aberrations in focussing experiments related to helium microscopy or for precise measurements of surface phonons and surface structures in molecular beam scattering experiments. For both of these applications, it is desirable to have as high a speed ratio as possible. Molecular beam TOF measurements are typically performed by chopping the beammore » using a rotating chopper with one or more slit openings. The TOF spectra are evaluated using a standard deconvolution method. However, for higher speed ratios, this method is very sensitive to errors related to the determination of the slit width and the beam diameter. The exact sensitivity depends on the beam diameter, the number of slits, the chopper radius, and the chopper rotation frequency. We present a modified method suitable for the evaluation of TOF measurements of high speed ratio beams. The modified method is based on a systematic variation of the chopper convolution parameters so that a set of independent measurements that can be fitted with an appropriate function are obtained. We show that with this modified method, it is possible to reduce the error by typically one order of magnitude compared to the standard method.« less

Validation of a next-generation sequencing assay for clinical molecular oncology.

PubMed

Cottrell, Catherine E; Al-Kateb, Hussam; Bredemeyer, Andrew J; Duncavage, Eric J; Spencer, David H; Abel, Haley J; Lockwood, Christina M; Hagemann, Ian S; O'Guin, Stephanie M; Burcea, Lauren C; Sawyer, Christopher S; Oschwald, Dayna M; Stratman, Jennifer L; Sher, Dorie A; Johnson, Mark R; Brown, Justin T; Cliften, Paul F; George, Bijoy; McIntosh, Leslie D; Shrivastava, Savita; Nguyen, Tudung T; Payton, Jacqueline E; Watson, Mark A; Crosby, Seth D; Head, Richard D; Mitra, Robi D; Nagarajan, Rakesh; Kulkarni, Shashikant; Seibert, Karen; Virgin, Herbert W; Milbrandt, Jeffrey; Pfeifer, John D

2014-01-01

Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (≥ 1000× average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4-100.0 for sensitivity and 94.2-100.0 for specificity) or whole-genome sequencing (95% CI = 89.1-100.0 for sensitivity and 99.9-100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2-100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of ≥ 15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Surface Plasmon Resonance-Based Fiber Optic Sensors Utilizing Molecular Imprinting

PubMed Central

Gupta, Banshi D.; Shrivastav, Anand M.; Usha, Sruthi P.

2016-01-01

Molecular imprinting is earning worldwide attention from researchers in the field of sensing and diagnostic applications, due to its properties of inevitable specific affinity for the template molecule. The fabrication of complementary template imprints allows this technique to achieve high selectivity for the analyte to be sensed. Sensors incorporating this technique along with surface plasmon or localized surface plasmon resonance (SPR/LSPR) provide highly sensitive real time detection with quick response times. Unfolding these techniques with optical fiber provide the additional advantages of miniaturized probes with ease of handling, online monitoring and remote sensing. In this review a summary of optical fiber sensors using the combined approaches of molecularly imprinted polymer (MIP) and the SPR/LSPR technique is discussed. An overview of the fundamentals of SPR/LSPR implementation on optical fiber is provided. The review also covers the molecular imprinting technology (MIT) with its elementary study, synthesis procedures and its applications for chemical and biological anlayte detection with different sensing methods. In conclusion, we explore the advantages, challenges and the future perspectives of developing highly sensitive and selective methods for the detection of analytes utilizing MIT with the SPR/LSPR phenomenon on optical fiber platforms. PMID:27589746

Molecular Imprinting of Macromolecules for Sensor Applications

PubMed Central

Saylan, Yeşeren; Yilmaz, Fatma; Özgür, Erdoğan; Derazshamshir, Ali; Yavuz, Handan; Denizli, Adil

2017-01-01

Molecular recognition has an important role in numerous living systems. One of the most important molecular recognition methods is molecular imprinting, which allows host compounds to recognize and detect several molecules rapidly, sensitively and selectively. Compared to natural systems, molecular imprinting methods have some important features such as low cost, robustness, high recognition ability and long term durability which allows molecularly imprinted polymers to be used in various biotechnological applications, such as chromatography, drug delivery, nanotechnology, and sensor technology. Sensors are important tools because of their ability to figure out a potentially large number of analytical difficulties in various areas with different macromolecular targets. Proteins, enzymes, nucleic acids, antibodies, viruses and cells are defined as macromolecules that have wide range of functions are very important. Thus, macromolecules detection has gained great attention in concerning the improvement in most of the studies. The applications of macromolecule imprinted sensors will have a spacious exploration according to the low cost, high specificity and stability. In this review, macromolecules for molecularly imprinted sensor applications are structured according to the definition of molecular imprinting methods, developments in macromolecular imprinting methods, macromolecular imprinted sensors, and conclusions and future perspectives. This chapter follows the latter strategies and focuses on the applications of macromolecular imprinted sensors. This allows discussion on how sensor strategy is brought to solve the macromolecules imprinting. PMID:28422082

Molecular Imprinting of Macromolecules for Sensor Applications.

PubMed

Saylan, Yeşeren; Yilmaz, Fatma; Özgür, Erdoğan; Derazshamshir, Ali; Yavuz, Handan; Denizli, Adil

2017-04-19

Molecular recognition has an important role in numerous living systems. One of the most important molecular recognition methods is molecular imprinting, which allows host compounds to recognize and detect several molecules rapidly, sensitively and selectively. Compared to natural systems, molecular imprinting methods have some important features such as low cost, robustness, high recognition ability and long term durability which allows molecularly imprinted polymers to be used in various biotechnological applications, such as chromatography, drug delivery, nanotechnology, and sensor technology. Sensors are important tools because of their ability to figure out a potentially large number of analytical difficulties in various areas with different macromolecular targets. Proteins, enzymes, nucleic acids, antibodies, viruses and cells are defined as macromolecules that have wide range of functions are very important. Thus, macromolecules detection has gained great attention in concerning the improvement in most of the studies. The applications of macromolecule imprinted sensors will have a spacious exploration according to the low cost, high specificity and stability. In this review, macromolecules for molecularly imprinted sensor applications are structured according to the definition of molecular imprinting methods, developments in macromolecular imprinting methods, macromolecular imprinted sensors, and conclusions and future perspectives. This chapter follows the latter strategies and focuses on the applications of macromolecular imprinted sensors. This allows discussion on how sensor strategy is brought to solve the macromolecules imprinting.

Novel Methods at Molecular Level for Neurotoxicity Testing in 21st Century-Utility of Structure-Activity Relationship

EPA Science Inventory

Current neurotoxicity and developmental neurotoxicity testing methods for hazard identification rely on in vivo neurobehavior, neurophysiological, and gross pathology of the nervous system. These measures may not be sensitive enough to detect small changes caused by realistic ex...

Performance of Four Transport and Storage Systems for Molecular Detection of Multidrug-Resistant Tuberculosis

PubMed Central

Rabodoarivelo, Marie Sylvianne; Imperiale, Bélen; andrianiavomikotroka, Rina; Brandao, Angela; Kumar, Parveen; Singh, Sarman; Ferrazoli, Lucilaine; Morcillo, Nora; Rasolofo, Voahangy; Palomino, Juan Carlos; Vandamme, Peter; Martin, Anandi

2015-01-01

Background Detection of drug-resistant tuberculosis is essential for the control of the disease but it is often hampered by the limitation of transport and storage of samples from remote locations to the reference laboratory. We performed a retrospective field study to evaluate the performance of four supports enabling the transport and storage of samples to be used for molecular detection of drug resistance using the GenoType MTBDRplus. Methods Two hundred Mycobacterium tuberculosis strains were selected and spotted on slides, FTA cards, GenoCards, and in ethanol. GenoType MTBDRplus was subsequently performed with the DNA extracted from these supports. Sensitivity and specificity were calculated and compared to the results obtained by drug susceptibility testing. Results For all supports, the overall sensitivity and specificity for detection of resistance to RIF was between 95% and 100%, and for INH between 95% and 98%. Conclusion The four transport and storage supports showed a good sensitivity and specificity for the detection of resistance to RIF and INH in M. tuberculosis strains using the GenoType MTBDRplus. These supports can be maintained at room temperature and could represent an important alternative cost-effective method useful for rapid molecular detection of drug-resistant TB in low-resource settings. PMID:26431352

Visualizing BPA by molecularly imprinted ratiometric fluorescence sensor based on dual emission nanoparticles.

PubMed

Lu, Hongzhi; Xu, Shoufang

2017-06-15

Construction of ratiometric fluorescent probe often involved in tedious multistep preparation or complicated coupling or chemical modification process. The emergence of dual emission fluorescent nanoparticles would simplify the construction process and avoids the tedious chemical coupling. Herein, we reported a facile strategy to prepare ratiometric fluorescence molecularly imprinted sensor based on dual emission nanoparticles (d-NPs) which comprised of carbon dots and gold nanoclusters for detection of Bisphenol A (BPA). D-NPs emission at 460nm and 580nm were first prepared by seed growth co-microwave method using gold nanoparticles as seeds and glucose as precursor for carbon dots. When they were applied to propose ratiometric fluorescence molecularly imprinted sensor, the preparation process was simplified, and the sensitivity of sensor was improved with detection limit of 29nM, and visualizing BPA was feasible based on the distinguish fluorescence color change. The feasibility of the developed method in real samples was successfully evaluated through the analysis of BPA in water samples with satisfactory recoveries of 95.9-98.9% and recoveries ranging from 92.6% to 98.6% in canned food samples. When detection BPA in positive feeding bottles, the results agree well with those obtained by accredited method. The developed method proposed in this work to prepare ratiometric fluorescence molecularly imprinted sensor based on dual emission nanoparticles proved to be a convenient, reliable and practical way to prepared high sensitive and selective fluorescence sensors. Copyright © 2017 Elsevier B.V. All rights reserved.

Monitoring of occupational and environmental aeroallergens-- EAACI Position Paper. Concerted action of the EAACI IG Occupational Allergy and Aerobiology & Air Pollution.

PubMed

Raulf, M; Buters, J; Chapman, M; Cecchi, L; de Blay, F; Doekes, G; Eduard, W; Heederik, D; Jeebhay, M F; Kespohl, S; Krop, E; Moscato, G; Pala, G; Quirce, S; Sander, I; Schlünssen, V; Sigsgaard, T; Walusiak-Skorupa, J; Wiszniewska, M; Wouters, I M; Annesi-Maesano, I

2014-10-01

Exposure to high molecular weight sensitizers of biological origin is an important risk factor for the development of asthma and rhinitis. Most of the causal allergens have been defined based on their reactivity with IgE antibodies, and in many cases, the molecular structure and function of the allergens have been established. Significant information on allergen levels that cause sensitization and allergic symptoms for several major environmental and occupational allergens has been reported. Monitoring of high molecular weight allergens and allergen carrier particles is an important part of the management of allergic respiratory diseases and requires standardized allergen assessment methods for occupational and environmental (indoor and outdoor) allergen exposure. The aim of this EAACI task force was to review the essential points for monitoring environmental and occupational allergen exposure including sampling strategies and methods, processing of dust samples, allergen analysis, and quantification. The paper includes a summary of different methods for sampling and allergen quantification, as well as their pros and cons for various exposure settings. Recommendations are being made for different exposure scenarios. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Molecular diagnosis of protozoan parasites by Recombinase Polymerase Amplification.

PubMed

Castellanos-Gonzalez, A; White, A C; Melby, P; Travi, B

2018-06-01

Infections caused by protozoan parasites affect millions of people around the world. Traditionally, diagnosis was made by microscopy, which is insensitive and in some cases not specific. Molecular methods are highly sensitive and specific, but equipment costs and personnel training limit its availability only to specialized centers, usually far from populations with the highest risk of infection. Inexpensive methods that can be applied at the point of care (POC), especially in places with limited health infrastructure, would be a major advantage. Isothermal amplification of nucleic acids does not require thermocyclers and is relatively inexpensive and easy to implement. Among isothermal methods, recombinase polymerase amplification (RPA) is sensitive and potentially applicable at POC. We and others have developed RPA diagnostic tests to detect protozoan parasites of medical importance. Overall, our results have shown high specificity with limits of detection similar to PCR. Currently, the optimization of RPA for use at the POC is under development, and in the near future the tests should become available to detect protozoan infections in the field. In this review we discuss the current status, challenges, and future of RPA in the field of molecular diagnosis of protozoan parasites. Copyright © 2018 Elsevier B.V. All rights reserved.

A new visually improved and sensitive loop mediated isothermal amplification (LAMP) for diagnosis of symptomatic falciparum malaria.

PubMed

Mohon, Abu Naser; Elahi, Rubayet; Khan, Wasif A; Haque, Rashidul; Sullivan, David J; Alam, Mohammad Shafiul

2014-06-01

Molecular diagnosis of malaria by nucleotide amplification requires sophisticated and expensive instruments, typically found only in well-established laboratories. Loop-mediated isothermal amplification (LAMP) has provided a new platform for an easily adaptable molecular technique for molecular diagnosis of malaria without the use of expensive instruments. A new primer set has been designed targeting the 18S rRNA gene for the detection of Plasmodium falciparum in whole blood samples. The efficacy of LAMP using the new primer set was assessed in this study in comparison to that of a previously described set of LAMP primers as well as with microscopy and real-time PCR as reference methods for detecting P. falciparum. Pre-addition of hydroxy napthol blue (HNB) in the LAMP reaction caused a distinct color change, thereby improving the visual detection system. The new LAMP assay was found to be 99.1% sensitive compared to microscopy and 98.1% when compared to real-time PCR. Meanwhile, its specificity was 99% and 100% in contrast to microscopy and real-time PCR, respectively. Moreover, the LAMP method was in very good agreement with microscopy and real-time PCR (0.94 and 0.98, respectively). This new LAMP method can detect at least 5parasites/μL of infected blood within 35min, while the other LAMP method tested in this study, could detect a minimum of 100parasites/μL of human blood after 60min of amplification. Thus, the new method is sensitive and specific, can be carried out in a very short time, and can substitute PCR in healthcare clinics and standard laboratories. Copyright © 2014 Elsevier B.V. All rights reserved.

High-resolution melting analysis for bird sexing: a successful approach to molecular sex identification using different biological samples.

PubMed

Morinha, Francisco; Travassos, Paulo; Seixas, Fernanda; Santos, Nuno; Sargo, Roberto; Sousa, Luís; Magalhães, Paula; Cabral, João A; Bastos, Estela

2013-05-01

High-resolution melting (HRM) analysis is a very attractive and flexible advanced post-PCR method with high sensitivity/specificity for simple, fast and cost-effective genotyping based on the detection of specific melting profiles of PCR products. Next generation real-time PCR systems, along with improved saturating DNA-binding dyes, enable the direct acquisition of HRM data after quantitative PCR. Melting behaviour is particularly influenced by the length, nucleotide sequence and GC content of the amplicons. This method is expanding rapidly in several research areas such as human genetics, reproductive biology, microbiology and ecology/conservation of wild populations. Here we have developed a successful HRM protocol for avian sex identification based on the amplification of sex-specific CHD1 fragments. The melting curve patterns allowed efficient sexual differentiation of 111 samples analysed (plucked feathers, muscle tissues, blood and oral cavity epithelial cells) of 14 bird species. In addition, we sequenced the amplified regions of the CHD1 gene and demonstrated the usefulness of this strategy for the genotype discrimination of various amplicons (CHD1Z and CHD1W), which have small size differences, ranging from 2 bp to 44 bp. The established methodology clearly revealed the advantages (e.g. closed-tube system, high sensitivity and rapidity) of a simple HRM assay for accurate sex differentiation of the species under study. The requirements, strengths and limitations of the method are addressed to provide a simple guide for its application in the field of molecular sexing of birds. The high sensitivity and resolution relative to previous real-time PCR methods makes HRM analysis an excellent approach for improving advanced molecular methods for bird sexing. © 2013 Blackwell Publishing Ltd.

Emerging commercial molecular tests for the diagnosis of bloodstream infection.

PubMed

Mwaigwisya, Solomon; Assiri, Rasha Assad M; O'Grady, Justin

2015-05-01

Bloodstream infection (BSI) by microorganisms can lead to sepsis. This condition has a high mortality rate, which rises significantly with delays in initiation of appropriate antimicrobial treatment. Current culture methods for diagnosing BSI have long turnaround times and poor clinical sensitivity. While clinicians wait for culture diagnosis, patients are treated empirically, which can result in inappropriate treatment, undesirable side effects and contribute to drug resistance development. Molecular diagnostics assays that target pathogen DNA can identify pathogens and resistance markers within hours. Early diagnosis improves antibiotic stewardship and is associated with favorable clinical outcomes. Nonetheless, limitations of current molecular diagnostic methods are substantial. This article reviews recent commercially available molecular methods that use pathogen DNA to diagnose BSI, either by testing positive blood cultures or directly testing patient blood. We critically assess these tests and their application in clinical microbiology. A view of future directions in BSI diagnosis is also provided.

[Usefulness of the molecular techniques for detecting and/or identifing of parasites and fungi in humans and animals or pathogens transmitted by ticks (Part I)].

PubMed

Myjak, P; Majewska, A C; Bajer, A; Siński, E; Wedrychowicz, H; Gołab, E; Budak, A; Stańczak, J

2001-01-01

After a long period of using basic microscopic, immunological and biochemical methods for diagnosis, rapid development of nucleic acids investigation enabled introduction of specific and sensitive methods of detection of pathogenic agents on the molecular level. Among others, polymerase chain reaction (PCR), discovered in mid of 80'ies and then automatized, offered an attractive alternative to conventional testing systems. In this paper we describe reliable diagnostic tests widely used in the world, including Poland, and capable of detecting different disease agents as parasites and fungi in clinical specimens and pathogens of emerging zoonotic diseases in ticks. The possibilities of using molecular methods for determination of Plasmodium falciparum drug resistance is also discussed. Moreover, the report offers information concerning kinds of molecular tests and institutions in which there are executed.

Observing phthalate leaching from plasticized polymer films at the molecular level.

PubMed

Zhang, Xiaoxian; Chen, Zhan

2014-05-06

Phthalates, the most widely used plasticizers in poly(vinyl chloride) (PVC), have been extensively studied. In this paper, a highly sensitive, easy, and effective method was developed to examine short-term phthalate leaching from PVC/phthalate films at the molecular level using sum frequency generation vibrational spectroscopy (SFG). Combining SFG and Fourier transform infrared spectroscopy (FTIR), surface and bulk molecular structures of PVC/phthalate films were also comprehensively evaluated during the phthalate leaching process under various environments. The leaching processes of two phthalates, diethyl phthalate (DEP) and dibutyl phthalate (DBP), from the PVC/phthalate films with various weight ratios were studied. Oxygen plasma was applied to treat the PVC/phthalate film surfaces to verify its efficacy on preventing/reducing phthalate leaching from PVC. Our results show that DBP is more stable than DEP in PVC/phthalate films. Even so, DBP molecules were still found to very slowly leach to the environment from PVC at 30 °C, at a rate much slower than DEP. Also, the bulk DBP content substantially influences the DBP leaching. Higher DBP bulk concentration yields less stable DBP molecules in the PVC matrix, allowing molecules to leach from the polymer film more easily. Additionally, DBP leaching is very sensitive to temperature changes; higher temperature can strongly enhance the leaching process. For most cases, the oxygen plasma treatment can effectively prevent phthalate leaching from PVC films (e.g., for samples with low bulk concentrations of DBP-5 and 30 wt %). It is also capable of reducing phthalate leaching from high DBP bulk concentration PVC samples (e.g., 70 wt % DBP in PVC/DBP mixture). This research develops a highly sensitive method to detect chemicals at the molecular level as well as provides surface and bulk molecular structural changes. The method developed here is general and can be applied to detect small amounts of chemical/biological environmental contaminants.

Molecular diagnostic methods for invasive fungal disease: the horizon draws nearer?

PubMed

Halliday, C L; Kidd, S E; Sorrell, T C; Chen, S C-A

2015-04-01

Rapid, accurate diagnostic laboratory tests are needed to improve clinical outcomes of invasive fungal disease (IFD). Traditional direct microscopy, culture and histological techniques constitute the 'gold standard' against which newer tests are judged. Molecular diagnostic methods, whether broad-range or fungal-specific, have great potential to enhance sensitivity and speed of IFD diagnosis, but have varying specificities. The use of PCR-based assays, DNA sequencing, and other molecular methods including those incorporating proteomic approaches such as matrix-assisted laser desorption ionisation-time of flight mass spectroscopy (MALDI-TOF MS) have shown promising results. These are used mainly to complement conventional methods since they require standardisation before widespread implementation can be recommended. None are incorporated into diagnostic criteria for defining IFD. Commercial assays may assist standardisation. This review provides an update of molecular-based diagnostic approaches applicable to biological specimens and fungal cultures in microbiology laboratories. We focus on the most common pathogens, Candida and Aspergillus, and the mucormycetes. The position of molecular-based approaches in the detection of azole and echinocandin antifungal resistance is also discussed.

Current molecular and emerging nanobiotechnology approaches for the detection of microbial pathogens.

PubMed

Theron, Jacques; Eugene Cloete, Thomas; de Kwaadsteniet, Michele

2010-11-01

Waterborne microbial diseases are escalating worldwide increasing the need for powerful and sensitive diagnostics tools. Molecular methodologies, including immunological and nucleic acid-based methods, have only recently been applied in the water sector. Advances in nanotechnology and nanomaterials have opened the door for the development of new diagnostic tools with increased sensitivity and speed, and reduced cost and labor. Quantum dots, flo dots, gold nanoparticles, magnetic nanoparticles, carbon nanotubes, nanowires, and nanocantilevers, with their unique optical and physical properties, have already been applied in nanodiagnostics. Nanobiotechnology, once remaining technical and practical problems has been addressed, will play an important role in the detection of microbial pathogens.

Photoelectrochemical molecular comb

DOEpatents

Thundat, Thomas G [Knoxville, TN; Ferrell, Thomas L [Knoxville, TN; Brown,; Gilbert, M [Knoxville, TN

2007-05-01

A method, system, and apparatus are provided for separating molecules, such as biomolecules. The method, system, and apparatus utilize an electrochemical cell having at least to electrodes, one electrode comprising a photo-sensitive material capable of generating a photopotential. Molecules are moved through an electrolyte medium between the at least two electrodes based upon localized photopotentials.

Photoelectrochemical molecular comb

DOEpatents

Thundat, Thomas G [Knoxville, TN; Ferrell, Thomas L [Knoxville, TN; Brown, Gilbert M [Knoxville, TN

2012-02-07

A method, system, and apparatus are provided for separating molecules, such as biomolecules. The method, system, and apparatus utilize an electrochemical cell having at least two electrodes, one electrode comprising a photo-sensitive material capable of generating a photopotential. Molecules are moved through an electrolyte medium between the at least two electrodes based upon localized photopotentials.

Novel Methods at Molecular Level for Developmental Neurotoxicity Testing in 21st Century-Utility of Structure-Activity Relationship

EPA Science Inventory

Current neurotoxicity and developmental neurotoxicity testing methods for hazard identification rely on in vivo neurobehavior, neurophysiological, and gross pathology of the nervous system. These measures may not be sensitive enough to detect small changes caused by realistic ex...

MOLECULAR TRACKING FECAL CONTAMINATION IN SURFACE WATERS: 16S RDNA VERSUS METAGENOMICS APPROACHES

EPA Science Inventory

Microbial source tracking methods need to be sensitive and exhibit temporal and geographic stability in order to provide meaningful data in field studies. The objective of this study was to use a combination of PCR-based methods to track cow fecal contamination in two watersheds....

Molecular Modeling of Thermodynamic and Transport Properties for CO 2 and Aqueous Brines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Hao; Economou, Ioannis G.; Panagiotopoulos, Athanassios Z.

Molecular simulation techniques using classical force-fields occupy the space between ab initio quantum mechanical methods and phenomenological correlations. In particular, Monte Carlo and molecular dynamics algorithms can be used to provide quantitative predictions of thermodynamic and transport properties of fluids relevant for geologic carbon sequestration at conditions for which experimental data are uncertain or not available. These methods can cover time and length scales far exceeding those of quantum chemical methods, while maintaining transferability and predictive power lacking from phenomenological correlations. The accuracy of predictions depends sensitively on the quality of the molecular models used. Many existing fixed-point-charge models formore » water and aqueous mixtures fail to represent accurately these fluid properties, especially when descriptions covering broad ranges of thermodynamic conditions are needed. Recent work on development of accurate models for water, CO 2, and dissolved salts, as well as their mixtures, is summarized in this Account. Polarizable models that can respond to the different dielectric environments in aqueous versus nonaqueous phases are necessary for predictions of properties over extended ranges of temperatures and pressures. Phase compositions and densities, activity coefficients of the dissolved salts, interfacial tensions, viscosities and diffusivities can be obtained in near-quantitative agreement to available experimental data, using relatively modest computational resources. In some cases, for example, for the composition of the CO 2-rich phase in coexistence with an aqueous phase, recent results from molecular simulations have helped discriminate among conflicting experimental data sets. The sensitivity of properties on the quality of the intermolecular interaction model varies significantly. Properties such as the phase compositions or electrolyte activity coefficients are much more sensitive than phase densities, viscosities, or component diffusivities. Strong confinement effects on physical properties in nanoscale media can also be directly obtained from molecular simulations. Future work on molecular modeling for CO 2 and aqueous brines is likely to be focused on more systematic generation of interaction models by utilizing quantum chemical as well as direct experimental measurements. New ion models need to be developed for use with the current generation of polarizable water models, including ion–ion interactions that will allow for accurate description of dense, mixed brines. Methods will need to be devised that go beyond the use of effective potentials for incorporation of quantum effects known to be important for water, and reactive force fields developed that can handle bond creation and breaking in systems with carbonate and silicate minerals. Lastly, another area of potential future work is the integration of molecular simulation methods in multiscale models for the chemical reactions leading to mineral dissolution and flow within the porous media in underground formations.« less

Molecular Modeling of Thermodynamic and Transport Properties for CO2 and Aqueous Brines.

PubMed

Jiang, Hao; Economou, Ioannis G; Panagiotopoulos, Athanassios Z

2017-04-18

Molecular simulation techniques using classical force-fields occupy the space between ab initio quantum mechanical methods and phenomenological correlations. In particular, Monte Carlo and molecular dynamics algorithms can be used to provide quantitative predictions of thermodynamic and transport properties of fluids relevant for geologic carbon sequestration at conditions for which experimental data are uncertain or not available. These methods can cover time and length scales far exceeding those of quantum chemical methods, while maintaining transferability and predictive power lacking from phenomenological correlations. The accuracy of predictions depends sensitively on the quality of the molecular models used. Many existing fixed-point-charge models for water and aqueous mixtures fail to represent accurately these fluid properties, especially when descriptions covering broad ranges of thermodynamic conditions are needed. Recent work on development of accurate models for water, CO 2 , and dissolved salts, as well as their mixtures, is summarized in this Account. Polarizable models that can respond to the different dielectric environments in aqueous versus nonaqueous phases are necessary for predictions of properties over extended ranges of temperatures and pressures. Phase compositions and densities, activity coefficients of the dissolved salts, interfacial tensions, viscosities and diffusivities can be obtained in near-quantitative agreement to available experimental data, using relatively modest computational resources. In some cases, for example, for the composition of the CO 2 -rich phase in coexistence with an aqueous phase, recent results from molecular simulations have helped discriminate among conflicting experimental data sets. The sensitivity of properties on the quality of the intermolecular interaction model varies significantly. Properties such as the phase compositions or electrolyte activity coefficients are much more sensitive than phase densities, viscosities, or component diffusivities. Strong confinement effects on physical properties in nanoscale media can also be directly obtained from molecular simulations. Future work on molecular modeling for CO 2 and aqueous brines is likely to be focused on more systematic generation of interaction models by utilizing quantum chemical as well as direct experimental measurements. New ion models need to be developed for use with the current generation of polarizable water models, including ion-ion interactions that will allow for accurate description of dense, mixed brines. Methods will need to be devised that go beyond the use of effective potentials for incorporation of quantum effects known to be important for water, and reactive force fields developed that can handle bond creation and breaking in systems with carbonate and silicate minerals. Another area of potential future work is the integration of molecular simulation methods in multiscale models for the chemical reactions leading to mineral dissolution and flow within the porous media in underground formations.

Molecular Modeling of Thermodynamic and Transport Properties for CO 2 and Aqueous Brines

DOE PAGES

Jiang, Hao; Economou, Ioannis G.; Panagiotopoulos, Athanassios Z.

2017-02-24

Molecular simulation techniques using classical force-fields occupy the space between ab initio quantum mechanical methods and phenomenological correlations. In particular, Monte Carlo and molecular dynamics algorithms can be used to provide quantitative predictions of thermodynamic and transport properties of fluids relevant for geologic carbon sequestration at conditions for which experimental data are uncertain or not available. These methods can cover time and length scales far exceeding those of quantum chemical methods, while maintaining transferability and predictive power lacking from phenomenological correlations. The accuracy of predictions depends sensitively on the quality of the molecular models used. Many existing fixed-point-charge models formore » water and aqueous mixtures fail to represent accurately these fluid properties, especially when descriptions covering broad ranges of thermodynamic conditions are needed. Recent work on development of accurate models for water, CO 2, and dissolved salts, as well as their mixtures, is summarized in this Account. Polarizable models that can respond to the different dielectric environments in aqueous versus nonaqueous phases are necessary for predictions of properties over extended ranges of temperatures and pressures. Phase compositions and densities, activity coefficients of the dissolved salts, interfacial tensions, viscosities and diffusivities can be obtained in near-quantitative agreement to available experimental data, using relatively modest computational resources. In some cases, for example, for the composition of the CO 2-rich phase in coexistence with an aqueous phase, recent results from molecular simulations have helped discriminate among conflicting experimental data sets. The sensitivity of properties on the quality of the intermolecular interaction model varies significantly. Properties such as the phase compositions or electrolyte activity coefficients are much more sensitive than phase densities, viscosities, or component diffusivities. Strong confinement effects on physical properties in nanoscale media can also be directly obtained from molecular simulations. Future work on molecular modeling for CO 2 and aqueous brines is likely to be focused on more systematic generation of interaction models by utilizing quantum chemical as well as direct experimental measurements. New ion models need to be developed for use with the current generation of polarizable water models, including ion–ion interactions that will allow for accurate description of dense, mixed brines. Methods will need to be devised that go beyond the use of effective potentials for incorporation of quantum effects known to be important for water, and reactive force fields developed that can handle bond creation and breaking in systems with carbonate and silicate minerals. Lastly, another area of potential future work is the integration of molecular simulation methods in multiscale models for the chemical reactions leading to mineral dissolution and flow within the porous media in underground formations.« less

Rotation Detection Using the Precession of Molecular Electric Dipole Moment

NASA Astrophysics Data System (ADS)

Ke, Yi; Deng, Xiao-Bing; Hu, Zhong-Kun

2017-11-01

We present a method to detect the rotation by using the precession of molecular electric dipole moment in a static electric field. The molecular electric dipole moments are polarized under the static electric field and a nonzero electric polarization vector emerges in the molecular gas. A resonant radio-frequency pulse electric field is applied to realize a 90° flip of the electric polarization vector of a particular rotational state. After the pulse electric field, the electric polarization vector precesses under the static electric field. The rotation induces a shift in the precession frequency which is measured to deduce the angular velocity of the rotation. The fundamental sensitivity limit of this method is estimated. This work is only a proposal and does not involve experimental results.

Reflection mass spectrometry technique for monitoring and controlling composition during molecular beam epitaxy

DOEpatents

Brennan, T.M.; Hammons, B.E.; Tsao, J.Y.

1992-12-15

A method for on-line accurate monitoring and precise control of molecular beam epitaxial growth of Groups III-III-V or Groups III-V-V layers in an advanced semiconductor device incorporates reflection mass spectrometry. The reflection mass spectrometry is responsive to intentional perturbations in molecular fluxes incident on a substrate by accurately measuring the molecular fluxes reflected from the substrate. The reflected flux is extremely sensitive to the state of the growing surface and the measurements obtained enable control of newly forming surfaces that are dynamically changing as a result of growth. 3 figs.

Reflection mass spectrometry technique for monitoring and controlling composition during molecular beam epitaxy

DOEpatents

Brennan, Thomas M.; Hammons, B. Eugene; Tsao, Jeffrey Y.

1992-01-01

A method for on-line accurate monitoring and precise control of molecular beam epitaxial growth of Groups III-III-V or Groups III-V-V layers in an advanced semiconductor device incorporates reflection mass spectrometry. The reflection mass spectrometry is responsive to intentional perturbations in molecular fluxes incident on a substrate by accurately measuring the molecular fluxes reflected from the substrate. The reflected flux is extremely sensitive to the state of the growing surface and the measurements obtained enable control of newly forming surfaces that are dynamically changing as a result of growth.

Development and assessment of molecular diagnostic tests for 15 enteropathogens causing childhood diarrhoea: a multicentre study.

PubMed

Liu, Jie; Kabir, Furqan; Manneh, Jainaba; Lertsethtakarn, Paphavee; Begum, Sharmin; Gratz, Jean; Becker, Steve M; Operario, Darwin J; Taniuchi, Mami; Janaki, Lalitha; Platts-Mills, James A; Haverstick, Doris M; Kabir, Mamun; Sobuz, Shihab U; Nakjarung, Kaewkanya; Sakpaisal, Pimmada; Silapong, Sasikorn; Bodhidatta, Ladaporn; Qureshi, Shahida; Kalam, Adil; Saidi, Queen; Swai, Ndealilia; Mujaga, Buliga; Maro, Athanasia; Kwambana, Brenda; Dione, Michel; Antonio, Martin; Kibiki, Gibson; Mason, Carl J; Haque, Rashidul; Iqbal, Najeeha; Zaidi, Anita K M; Houpt, Eric R

2014-08-01

Childhood diarrhoea can be caused by many pathogens that are difficult to assay in the laboratory. Molecular diagnostic techniques provide a uniform method to detect and quantify candidate enteropathogens. We aimed to develop and assess molecular tests for identification of enteropathogens and their association with disease. We developed and assessed molecular diagnostic tests for 15 enteropathogens across three platforms-PCR-Luminex, multiplex real-time PCR, and TaqMan array card-at five laboratories worldwide. We judged the analytical and clinical performance of these molecular techniques against comparator methods (bacterial culture, ELISA, and PCR) using 867 diarrhoeal and 619 non-diarrhoeal stool specimens. We also measured molecular quantities of pathogens to predict the association with diarrhoea, by univariate logistic regression analysis. The molecular tests showed very good analytical and clinical performance at all five laboratories. Comparator methods had limited sensitivity compared with the molecular techniques (20-85% depending on the target) but good specificity (median 97·3%, IQR 96·5-98·9; mean 95·2%, SD 9·1). Positive samples by comparator methods usually had higher molecular quantities of pathogens than did negative samples, across almost all platforms and for most pathogens (p<0·05). The odds ratio for diarrhoea at a given quantity (measured by quantification cycle, Cq) showed that for most pathogens associated with diarrhoea-including Campylobacter jejuni and Campylobacter coli, Cryptosporidium spp, enteropathogenic Escherichia coli, heat-stable enterotoxigenic E coli, rotavirus, Shigella spp and enteroinvasive E coli, and Vibrio cholerae-the strength of association with diarrhoea increased at higher pathogen loads. For example, Shigella spp at a Cq range of 15-20 had an odds ratio of 8·0 (p<0·0001), but at a Cq range of 25-30 the odds ratio fell to 1·7 (p=0·043). Molecular diagnostic tests can be implemented successfully and with fidelity across laboratories around the world. In the case of diarrhoea, these techniques can detect pathogens with high sensitivity and ascribe diarrhoeal associations based on quantification, including in mixed infections, providing rich and unprecedented measurements of infectious causes. Bill & Melinda Gates Foundation Next Generation Molecular Diagnostics Project. Copyright © 2014 Elsevier Ltd. All rights reserved.

Molecular Signature of Indeterminate Thyroid Lesions: Current Methods to Improve Fine Needle Aspiration Cytology (FNAC) Diagnosis

PubMed Central

Cantara, Silvia; Marzocchi, Carlotta; Pilli, Tania; Cardinale, Sandro; Forleo, Raffaella; Castagna, Maria Grazia; Pacini, Furio

2017-01-01

Fine needle aspiration cytology (FNAC) represents the gold standard for determining the nature of thyroid nodules. It is a reliable method with good sensitivity and specificity. However, indeterminate lesions remain a diagnostic challenge and researchers have contributed molecular markers to search for in cytological material to refine FNAC diagnosis and avoid unnecessary surgeries. Nowadays, several “home-made” methods as well as commercial tests are available to investigate the molecular signature of an aspirate. Moreover, other markers (i.e., microRNA, and circulating tumor cells) have been proposed to discriminate benign from malignant thyroid lesions. Here, we review the literature and provide data from our laboratory on mutational analysis of FNAC material and circulating microRNA expression obtained in the last 6 years. PMID:28383480

Single-molecule spectroscopic methods.

PubMed

Haustein, Elke; Schwille, Petra

2004-10-01

Being praised for the mere fact of enabling the detection of individual fluorophores a dozen years ago, single-molecule techniques nowadays represent standard methods for the elucidation of the structural rearrangements of biologically relevant macromolecules. Single-molecule-sensitive techniques, such as fluorescence correlation spectroscopy, allow real-time access to a multitude of molecular parameters (e.g. diffusion coefficients, concentration and molecular interactions). As a result of various recent advances, this technique shows promise even for intracellular applications. Fluorescence imaging can reveal the spatial localization of fluorophores on nanometer length scales, whereas fluorescence resonance energy transfer supports a wide range of different applications, including real-time monitoring of conformational rearrangements (as in protein folding). Still in their infancy, single-molecule spectroscopic methods thus provide unprecedented insights into basic molecular mechanisms. Copyright 2004 Elsevier Ltd.

Total internal reflection ellipsometry and SPR detection of low molecular weight environmental toxins

NASA Astrophysics Data System (ADS)

Nabok, A. V.; Tsargorodskaya, A.; Hassan, A. K.; Starodub, N. F.

2005-06-01

The environmental toxins, such as herbicides simazine and atrazine, and T2 mycotoxin were registered with the optical methods of surface plasmon resonance (SPR) and recently developed total internal reflection ellipsometry (TIRE). The immune assay approach was exploited for in situ registration of the above low molecular weight toxins with specific antibodies immobilised onto the gold surface via (poly)allylamine hydrochloride layer using electrostatic self-assembly (ESA) technique. The comparison of two methods of SPR and TIRE shows a higher sensitivity of the latter.

Sensitive DNA detection and SNP discrimination using ultrabright SERS nanorattles and magnetic beads for malaria diagnostics.

PubMed

Ngo, Hoan T; Gandra, Naveen; Fales, Andrew M; Taylor, Steve M; Vo-Dinh, Tuan

2016-07-15

One of the major obstacles to implement nucleic acid-based molecular diagnostics at the point-of-care (POC) and in resource-limited settings is the lack of sensitive and practical DNA detection methods that can be seamlessly integrated into portable platforms. Herein we present a sensitive yet simple DNA detection method using a surface-enhanced Raman scattering (SERS) nanoplatform: the ultrabright SERS nanorattle. The method, referred to as the nanorattle-based method, involves sandwich hybridization of magnetic beads that are loaded with capture probes, target sequences, and ultrabright SERS nanorattles that are loaded with reporter probes. Upon hybridization, a magnet was applied to concentrate the hybridization sandwiches at a detection spot for SERS measurements. The ultrabright SERS nanorattles, composed of a core and a shell with resonance Raman reporters loaded in the gap space between the core and the shell, serve as SERS tags for signal detection. Using this method, a specific DNA sequence of the malaria parasite Plasmodium falciparum could be detected with a detection limit of approximately 100 attomoles. Single nucleotide polymorphism (SNP) discrimination of wild type malaria DNA and mutant malaria DNA, which confers resistance to artemisinin drugs, was also demonstrated. These test models demonstrate the molecular diagnostic potential of the nanorattle-based method to both detect and genotype infectious pathogens. Furthermore, the method's simplicity makes it a suitable candidate for integration into portable platforms for POC and in resource-limited settings applications. Copyright © 2016. Published by Elsevier B.V.

Improving statistical inference on pathogen densities estimated by quantitative molecular methods: malaria gametocytaemia as a case study.

PubMed

Walker, Martin; Basáñez, María-Gloria; Ouédraogo, André Lin; Hermsen, Cornelus; Bousema, Teun; Churcher, Thomas S

2015-01-16

Quantitative molecular methods (QMMs) such as quantitative real-time polymerase chain reaction (q-PCR), reverse-transcriptase PCR (qRT-PCR) and quantitative nucleic acid sequence-based amplification (QT-NASBA) are increasingly used to estimate pathogen density in a variety of clinical and epidemiological contexts. These methods are often classified as semi-quantitative, yet estimates of reliability or sensitivity are seldom reported. Here, a statistical framework is developed for assessing the reliability (uncertainty) of pathogen densities estimated using QMMs and the associated diagnostic sensitivity. The method is illustrated with quantification of Plasmodium falciparum gametocytaemia by QT-NASBA. The reliability of pathogen (e.g. gametocyte) densities, and the accompanying diagnostic sensitivity, estimated by two contrasting statistical calibration techniques, are compared; a traditional method and a mixed model Bayesian approach. The latter accounts for statistical dependence of QMM assays run under identical laboratory protocols and permits structural modelling of experimental measurements, allowing precision to vary with pathogen density. Traditional calibration cannot account for inter-assay variability arising from imperfect QMMs and generates estimates of pathogen density that have poor reliability, are variable among assays and inaccurately reflect diagnostic sensitivity. The Bayesian mixed model approach assimilates information from replica QMM assays, improving reliability and inter-assay homogeneity, providing an accurate appraisal of quantitative and diagnostic performance. Bayesian mixed model statistical calibration supersedes traditional techniques in the context of QMM-derived estimates of pathogen density, offering the potential to improve substantially the depth and quality of clinical and epidemiological inference for a wide variety of pathogens.

Advances in Molecular Rotational Spectroscopy for Applied Science

NASA Astrophysics Data System (ADS)

Harris, Brent; Fields, Shelby S.; Pulliam, Robin; Muckle, Matt; Neill, Justin L.

2017-06-01

Advances in chemical sensitivity and robust, solid-state designs for microwave/millimeter-wave instrumentation compel the expansion of molecular rotational spectroscopy as research tool into applied science. It is familiar to consider molecular rotational spectroscopy for air analysis. Those techniques for molecular rotational spectroscopy are included in our presentation of a more broad application space for materials analysis using Fourier Transform Molecular Rotational Resonance (FT-MRR) spectrometers. There are potentially transformative advantages for direct gas analysis of complex mixtures, determination of unknown evolved gases with parts per trillion detection limits in solid materials, and unambiguous chiral determination. The introduction of FT-MRR as an alternative detection principle for analytical chemistry has created a ripe research space for the development of new analytical methods and sampling equipment to fully enable FT-MRR. We present the current state of purpose-built FT-MRR instrumentation and the latest application measurements that make use of new sampling methods.

Imaging-based molecular barcoding with pixelated dielectric metasurfaces

NASA Astrophysics Data System (ADS)

Tittl, Andreas; Leitis, Aleksandrs; Liu, Mingkai; Yesilkoy, Filiz; Choi, Duk-Yong; Neshev, Dragomir N.; Kivshar, Yuri S.; Altug, Hatice

2018-06-01

Metasurfaces provide opportunities for wavefront control, flat optics, and subwavelength light focusing. We developed an imaging-based nanophotonic method for detecting mid-infrared molecular fingerprints and implemented it for the chemical identification and compositional analysis of surface-bound analytes. Our technique features a two-dimensional pixelated dielectric metasurface with a range of ultrasharp resonances, each tuned to a discrete frequency; this enables molecular absorption signatures to be read out at multiple spectral points, and the resulting information is then translated into a barcode-like spatial absorption map for imaging. The signatures of biological, polymer, and pesticide molecules can be detected with high sensitivity, covering applications such as biosensing and environmental monitoring. Our chemically specific technique can resolve absorption fingerprints without the need for spectrometry, frequency scanning, or moving mechanical parts, thereby paving the way toward sensitive and versatile miniaturized mid-infrared spectroscopy devices.

A Highly Sensitive Method for Quantitative Determination of Abscisic Acid 1

PubMed Central

Michler, Charles H.; Lineberger, R. Daniel; Chism, Grady W.

1986-01-01

An abscisic acid derivative was formed by reaction with pentafluorobenzyl bromide which allowed highly sensitive detection by gas-liquid chromatography with electron capture detection. In comparison to the methyl ester derivative, the pentafluorobenzyl derivative of abscisic acid was four times more sensitive to electron capture detection and was stable at room temperature in the presence of ultraviolet light. Derivatization was rapid and the molecular weight of the new compound was confirmed by gas-liquid chromatography-mass spectrometry. PMID:16665076

Specific and Sensitive Isothermal Electrochemical Biosensor for Plant Pathogen DNA Detection with Colloidal Gold Nanoparticles as Probes

NASA Astrophysics Data System (ADS)

Lau, Han Yih; Wu, Haoqi; Wee, Eugene J. H.; Trau, Matt; Wang, Yuling; Botella, Jose R.

2017-01-01

Developing quick and sensitive molecular diagnostics for plant pathogen detection is challenging. Herein, a nanoparticle based electrochemical biosensor was developed for rapid and sensitive detection of plant pathogen DNA on disposable screen-printed carbon electrodes. This 60 min assay relied on the rapid isothermal amplification of target pathogen DNA sequences by recombinase polymerase amplification (RPA) followed by gold nanoparticle-based electrochemical assessment with differential pulse voltammetry (DPV). Our method was 10,000 times more sensitive than conventional polymerase chain reaction (PCR)/gel electrophoresis and could readily identify P. syringae infected plant samples even before the disease symptoms were visible. On the basis of the speed, sensitivity, simplicity and portability of the approach, we believe the method has potential as a rapid disease management solution for applications in agriculture diagnostics.

Specific and Sensitive Isothermal Electrochemical Biosensor for Plant Pathogen DNA Detection with Colloidal Gold Nanoparticles as Probes.

PubMed

Lau, Han Yih; Wu, Haoqi; Wee, Eugene J H; Trau, Matt; Wang, Yuling; Botella, Jose R

2017-01-17

Developing quick and sensitive molecular diagnostics for plant pathogen detection is challenging. Herein, a nanoparticle based electrochemical biosensor was developed for rapid and sensitive detection of plant pathogen DNA on disposable screen-printed carbon electrodes. This 60 min assay relied on the rapid isothermal amplification of target pathogen DNA sequences by recombinase polymerase amplification (RPA) followed by gold nanoparticle-based electrochemical assessment with differential pulse voltammetry (DPV). Our method was 10,000 times more sensitive than conventional polymerase chain reaction (PCR)/gel electrophoresis and could readily identify P. syringae infected plant samples even before the disease symptoms were visible. On the basis of the speed, sensitivity, simplicity and portability of the approach, we believe the method has potential as a rapid disease management solution for applications in agriculture diagnostics.

Pretreatment evaluation of fluorescence resonance energy transfer-based drug sensitivity test for patients with chronic myelogenous leukemia treated with dasatinib.

PubMed

Kondo, Takeshi; Fujioka, Mari; Tsuda, Masumi; Murai, Kazunori; Yamaguchi, Kohei; Miyagishima, Takuto; Shindo, Motohiro; Nagashima, Takahiro; Wakasa, Kentaro; Fujimoto, Nozomu; Yamamoto, Satoshi; Yonezumi, Masakatsu; Saito, Souichi; Sato, Shinji; Ogawa, Kazuei; Chou, Takaaki; Watanabe, Reiko; Kato, Yuichi; Takahashi, Shuichiro; Okano, Yoshiaki; Yamamoto, Joji; Ohta, Masatsugu; Iijima, Hiroaki; Oba, Koji; Kishino, Satoshi; Sakamoto, Junichi; Ishida, Yoji; Ohba, Yusuke; Teshima, Takanori

2018-05-02

Tyrosine kinase inhibitors (TKI) are used for primary therapy in patients with newly diagnosed CML. However, a reliable method for optimal selection of a TKI from the viewpoint of drug sensitivity of CML cells has not been established. We have developed a FRET-based drug sensitivity test in which a CrkL-derived fluorescent biosensor efficiently quantifies the kinase activity of BCR-ABL of living cells and sensitively evaluates the inhibitory activity of a TKI against BCR-ABL. Here, we validated the utility of the FRET-based drug sensitivity test carried out at diagnosis for predicting the molecular efficacy. Sixty-two patients with newly diagnosed chronic phase CML were enrolled in this study and treated with dasatinib. Bone marrow cells at diagnosis were subjected to FRET analysis. The ΔFRET value was calculated by subtraction of FRET efficiency in the presence of dasatinib from that in the absence of dasatinib. Treatment response was evaluated every 3 months by the BCR-ABL1 International Scale. Based on the ΔFRET value and molecular response, a threshold of the ΔFRET value in the top 10% of FRET efficiency was set to 0.31. Patients with ΔFRET value ≥0.31 had significantly superior molecular responses (MMR at 6 and 9 months and both MR4 and MR4.5 at 6, 9, and 12 months) compared with the responses in patients with ΔFRET value <0.31. These results suggest that the FRET-based drug sensitivity test at diagnosis can predict early and deep molecular responses. This study is registered with UMIN Clinical Trials Registry (UMIN000006358). © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Easy, fast and environmental friendly method for the simultaneous extraction of the 16 EPA PAHs using magnetic molecular imprinted polymers (mag-MIPs).

PubMed

Villar-Navarro, Mercedes; Martín-Valero, María Jesús; Fernández-Torres, Rut Maria; Callejón-Mochón, Manuel; Bello-López, Miguel Ángel

2017-02-15

An easy and environmental friendly method, based on the use of magnetic molecular imprinted polymers (mag-MIPs) is proposed for the simultaneous extraction of the 16 U.S. EPA polycyclic aromatic hydrocarbons (PAHs) priority pollutants. The mag-MIPs based extraction protocol is simple, more sensitive and low organic solvent consuming compared to official methods and also adequate for those PAHs more retained in the particulate matter. The new proposed extraction method followed by HPLC determination has been validated and applied to different types of water samples: tap water, river water, lake water and mineral water. Copyright © 2017 Elsevier B.V. All rights reserved.

Diagnostic algorithm for detection of targetable driver mutations in lung adenocarcinomas: Comprehensive analyses of 205 cases with immunohistochemistry, real-time PCR and fluorescence in situ hybridization methods.

PubMed

Kao, Hua-Lin; Yeh, Yi-Chen; Lin, Chin-Hsuan; Hsu, Wei-Fang; Hsieh, Wen-Yu; Ho, Hsiang-Ling; Chou, Teh-Ying

2016-11-01

Analysis of the targetable driver mutations is now recommended in all patients with advanced lung adenocarcinoma. Molecular-based methods are usually adopted, however, along with the implementation of highly sensitive and/or mutation-specific antibodies, immunohistochemistry (IHC) has been considered an alternative method for identifying driver mutations in lung adenocarcinomas. A total of 205 lung adenocarcinomas were examined for EGFR mutations and ALK and ROS1 rearrangements using real-time PCR, fluorescence in situ hybridization (FISH) and IHC in parallel. The performance of different commercially available IHC antibody clones toward targetable driver mutations was evaluated. The association between these driver mutations and clinicopathological characteristics was also analyzed. In 205 cases we studied, 58.5% were found to harbor EGFR mutations, 6.3% ALK rearrangements and 1.0% ROS1 rearrangements. Compared to molecular-based methods, IHC of EGFR mutations showed an excellent specificity but the sensitivity is suboptimal, while IHC of ALK and ROS1 rearrangements demonstrated high sensitivity and specificity. No significant difference regarding the performance of different antibody clones toward these driver mutations was observed, except that clone SP125 showed a higher sensitivity than 43B2 in the detection of p.L858R of EGFR. In circumstances such as poor quality of nucleic acids or low content of tumor cells, IHC of EGFR mutation-specific antibodies could be used as an alternative method. Patients negative for EGFR mutations are subjected to further analysis on ALK and ROS1 rearrangements using IHC methods. Herein, we proposed a lung adenocarcinoma testing algorithm for the application of IHC in therapeutic diagnosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The role of rapid antigen testing for influenza in the era of molecular diagnostics.

PubMed

Dale, Suzanne E

2010-08-01

Rapid antigen testing for influenza has been both maligned and revered since its conception. Microbiologists have long lamented the lack of sensitivity of commercial rapid influenza detection tests (RIDTs), whereas many clinicians have eschewed their utility by emphasizing the value of definitely diagnosing influenza at the patient's bedside. RIDTs, although quick and easy to perform, are widely accepted as being less sensitive than traditional culture techniques and newer molecular methods, including reverse-transcription polymerase chain reaction (RT-PCR). Moreover, the performance characteristics of RIDTs vary widely, and their applications as clinical diagnostic tools are not well understood. In contrast, traditional techniques are time consuming and require significant expertise to perform. Often, the delay in diagnosing influenza through these methods has little impact on patient care. The benefits of achieving a diagnosis of influenza at the point of care are numerous and include increased access to appropriate antivirals, appropriate patient cohorting for infection control purposes, and better resource utilization. Therefore, it behooves the microbiology community to communicate these issues to clinicians and to work to improve the sensitivity of RIDTs.

Development of an immunomagnetic separation-polymerase chain reaction (IMS-PCR) assay specific for Enterocytozoon bieneusi in water samples.

PubMed

Sorel, N; Guillot, E; Thellier, M; Accoceberry, I; Datry, A; Mesnard-Rouiller, L; Miégeville, M

2003-01-01

Microsporidia have become widely recognized as important human pathogens. Among Microsporidia, Enterocytozoon bieneusi is responsible for severe gastrointestinal disease. To date, no current therapy has been proven effective. Their mode of transmission and environmental occurrence are poorly documented because of the lack of detection methods that are both species-specific and sensitive. In this study, we developed a sensitive and specific molecular method to detect E. bieneusi spores in water samples. The molecular assay combined immunomagnetic separation (IMS) and polymerase chain reaction (PCR) amplification to detect E. bieneusi spores. A comparison was made of IMS magnetic beads coated with two different monoclonal antibodies, one specific for the Encephalitozoon genus that cross-reacts with E. bieneusi and the other specific only for the E. bieneusi species itself. Immunotech beads coated with the antibody specific for E. bieneusi were found to be the most effective combination. The highly specific IMS-PCR assay developed in this study provides a rapid and sensitive means of screening water samples for the presence of E. bieneusi spores.

Rapid Diagnosis of Bloodstream Infections with PCR Followed by Mass Spectrometry

PubMed Central

Jordana-Lluch, Elena; Carolan, Heather E.; Giménez, Montserrat; Sampath, Rangarajan; Ecker, David J.; Quesada, M. Dolores; Mòdol, Josep M.; Arméstar, Fernando; Blyn, Lawrence B.; Cummins, Lendell L.; Ausina, Vicente; Martró, Elisa

2013-01-01

Achieving a rapid microbiological diagnosis is crucial for decreasing morbidity and mortality of patients with a bloodstream infection, as it leads to the administration of an appropriate empiric antimicrobial therapy. Molecular methods may offer a rapid alternative to conventional microbiological diagnosis involving blood culture. In this study, the performance of a new technology that uses broad-spectrum PCR coupled with mass spectrometry (PCR/ESI-MS) was evaluated for the detection of microorganisms directly from whole blood. A total of 247 whole blood samples and paired blood cultures were prospectively obtained from 175 patients with a suspicion of sepsis. Both sample types were analyzed using the PCR/ESI-MS technology, and the results were compared with those obtained by conventional identification methods. The overall agreement between conventional methods and PCR/ESI-MS performed in blood culture aliquots was 94.2% with 96.8% sensitivity and 98.5% specificity for the molecular method. When comparing conventional methods with PCR/ESI-MS performed in whole blood specimens, the overall agreement was 77.1% with 50% sensitivity and 93.8% specificity for the molecular method. Interestingly, the PCR/ESI-MS technology led to the additional identification of 13 pathogens that were not found by conventional methods. Using the PCR/ESI-MS technology the microbiological diagnosis of bloodstream infections could be anticipated in about half of the patients in our setting, including a small but significant proportion of patients newly diagnosed. Thus, this promising technology could be very useful for the rapid diagnosis of sepsis in combination with traditional methods. PMID:23626775

Discovery and utilization of sorghum genes (Ma5/Ma6)

DOEpatents

Mullet, John E; Rooney, William L; Klein, Patricia E; Morishige, Daryl; Murphy, Rebecca; Brady, Jeff A

2012-11-13

Methods and composition for the production of non-flowering or late flowering sorghum hybrid. For example, in certain aspects methods for use of molecular markers that constitute the Ma5/Ma6 pathway to modulate photoperiod sensitivity are described. The invention allows the production of plants having improved productivity and biomass generation.

Specific detection of common pathogens of acute bacterial meningitis using an internally controlled tetraplex-PCR assay.

PubMed

Farahani, Hamidreza; Ghaznavi-Rad, Ehsanollah; Mondanizadeh, Mahdieh; MirabSamiee, Siamak; Khansarinejad, Behzad

2016-08-01

Accurate and timely diagnosis of acute bacterial meningitis is critical for antimicrobial treatment of patients. Although PCR-based methods have been widely used for the diagnosis of acute meningitis caused by bacterial pathogens, the main disadvantage of these methods is their high cost. This disadvantage has hampered the widespread use of molecular assays in many developing countries. The application of multiplex assays and "in-house" protocols are two main approaches that can reduce the overall cost of a molecular test. In the present study, an internally controlled tetraplex-PCR was developed and validated for the specific detection of Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae in cerebrospinal fluid (CSF) samples. The analysis of a panel of other human pathogens showed no cross-reactivity in the assay. The analytical sensitivity of the in-house assay was 792.3 copies/ml, when all three bacteria were presentin the specimens. This value was calculated as 444.5, 283.7, 127.8 copies/ml when only S. pneumoniae, N. meningitidis and H. influenzae, respectively, were present. To demonstrate the diagnostic performance of the assay, a total of 150 archival CSF samples were tested and compared with a commercial multiplex real-time PCR kit. A diagnostic sensitivity of 92.8% and a specificity of 95.1% were determined for the present tetraplex-PCR assay. The results indicate that the established method is sensitive, specific and cost-effective, and can be used particularly in situations where the high cost of commercial kits prevents the use of molecular methods for the diagnosis of bacterial meningitis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular diagnosis and immunotherapy.

PubMed

Sastre, Joaquín; Sastre-Ibañez, Marina

2016-12-01

To describe recent insights into how molecular diagnosis can improve indication and selection of suitable allergens for specific immunotherapy and increase the safety of this therapy. As specific allergen immunotherapy targets specific allergens, identification of the disease-eliciting allergen is a prerequisite for accurate prescription of treatment. In areas of complex sensitization to aeroallergens or in cases of hymenoptera venom allergy, the use of molecular diagnosis has demonstrated that it may lead to a change in indication and selection of allergens for immunotherapy in a large proportion of patients when compared with diagnosis based on skin prick testing and/or specific IgE determination with commercial extracts. These changes in immunotherapy prescription aided by molecular diagnosis have been demonstrated to be cost-effective in some scenarios. Certain patterns of sensitization to grass or olive pollen and bee allergens may identify patients with higher risk of adverse reaction during immunotherapy. Molecular diagnosis, when used with other tools and patients' clinical records, can help clinicians better to select the most appropriate patients and allergens for specific immunotherapy and, in some cases, predict the risk of adverse reactions. The pattern of sensitization to allergens could potentially predict the efficacy of allergen immunotherapy provided that these immunotherapy products contain a sufficient amount of these allergens. Nevertheless, multiplex assay remains a third-level approach, not to be used as screening method in current practice.

Validation and clinical application of a molecular method for the identification of Cryptococcus neoformans/Cryptococcus gattii complex DNA in human clinical specimens.

PubMed

Rivera, Vanessa; Gaviria, Marcela; Muñoz-Cadavid, Cesar; Cano, Luz; Naranjo, Tonny

2015-01-01

The diagnosis of cryptococcosis is usually performed based on cultures of tissue or body fluids and isolation of the fungus, but this method may require several days. Direct microscopic examination, although rapid, is relatively insensitive. Biochemical and immunodiagnostic rapid tests are also used. However, all of these methods have limitations that may hinder final diagnosis. The increasing incidence of fungal infections has focused attention on tools for rapid and accurate diagnosis using molecular biological techniques. Currently, PCR-based methods, particularly nested, multiplex and real-time PCR, provide both high sensitivity and specificity. In the present study, we evaluated a nested PCR targeting the gene encoding the ITS-1 and ITS-2 regions of rDNA in samples from a cohort of patients diagnosed with cryptococcosis. The results showed that in our hands, this Cryptococcus nested PCR assay has 100% specificity and 100% sensitivity and was able to detect until 2 femtograms of Cryptococcus DNA. Copyright © 2015 Elsevier Editora Ltda. All rights reserved.

Advances in serological, imaging techniques and molecular diagnosis of Toxoplasma gondii infection.

PubMed

Rostami, Ali; Karanis, Panagiotis; Fallahi, Shirzad

2018-06-01

Toxoplasmosis is worldwide distributed zoonotic infection disease with medical importance in immunocompromised patients, pregnant women and congenitally infected newborns. Having basic information on the traditional and new developed methods is essential for general physicians and infectious disease specialists for choosing a suitable diagnostic approach for rapid and accurate diagnosis of the disease and, consequently, timely and effective treatment. We conducted English literature searches in PubMed from 1989 to 2016 using relevant keywords and summarized the recent advances in diagnosis of toxoplasmosis. Enzyme-linked immunosorbent assay (ELISA) was most used method in past century. Recently advanced ELISA-based methods including chemiluminescence assays (CLIA), enzyme-linked fluorescence assay (ELFA), immunochromatographic test (ICT), serum IgG avidity test and immunosorbent agglutination assays (ISAGA) have shown high sensitivity and specificity. Recent studies using recombinant or chimeric antigens and multiepitope peptides method demonstrated very promising results to development of new strategies capable of discriminating recently acquired infections from chronic infection. Real-time PCR and loop-mediated isothermal amplification (LAMP) are two recently developed PCR-based methods with high sensitivity and specificity and could be useful to early diagnosis of infection. Computed tomography, magnetic resonance imaging, nuclear imaging and ultrasonography could be useful, although their results might be not specific alone. This review provides a summary of recent developed methods and also attempts to improve their sensitivity for diagnosis of toxoplasmosis. Serology, molecular and imaging technologies each has their own advantages and limitations which can certainly achieve definitive diagnosis of toxoplasmosis by combining these diagnostic techniques.

Quantitative molecular analysis in mantle cell lymphoma.

PubMed

Brízová, H; Hilská, I; Mrhalová, M; Kodet, R

2011-07-01

A molecular analysis has three major roles in modern oncopathology--as an aid in the differential diagnosis, in molecular monitoring of diseases, and in estimation of the potential prognosis. In this report we review the application of the molecular analysis in a group of patients with mantle cell lymphoma (MCL). We demonstrate that detection of the cyclin D1 mRNA level is a molecular marker in 98% of patients with MCL. Cyclin D1 quantitative monitoring is specific and sensitive for the differential diagnosis and for the molecular monitoring of the disease in the bone marrow. Moreover, the dynamics of cyclin D1 in bone marrow reflects the disease development and it predicts the clinical course. We employed the molecular analysis for a precise quantitative detection of proliferation markers, Ki-67, topoisomerase IIalpha, and TPX2, that are described as effective prognostic factors. Using the molecular approach it is possible to measure the proliferation rate in a reproducible, standard way which is an essential prerequisite for using the proliferation activity as a routine clinical tool. Comparing with immunophenotyping we may conclude that the quantitative PCR-based analysis is a useful, reliable, rapid, reproducible, sensitive and specific method broadening our diagnostic tools in hematopathology. In comparison to interphase FISH in paraffin sections quantitative PCR is less technically demanding and less time-consuming and furthermore it is more sensitive in detecting small changes in the mRNA level. Moreover, quantitative PCR is the only technology which provides precise and reproducible quantitative information about the expression level. Therefore it may be used to demonstrate the decrease or increase of a tumor-specific marker in bone marrow in comparison with a previously aspirated specimen. Thus, it has a powerful potential to monitor the course of the disease in correlation with clinical data.

Molecular analysis of breast sentinel lymph nodes.

PubMed

Blumencranz, Peter W; Pieretti, Maura; Allen, Kathleen G; Blumencranz, Lisa E

2011-07-01

Lymphatic mapping and sentinel lymph node (SLN) biopsy have become the standard of care for staging the axilla in patients with invasive breast cancer. Current histologic methods for SLN evaluation have limitations, including subjectivity, limited sensitivity, and lack of standardization. The discovery of molecular markers to detect metastases has been reported over the last 2 decades. The authors review the historical development of these markers and the clinical use of one of the molecular platforms in 478 patients at their institution. Controversies and future directions are discussed. Copyright © 2011 Elsevier Inc. All rights reserved.

Phase-Sensitive Surface Plasmon Resonance Sensors: Recent Progress and Future Prospects

PubMed Central

Deng, Shijie; Wang, Peng; Yu, Xinglong

2017-01-01

Surface plasmon resonance (SPR) is an optical sensing technique that is capable of performing real-time, label-free and high-sensitivity monitoring of molecular interactions. SPR biosensors can be divided according to their operating principles into angle-, wavelength-, intensity- and phase-interrogated devices. With their complex optical configurations, phase-interrogated SPR sensors generally provide higher sensitivity and throughput, and have thus recently emerged as prominent biosensing devices. To date, several methods have been developed for SPR phase interrogation, including heterodyne detection, polarimetry, shear interferometry, spatial phase modulation interferometry and temporal phase modulation interferometry. This paper summarizes the fundamentals of phase-sensitive SPR sensing, reviews the available methods for phase interrogation of these sensors, and discusses the future prospects for and trends in the development of this technology. PMID:29206182

Design and synthesis study of the thermo-sensitive poly (N-vinylpyrrolidone-b- N, N-diethylacrylamide).

PubMed

Zhang, Xiayun; Yang, Zhongduo; Xie, Dengmin; Liu, Donglei; Chen, Zhenbin; Li, Ke; Li, Zhizhong; Tichnell, Brandon; Liu, Zhen

2018-01-01

The reversible addition fragmentation chain transfer (RAFT) polymerization method was adopted here to prepare a series of thermo-sensitive copolymers, poly (N,N-diethyl- acrylamide-b-N-vinylpyrrolidone). Their structures, molecular weight distribution and temperature sensitivity performances were characterized by the nuclear magnetic resonance ( 1 HNMR), the gel permeation chromatography (GPC) and the fluorescence spectrophotometer, respectively. It has been identified that the synthesis reaction of the block copolymer was living polymerization. The thermo-sensitivity study suggested that N-vinylpyrrolidone (NVP), played a key role on the lower critical solution temperature (LCST) performance.

Polymerase chain reaction in the detection of tumor cells: new approaches in diagnosis and follow-up of patients with thyroid cancer.

PubMed

Bojunga, Jörg; Kusterer, Klaus; Schumm-Draeger, Petra-Maria; Usadel, Klaus-Henning

2002-12-01

Thyroid cancers are the most common endocrine malignancies and are being diagnosed with increasing frequency. In addition to other measures, diagnosis is based on fine-needle aspiration cytology examination. Recently, new assays using reverse transcription-polymerase chain reaction (PCR) are being tested to improve sensitivity and specificity of primary diagnosis and detection of recurrent thyroid cancer. In the preoperative diagnosis of thyroid cancer, several tissue- and/or tumor-specific mRNA have been described and in several cases, a higher sensitivity and specificity could be achieved using molecular techniques compared to conventional methods. In the postoperative follow-up of patients with thyroid cancer, conflicting data have been published and the use of PCR techniques revealed several problems of the molecular approach, which are based on some technical as well as biologic limitations. Despite these problems, which are discussed in detail in this review, molecular techniques may nevertheless improve the sensitivity and accuracy of fine-needle aspiration of thyroid nodules, fine-needle aspiration of metastases, and detection of recurrent disease in peripheral blood samples.

Detection of multidrug-resistant tuberculosis from stored DNA Samples: A multicenter study.

PubMed

Rabodoarivelo, Marie Sylvianne; Brandao, A; Cergole Novella, M C; C Bombonatte, A G; Imperiale, B; Rakotosamimanana, N; Morcillo, N; Rasolofo, V; Palomino, J C; Martin, A

2018-01-01

In low-income countries, rapid detection of tuberculosis (TB) drug resistance is often restricted by the difficulties of transporting and storing sputum samples from remote health centers to the reference laboratories where molecular tests are available. The aim of this study was to evaluate the performance of four transport and storage systems for molecular detection of rifampicin (RIF) and isoniazid (INH) resistance. This was a multicenter study. Molecular detection of RIF and INH resistance was performed directly from smear-positive TB sputa spotted on a slide, FTA card, GenoCard, and ethanol using the Genotype MTBDRplus assay. The performance of the DNA extraction method from each storage support to detect drug resistance was assessed by calculating their sensitivity and specificity compared to the phenotypic method. From all sites, the overall sensitivity and specificity for RIF-resistance detection was 88% and 85%, respectively, for slides, 86% and 92%, respectively, for GenoCard, 87% and 89%, respectively, for FTA card, and 88% and 92%, respectively, for ethanol. For INH-resistance detection, the overall sensitivity and specificity was 82% and 90%, respectively, for slides, 85% and 96%, respectively, for GenoCard, 86% and 92%, respectively, for FTA card, and 86% and 94%, respectively, for ethanol. Smear slides and filter cards showed to be very useful tools to facilitate DNA extraction from sputum samples with the potential to accelerate the detection of drug resistance in remote areas.

Phenotypic detection of broad-spectrum beta-lactamases in microbiological practice

PubMed Central

Sedlakova, Miroslava Htoutou; Hanulik, Vojtech; Chroma, Magdalena; Hricova, Kristyna; Kolar, Milan; Latal, Tomas; Schaumann, Reiner; Rodloff, Arne C.

2011-01-01

Summary Background Enterobacteriaceae producing ESBL and AmpC enzymes can be associated with failure of antibiotic therapy and related morbidity and mortality. Their routine detection in microbiology laboratories is still a problem. The aim of this study was to compare the sensitivity of selected phenotypic methods. Material/Methods A total of 106 strains of the Enterobacteriaceae family were tested, in which molecular biology methods confirmed the presence of genes encoding ESBL or AmpC. In ESBL-positive strains, the sensitivity of the ESBL Etest (AB Biodisk) and a modified double-disk synergy test (DDST) were evaluated. AmpC strains were tested by a modified AmpC disk method using 3-aminophenylboronic acid. For simultaneous detection of ESBL and AmpC, the microdilution method with a modified set of antimicrobial agents was used. Results The sensitivity of the ESBL Etest was 95%; the modified DDST yielded 100% sensitivity for ESBL producers and the AmpC test correctly detected 95% of AmpC-positive strains. The sensitivity of the modified microdilution method was 87% and 95% for ESBL and AmpC beta lactamases, respectively. Conclusions The detection of ESBL and AmpC beta lactamases should be based on specific phenotypic methods such as the modified DDST, ESBL Etest, AmpC disk test and the modified microdilution method. PMID:21525803

Nested-PCR real time as alternative molecular tool for detection of Borrelia burgdorferi compared to the classical serological diagnosis of the blood.

PubMed

Sroka-Oleksiak, Agnieszka; Ufir, Krzysztof; Salamon, Dominika; Bulanda, Malgorzata; Gosiewski, Tomasz

Lyme disease, caused by Borrelia burgdorferi, is a multisystem disease that often makes difficulties to recognize caused by their genetic heterogenity. Currently, the gold standard for the detection of Lyme disease (LD) is serologic diagnostics based mainly on tests: ELISA and Western blot (WB). These methods, however, are subject to consider- able defect, especially in the initial phase of infection due to the occurrence of so-called serological window period and low specificity. For this reason, they might be replaced by molecular methods, for example polymerase chain reaction (PCR), which should be more sensitivity and specificity. In the present study we attempt to optimize the PCR reaction conditions and enhance existing test sensitivity by applying the equivalent of real time PCR - nested PCR for detection B. burgdorferi DNA in the patient's blood. The study involved 94 blood samples of patients with suspected LD. From each sample, 1.5 ml of blood was used for the isolation of bacterial DNA and PCR real time am- plification and its equivalent, in nested version. The remaining part earmarked for serologi- cal testing. Optimization of the reaction conditions made experimentally, using gradient of the temperature and gradient of the magnesium ions concentration for reaction real time in nested-PCR and PCR version. The results show that the nested-PCR real time, has a much higher sensitivity 45 (47.8%) of positive results for the detection of B. burgdorferi compared to the single- variety, without a preceding pre-amplification 2 (2.1%). Serological methods allowed the detection of infection in 41 (43.6%) samples. These results support of the nested PCR method as a better molecular tool for the detection of B. burgdorferi infection than classical PCR real time reaction. The nested-PCR real time method may be considered as a complement to ELISA and WB mainly in the early stages of infection, when in the blood circulating B. burgdorferi cells. By contrast, the results of serological and molecular tests should always be carried out tak- ing into account the patient's clinical status.

Microscopy outperformed in a comparison of five methods for detecting Trichomonas vaginalis in symptomatic women.

PubMed

Nathan, B; Appiah, J; Saunders, P; Heron, D; Nichols, T; Brum, R; Alexander, S; Baraitser, P; Ison, C

2015-03-01

In the UK, despite its low sensitivity, wet mount microscopy is often the only method of detecting Trichomonas vaginalis infection. A study was conducted in symptomatic women to compare the performance of five methods for detecting T. vaginalis: an in-house polymerase chain reaction (PCR); Aptima T. vaginalis kit; OSOM ®Trichomonas Rapid Test; culture and microscopy. Symptomatic women underwent routine testing; microscopy and further swabs were taken for molecular testing, OSOM and culture. A true positive was defined as a sample that was positive for T. vaginalis by two or more different methods. Two hundred and forty-six women were recruited: 24 patients were positive for T. vaginalis by two or more different methods. Of these 24 patients, 21 patients were detected by real-time PCR (sensitivity 88%); 22 patients were detected by the Aptima T. vaginalis kit (sensitivity 92%); 22 patients were detected by OSOM (sensitivity 92%); nine were detected by wet mount microscopy (sensitivity 38%); and 21 were detected by culture (sensitivity 88%). Two patients were positive by just one method and were not considered true positives. All the other detection methods had a sensitivity to detect T. vaginalis that was significantly greater than wet mount microscopy, highlighting the number of cases that are routinely missed even in symptomatic women if microscopy is the only diagnostic method available. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Proteomic Signatures of the Zebrafish (Danio rerio) Embryo: Sensitivity and Specificity in Toxicity Assessment of Chemicals.

PubMed

Hanisch, Karen; Küster, Eberhard; Altenburger, Rolf; Gündel, Ulrike

2010-01-01

Studies using embryos of the zebrafish Danio rerio (DarT) instead of adult fish for characterising the (eco-) toxic potential of chemicals have been proposed as animal replacing methods. Effect analysis at the molecular level might enhance sensitivity, specificity, and predictive value of the embryonal studies. The present paper aimed to test the potential of toxicoproteomics with zebrafish eleutheroembryos for sensitive and specific toxicity assessment. 2-DE-based toxicoproteomics was performed applying low-dose (EC(10)) exposure for 48 h with three-model substances Rotenone, 4,6-dinitro-o-cresol (DNOC) and Diclofenac. By multivariate "pattern-only" PCA and univariate statistical analyses, alterations in the embryonal proteome were detectable in nonetheless visibly intact organisms and treatment with the three substances was distinguishable at the molecular level. Toxicoproteomics enabled the enhancement of sensitivity and specificity of the embryonal toxicity assay and bear the potency to identify protein markers serving as general stress markers and early diagnosis of toxic stress.

Proteomic Signatures of the Zebrafish (Danio rerio) Embryo: Sensitivity and Specificity in Toxicity Assessment of Chemicals

PubMed Central

Hanisch, Karen; Küster, Eberhard; Altenburger, Rolf; Gündel, Ulrike

2010-01-01

Studies using embryos of the zebrafish Danio rerio (DarT) instead of adult fish for characterising the (eco-) toxic potential of chemicals have been proposed as animal replacing methods. Effect analysis at the molecular level might enhance sensitivity, specificity, and predictive value of the embryonal studies. The present paper aimed to test the potential of toxicoproteomics with zebrafish eleutheroembryos for sensitive and specific toxicity assessment. 2-DE-based toxicoproteomics was performed applying low-dose (EC10) exposure for 48 h with three-model substances Rotenone, 4,6-dinitro-o-cresol (DNOC) and Diclofenac. By multivariate “pattern-only” PCA and univariate statistical analyses, alterations in the embryonal proteome were detectable in nonetheless visibly intact organisms and treatment with the three substances was distinguishable at the molecular level. Toxicoproteomics enabled the enhancement of sensitivity and specificity of the embryonal toxicity assay and bear the potency to identify protein markers serving as general stress markers and early diagnosis of toxic stress. PMID:22084678

Norwalk virus-associated gastroenteritis traced to ice consumption aboard a cruise ship in Hawaii: comparison and application of molecular method-based assays.

PubMed Central

Khan, A S; Moe, C L; Glass, R I; Monroe, S S; Estes, M K; Chapman, L E; Jiang, X; Humphrey, C; Pon, E; Iskander, J K

1994-01-01

Investigation of an outbreak of acute nonbacterial gastroenteritis on a cruise ship provided an opportunity to assess new molecular method-based diagnostic methods for Norwalk virus (NV) and the antibody response to NV infection. The outbreak began within 36 h of embarkation and affected 30% of 672 passengers and crew. No single meal, seating, or food item was implicated in the transmission of NV, but a passenger's risk of illness was associated with the amount of ice (but not water) consumed (chi-square for trend, P = 0.009). Of 19 fecal specimens examined, 7 were found to contain 27-nm NV-like particles by electron microscopy and 16 were positive by PCR with very sensitive NV-specific primers, but only 5 were positive by a new highly specific antigen enzyme immunoassay for NV. Ten of 12 serum specimen pairs demonstrated a fourfold or greater rise in antibody titer to recombinant baculovirus-expressed NV antigen. The amplified PCR band shared only 81% nucleotide sequence homology with the reference NV strain, which may explain the lack of utility of the fecal specimen enzyme immunoassay. This report, the first to document the use of these molecular method-based assays for investigation of an outbreak, demonstrates the importance of highly sensitive viral diagnostics such as PCR and serodiagnosis for the epidemiologic investigation of NV gastroenteritis. Images PMID:8150941

Leishmania infections: Molecular targets and diagnosis.

PubMed

Akhoundi, Mohammad; Downing, Tim; Votýpka, Jan; Kuhls, Katrin; Lukeš, Julius; Cannet, Arnaud; Ravel, Christophe; Marty, Pierre; Delaunay, Pascal; Kasbari, Mohamed; Granouillac, Bruno; Gradoni, Luigi; Sereno, Denis

2017-10-01

Progress in the diagnosis of leishmaniases depends on the development of effective methods and the discovery of suitable biomarkers. We propose firstly an update classification of Leishmania species and their synonymies. We demonstrate a global map highlighting the geography of known endemic Leishmania species pathogenic to humans. We summarize a complete list of techniques currently in use and discuss their advantages and limitations. The available data highlights the benefits of molecular markers in terms of their sensitivity and specificity to quantify variation from the subgeneric level to species complexes, (sub) species within complexes, and individual populations and infection foci. Each DNA-based detection method is supplied with a comprehensive description of markers and primers and proposal for a classification based on the role of each target and primer in the detection, identification and quantification of leishmaniasis infection. We outline a genome-wide map of genes informative for diagnosis that have been used for Leishmania genotyping. Furthermore, we propose a classification method based on the suitability of well-studied molecular markers for typing the 21 known Leishmania species pathogenic to humans. This can be applied to newly discovered species and to hybrid strains originating from inter-species crosses. Developing more effective and sensitive diagnostic methods and biomarkers is vital for enhancing Leishmania infection control programs. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Comparison of 16S rDNA-based PCR and checkerboard DNA-DNA hybridisation for detection of selected endodontic pathogens.

PubMed

Siqueira, José F; Rôças, Isabela N; De Uzeda, Milton; Colombo, Ana P; Santos, Kátia R N

2002-12-01

Molecular methods have been used recently to investigate the bacteria encountered in human endodontic infections. The aim of the present study was to compare the ability of a 16S rDNA-based PCR assay and checkerboard DNA-DNA hybridisation in detecting Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Peptostreptococcus micros, Porphyromonas endodontalis, Por. gingivalis and Treponema denticola directly from clinical samples. Specimens were obtained from 50 cases of endodontic infections and the presence of the target species was investigated by whole genomic DNA probes and checkerboard DNA-DNA hybridisation or taxon-specific oligonucleotides with PCR assay. Prevalence of the target species was based on data obtained by each method. The sensitivity and specificity of each molecular method was compared with the data generated by the other method as the reference--a value of 1.0 representing total agreement with the chosen standard. The methods were also compared with regard to the prevalence values for each target species. Regardless of the detection method used, T. denticola, Por. gingivalis, Por. endodontalis and B. forsythus were the most prevalent species. If the checkerboard data for these four species were used as the reference, PCR detection sensitivities ranged from 0.53 to 1.0, and specificities from 0.5 to 0.88, depending on the target bacterial species. When PCR data for the same species were used as the reference, the detection sensitivities for the checkerboard method ranged from 0.17 to 0.73, and specificities from 0.75 to 1.0. Accuracy values ranged from 0.6 to 0.74. On the whole, matching results between the two molecular methods ranged from 60% to 97.5%, depending on the target species. The major discrepancies between the methods comprised a number of PCR-positive but checkerboard-negative results. Significantly higher prevalence figures for Por. endodontalis and T. denticola were observed after PCR assessment. There was no further significant difference between the methods with regard to detection of the other target species.

Molecular barcodes detect redundancy and contamination in hairpin-bisulfite PCR

PubMed Central

Miner, Brooks E.; Stöger, Reinhard J.; Burden, Alice F.; Laird, Charles D.; Hansen, R. Scott

2004-01-01

PCR amplification of limited amounts of DNA template carries an increased risk of product redundancy and contamination. We use molecular barcoding to label each genomic DNA template with an individual sequence tag prior to PCR amplification. In addition, we include molecular ‘batch-stamps’ that effectively label each genomic template with a sample ID and analysis date. This highly sensitive method identifies redundant and contaminant sequences and serves as a reliable method for positive identification of desired sequences; we can therefore capture accurately the genomic template diversity in the sample analyzed. Although our application described here involves the use of hairpin-bisulfite PCR for amplification of double-stranded DNA, the method can readily be adapted to single-strand PCR. Useful applications will include analyses of limited template DNA for biomedical, ancient DNA and forensic purposes. PMID:15459281

The diagnosis of microorganism involved in infective endocarditis (IE) by polymerase chain reaction (PCR) and real-time PCR: A systematic review.

PubMed

Faraji, Reza; Behjati-Ardakani, Mostafa; Moshtaghioun, Seyed Mohammad; Kalantar, Seyed Mehdi; Namayandeh, Seyedeh Mahdieh; Soltani, Mohammadhossien; Emami, Mahmood; Zandi, Hengameh; Firoozabadi, Ali Dehghani; Kazeminasab, Mahmood; Ahmadi, Nastaran; Sarebanhassanabadi, Mohammadtaghi

2018-02-01

Broad-range bacterial rDNA polymerase chain reaction (PCR) followed by sequencing may be identified as the etiology of infective endocarditis (IE) from surgically removed valve tissue; therefore, we reviewed the value of molecular testing in identifying organisms' DNA in the studies conducted until 2016. We searched Google Scholar, Scopus, ScienceDirect, Cochrane, PubMed, and Medline electronic databases without any time limitations up to December 2016 for English studies reporting microorganisms involved in infective endocarditis microbiology using PCR and real-time PCR. Most studies were prospective. Eleven out of 12 studies used valve tissue samples and blood cultures while only 1 study used whole blood. Also, 10 studies used the molecular method of PCR while 2 studies used real-time PCR. Most studies used 16S rDNA gene as the target gene. The bacteria were identified as the most common microorganisms involved in infective endocarditis. Streptococcus spp. and Staphylococcus spp. were, by far, the most predominant bacteria detected. In all studies, PCR and real-time PCR identified more pathogens than blood and tissue cultures; moreover, the sensitivity and specificity of PCR and real-time PCR were more than cultures in most of the studies. The highest sensitivity and specificity were 96% and 100%, respectively. The gram positive bacteria were the most frequent cause of infective endocarditis. The molecular methods enjoy a greater sensitivity compared to the conventional blood culture methods; yet, they are applicable only to the valve tissue of the patients undergoing cardiac valve surgery. Copyright © 2017. Published by Elsevier Taiwan.

An Efficient Variable Screening Method for Effective Surrogate Models for Reliability-Based Design Optimization

DTIC Science & Technology

2014-04-01

surrogate model generation is difficult for high -dimensional problems, due to the curse of dimensionality. Variable screening methods have been...a variable screening model was developed for the quasi-molecular treatment of ion-atom collision [16]. In engineering, a confidence interval of...for high -level radioactive waste [18]. Moreover, the design sensitivity method can be extended to the variable screening method because vital

On simulation of local fluxes in molecular junctions

NASA Astrophysics Data System (ADS)

Cabra, Gabriel; Jensen, Anders; Galperin, Michael

2018-05-01

We present a pedagogical review of the current density simulation in molecular junction models indicating its advantages and deficiencies in analysis of local junction transport characteristics. In particular, we argue that current density is a universal tool which provides more information than traditionally simulated bond currents, especially when discussing inelastic processes. However, current density simulations are sensitive to the choice of basis and electronic structure method. We note that while discussing the local current conservation in junctions, one has to account for the source term caused by the open character of the system and intra-molecular interactions. Our considerations are illustrated with numerical simulations of a benzenedithiol molecular junction.

Intraoperative imaging-guided cancer surgery: from current fluorescence molecular imaging methods to future multi-modality imaging technology.

PubMed

Chi, Chongwei; Du, Yang; Ye, Jinzuo; Kou, Deqiang; Qiu, Jingdan; Wang, Jiandong; Tian, Jie; Chen, Xiaoyuan

2014-01-01

Cancer is a major threat to human health. Diagnosis and treatment using precision medicine is expected to be an effective method for preventing the initiation and progression of cancer. Although anatomical and functional imaging techniques such as radiography, computed tomography (CT), magnetic resonance imaging (MRI) and positron emission tomography (PET) have played an important role for accurate preoperative diagnostics, for the most part these techniques cannot be applied intraoperatively. Optical molecular imaging is a promising technique that provides a high degree of sensitivity and specificity in tumor margin detection. Furthermore, existing clinical applications have proven that optical molecular imaging is a powerful intraoperative tool for guiding surgeons performing precision procedures, thus enabling radical resection and improved survival rates. However, detection depth limitation exists in optical molecular imaging methods and further breakthroughs from optical to multi-modality intraoperative imaging methods are needed to develop more extensive and comprehensive intraoperative applications. Here, we review the current intraoperative optical molecular imaging technologies, focusing on contrast agents and surgical navigation systems, and then discuss the future prospects of multi-modality imaging technology for intraoperative imaging-guided cancer surgery.

Intraoperative Imaging-Guided Cancer Surgery: From Current Fluorescence Molecular Imaging Methods to Future Multi-Modality Imaging Technology

PubMed Central

Chi, Chongwei; Du, Yang; Ye, Jinzuo; Kou, Deqiang; Qiu, Jingdan; Wang, Jiandong; Tian, Jie; Chen, Xiaoyuan

2014-01-01

Cancer is a major threat to human health. Diagnosis and treatment using precision medicine is expected to be an effective method for preventing the initiation and progression of cancer. Although anatomical and functional imaging techniques such as radiography, computed tomography (CT), magnetic resonance imaging (MRI) and positron emission tomography (PET) have played an important role for accurate preoperative diagnostics, for the most part these techniques cannot be applied intraoperatively. Optical molecular imaging is a promising technique that provides a high degree of sensitivity and specificity in tumor margin detection. Furthermore, existing clinical applications have proven that optical molecular imaging is a powerful intraoperative tool for guiding surgeons performing precision procedures, thus enabling radical resection and improved survival rates. However, detection depth limitation exists in optical molecular imaging methods and further breakthroughs from optical to multi-modality intraoperative imaging methods are needed to develop more extensive and comprehensive intraoperative applications. Here, we review the current intraoperative optical molecular imaging technologies, focusing on contrast agents and surgical navigation systems, and then discuss the future prospects of multi-modality imaging technology for intraoperative imaging-guided cancer surgery. PMID:25250092

Immobilized enzymes: understanding enzyme - surface interactions at the molecular level.

PubMed

Hoarau, Marie; Badieyan, Somayesadat; Marsh, E Neil G

2017-11-22

Enzymes immobilized on solid supports have important and industrial and medical applications. However, their uses are limited by the significant reductions in activity and stability that often accompany the immobilization process. Here we review recent advances in our understanding of the molecular level interactions between proteins and supporting surfaces that contribute to changes in stability and activity. This understanding has been facilitated by the application of various surface-sensitive spectroscopic techniques that allow the structure and orientation of enzymes at the solid/liquid interface to be probed, often with monolayer sensitivity. An appreciation of the molecular interactions between enzyme and surface support has allowed the surface chemistry and method of enzyme attachement to be fine-tuned such that activity and stability can be greatly enhanced. These advances suggest that a much wider variety of enzymes may eventually be amenable to immobilization as green catalysts.

Molecular Machine Powered Surface Programmatic Chain Reaction for Highly Sensitive Electrochemical Detection of Protein.

PubMed

Zhu, Jing; Gan, Haiying; Wu, Jie; Ju, Huangxian

2018-04-17

A bipedal molecular machine powered surface programmatic chain reaction was designed for electrochemical signal amplification and highly sensitive electrochemical detection of protein. The bipedal molecular machine was built through aptamer-target specific recognition for the binding of one target protein with two DNA probes, which hybridized with surface-tethered hairpin DNA 1 (H1) via proximity effect to expose the prelocked toehold domain of H1 for the hybridization of ferrocene-labeled hairpin DNA 2 (H2-Fc). The toehold-mediated strand displacement reaction brought the electrochemical signal molecule Fc close to the electrode and meanwhile released the bipedal molecular machine to traverse the sensing surface by the surface programmatic chain reaction. Eventually, a large number of duplex structures of H1-H2 with ferrocene groups facing to the electrode were formed on the sensor surface to generate an amplified electrochemical signal. Using thrombin as a model target, this method showed a linear detection range from 2 pM to 20 nM with a detection limit of 0.76 pM. The proposed detection strategy was enzyme-free and allowed highly sensitive and selective detection of a variety of protein targets by using corresponding DNA-based affinity probes, showing potential application in bioanalysis.

Identifying Recent HIV Infections: From Serological Assays to Genomics.

PubMed

Moyo, Sikhulile; Wilkinson, Eduan; Novitsky, Vladimir; Vandormael, Alain; Gaseitsiwe, Simani; Essex, Max; Engelbrecht, Susan; de Oliveira, Tulio

2015-10-23

In this paper, we review serological and molecular based methods to identify HIV infection recency. The accurate identification of recent HIV infection continues to be an important research area and has implications for HIV prevention and treatment interventions. Longitudinal cohorts that follow HIV negative individuals over time are the current gold standard approach, but they are logistically challenging, time consuming and an expensive enterprise. Methods that utilize cross-sectional testing and biomarker information have become an affordable alternative to the longitudinal approach. These methods use well-characterized biological makers to differentiate between recent and established HIV infections. However, recent results have identified a number of limitations in serological based assays that are sensitive to the variability in immune responses modulated by HIV subtypes, viral load and antiretroviral therapy. Molecular methods that explore the dynamics between the timing of infection and viral evolution are now emerging as a promising approach. The combination of serological and molecular methods may provide a good solution to identify recent HIV infection in cross-sectional data. As part of this review, we present the advantages and limitations of serological and molecular based methods and their potential complementary role for the identification of HIV infection recency.

Method development for the determination of fluorine in toothpaste via molecular absorption of aluminum mono fluoride using a high-resolution continuum source nitrous oxide/acetylene flame atomic absorption spectrophotometer.

PubMed

Ozbek, Nil; Akman, Suleyman

2012-05-30

Fluorine was determined via the rotational molecular absorption line of aluminum mono fluoride (AlF) generated in C(2)H(2)/N(2)O flame at 227.4613 nm using a high-resolution continuum source flame atomic absorption spectrophotometer (HR-CS-FAAS). The effects of AlF wavelength, burner height, fuel rate (C(2)H(2)/N(2)O) and amount of Al on the accuracy, precision and sensitivity were investigated and optimized. The Al-F absorption band at 227.4613 nm was found to be the most suitable analytical line with respect to sensitivity and spectral interferences. Maximum sensitivity and a good linearity were obtained in acetylene-nitrous oxide flame at a flow rate of 210 L h(-1) and a burner height of 8mm using 3000 mg L(-1) of Al for 10-1000 mg L(-1)of F. The accuracy and precision of the method were tested by analyzing spiked samples and waste water certified reference material. The results were in good agreement with the certified and spiked amounts as well as the precision of several days during this study was satisfactory (RSD<10%). The limit of detection and characteristic concentration of the method were 5.5 mg L(-1) and 72.8 mg L(-1), respectively. Finally, the fluorine concentrations in several toothpaste samples were determined. The results found and given by the producers were not significantly different. The method was simple, fast, accurate and sensitive. Copyright © 2012 Elsevier B.V. All rights reserved.

Phenotypic detection of broad-spectrum beta-lactamases in microbiological practice.

PubMed

Htoutou Sedlakova, Miroslava; Hanulik, Vojtech; Chroma, Magdalena; Hricova, Kristyna; Kolar, Milan; Latal, Tomas; Schaumann, Reiner; Rodloff, Arne C

2011-05-01

Enterobacteriaceae producing ESBL and AmpC enzymes can be associated with failure of antibiotic therapy and related morbidity and mortality. Their routine detection in microbiology laboratories is still a problem. The aim of this study was to compare the sensitivity of selected phenotypic methods. A total of 106 strains of the Enterobacteriaceae family were tested, in which molecular biology methods confirmed the presence of genes encoding ESBL or AmpC. In ESBL-positive strains, the sensitivity of the ESBL Etest (AB Biodisk) and a modified double-disk synergy test (DDST) were evaluated. AmpC strains were tested by a modified AmpC disk method using 3-aminophenylboronic acid. For simultaneous detection of ESBL and AmpC, the microdilution method with a modified set of antimicrobial agents was used. The sensitivity of the ESBL Etest was 95%; the modified DDST yielded 100% sensitivity for ESBL producers and the AmpC test correctly detected 95% of AmpC-positive strains. The sensitivity of the modified microdilution method was 87% and 95% for ESBL and AmpC beta lactamases, respectively. The detection of ESBL and AmpC beta lactamases should be based on specific phenotypic methods such as the modified DDST, ESBL Etest, AmpC disk test and the modified microdilution method.

Identification and quantification of cardiac glycosides in blood and urine samples by HPLC/MS/MS.

PubMed

Guan, F; Ishii, A; Seno, H; Watanabe-Suzuki, K; Kumazawa, T; Suzuki, O

1999-09-15

Cardiac glycosides (CG) are of forensic importance because of their toxicity and the fact that very limited methods are available for identification of CG in biological samples. In this study, we have developed an identification and quantification method for digoxin, digitoxin, deslanoside, digoxigenin, and digitoxigenin by high-performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS). CG formed abundant [M + NH4]+ ions and much less abundant [M + H]+ ions as observed with electrospray ionization (ESI) source and ammonium formate buffer. Under mild conditions for collision-induced dissociation (CID), each [M + NH4]+ ion fragmented to produce a dominant daughter ion, which was essential to the sensitive method of selected reaction monitoring (SRM) quantification of CG achieved in this study. SRM was compared with selected ion monitoring (SIM) regarding the effects of sample matrixes on the methodology. SRM produced lower detection limits with biological samples than SIM, while both methods produced equal detection limits with CG standards. On the basis of the HPLC/MS/MS results for CG, we have proposed some generalized points for conducting sensitive SRM measurements, in view of the property of analytes as well as instrumental conditions such as the type of HPLC/MS interface and CID parameters. Analytes of which the molecular ion can produce one abundant daughter ion with high yield under CID conditions may be sensitively measured by SRM. ESI is the most soft ionization source developed so far and can afford formation of the fragile molecular ions that are necessary for sensitive SRM detection. Mild CID conditions such as low collision energy and low pressure of collision gas favor production of an abundant daughter ion that is essential to sensitive SRM detection. This knowledge may provide some guidelines for conducting sensitive SRM measurements of very low concentrations of drugs or toxicants in biological samples.

Diagnostics and Discovery in Viral Central Nervous System Infections.

PubMed

Lipkin, Walter Ian; Hornig, Mady

2015-09-01

The range of viruses implicated in central nervous system disease continues to grow with globalization of travel and trade, emergence and reemergence of zoonoses and investments in discovery science. Diagnosis of viral central nervous system infections is challenging in that brain tissue, where the pathogen concentration is likely to be highest, is not readily obtained and sensitive methods for molecular and serological detection of infection are not available in most clinical microbiology laboratories. Here we review these challenges and discuss how they may be addressed using advances in molecular, proteomic and immunological methods. © 2015 International Society of Neuropathology.

Multiplex detection of microRNAs by combining molecular beacon probes with T7 exonuclease-assisted cyclic amplification reaction.

PubMed

Liu, Yacui; Zhang, Jiangyan; Tian, Jingxiao; Fan, Xiaofei; Geng, Hao; Cheng, Yongqiang

2017-01-01

A simple, highly sensitive, and specific assay was developed for the homogeneous and multiplex detection of microRNAs (miRNAs) by combining molecular beacon (MB) probes and T7 exonuclease-assisted cyclic amplification. An MB probe with five base pairs in the stem region without special modification can effectively prevent the digestion by T7 exonuclease. Only in the presence of target miRNA is the MB probe hybridized with the target miRNA, and then digested by T7 exonuclease in the 5' to 3' direction. At the same time, the target miRNA is released and subsequently initiates the nuclease-assisted cyclic digestion process, generating enhanced fluorescence signal significantly. The results show that the combination of T7 exonuclease-assisted cyclic amplification reaction and MB probe possesses higher sensitivity for miRNA detection. Moreover, multiplex detection of miRNAs was successfully achieved by designing two MB probes labeled with FAM and Cy3, respectively. As a result, the method opens a new pathway for the sensitive and multiplex detection of miRNAs as well as clinical diagnosis. Graphical Abstract A simple, highly sensitive, and specific assay was developed for the detection of microRNAs by combining molecular beacon probes with T7 exonuclease-assisted cyclic amplification reaction.

Polarization Sensitive Coherent Anti-Stokes Raman Spectroscopy of DCVJ in Doped Polymer

NASA Astrophysics Data System (ADS)

Ujj, Laszlo

2014-05-01

Coherent Raman Microscopy is an emerging technic and method to image biological samples such as living cells by recording vibrational fingerprints of molecules with high spatial resolution. The race is on to record the entire image during the shortest time possible in order to increase the time resolution of the recorded cellular events. The electronically enhanced polarization sensitive version of Coherent anti-Stokes Raman scattering is one of the method which can shorten the recording time and increase the sharpness of an image by enhancing the signal level of special molecular vibrational modes. In order to show the effectiveness of the method a model system, a highly fluorescence sample, DCVJ in a polymer matrix is investigated. Polarization sensitive resonance CARS spectra are recorded and analyzed. Vibrational signatures are extracted with model independent methods. Details of the measurements and data analysis will be presented. The author gratefully acknowledge the UWF for financial support.

Molecular testing for clinical diagnosis and epidemiological investigations of intestinal parasitic infections.

PubMed

Verweij, Jaco J; Stensvold, C Rune

2014-04-01

Over the past few decades, nucleic acid-based methods have been developed for the diagnosis of intestinal parasitic infections. Advantages of nucleic acid-based methods are numerous; typically, these include increased sensitivity and specificity and simpler standardization of diagnostic procedures. DNA samples can also be stored and used for genetic characterization and molecular typing, providing a valuable tool for surveys and surveillance studies. A variety of technologies have been applied, and some specific and general pitfalls and limitations have been identified. This review provides an overview of the multitude of methods that have been reported for the detection of intestinal parasites and offers some guidance in applying these methods in the clinical laboratory and in epidemiological studies.

Molecular Testing for Clinical Diagnosis and Epidemiological Investigations of Intestinal Parasitic Infections

PubMed Central

Stensvold, C. Rune

2014-01-01

SUMMARY Over the past few decades, nucleic acid-based methods have been developed for the diagnosis of intestinal parasitic infections. Advantages of nucleic acid-based methods are numerous; typically, these include increased sensitivity and specificity and simpler standardization of diagnostic procedures. DNA samples can also be stored and used for genetic characterization and molecular typing, providing a valuable tool for surveys and surveillance studies. A variety of technologies have been applied, and some specific and general pitfalls and limitations have been identified. This review provides an overview of the multitude of methods that have been reported for the detection of intestinal parasites and offers some guidance in applying these methods in the clinical laboratory and in epidemiological studies. PMID:24696439

Natural dye sensitizer from cassava (Manihot utilissima) leaves extract and its adsorption onto TiO2 photo-anode

NASA Astrophysics Data System (ADS)

Nurlela; Wibowo, R.; Gunlazuardi, J.

2017-04-01

Interaction between TiO2 and dyes sensitizer have been studied. The chlorophyll presents in the crude leave extract (CLE-dye) from cassava (Manihot utilissima) was immobilized on to the photo-anode, consists of TiO2 supported by fluor doped Tin oxide (SnO2-F) Glass. The TiO2 was prepared by Rapid Breakdown Anodization (RBA) method then immobilized on to glass coated by SnO2-F using doctor blade technique, to give CLE-dye/TiO2/SnO2-F/Glass photo-anode. The prepared photo-anode was characterized by UV-Vis-DRS, FTIR, XRD, SEM, electrochemical and spectro-electrochemical systems. In this study, the HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) energy level of the CLE-dye were empirically determined by cyclic voltammetry method, while spectro-electro-chemistry method was used to determine the coefficient of degradation and formation of the dyes, and diffusion coefficient of the hole recombination as well. Good anchoring between TiO2 with dye extracts (CLE-dye) can be seen from value of dye LUMO energy level (-4.26 eV), which is approaching the conduction band of TiO2 (-4.3 eV). The coefficient of degradation and formation of the CLE-dye showed a quasi reversible and diffusion coefficient hole recombination values were small, indicated that it is quite suitable as a sensitizer in a dyes sensitized solar cell.

Fluorescence Molecular Tomography: Principles and Potential for Pharmaceutical Research

PubMed Central

Stuker, Florian; Ripoll, Jorge; Rudin, Markus

2011-01-01

Fluorescence microscopic imaging is widely used in biomedical research to study molecular and cellular processes in cell culture or tissue samples. This is motivated by the high inherent sensitivity of fluorescence techniques, the spatial resolution that compares favorably with cellular dimensions, the stability of the fluorescent labels used and the sophisticated labeling strategies that have been developed for selectively labeling target molecules. More recently, two and three-dimensional optical imaging methods have also been applied to monitor biological processes in intact biological organisms such as animals or even humans. These whole body optical imaging approaches have to cope with the fact that biological tissue is a highly scattering and absorbing medium. As a consequence, light propagation in tissue is well described by a diffusion approximation and accurate reconstruction of spatial information is demanding. While in vivo optical imaging is a highly sensitive method, the signal is strongly surface weighted, i.e., the signal detected from the same light source will become weaker the deeper it is embedded in tissue, and strongly depends on the optical properties of the surrounding tissue. Derivation of quantitative information, therefore, requires tomographic techniques such as fluorescence molecular tomography (FMT), which maps the three-dimensional distribution of a fluorescent probe or protein concentration. The combination of FMT with a structural imaging method such as X-ray computed tomography (CT) or Magnetic Resonance Imaging (MRI) will allow mapping molecular information on a high definition anatomical reference and enable the use of prior information on tissue's optical properties to enhance both resolution and sensitivity. Today many of the fluorescent assays originally developed for studies in cellular systems have been successfully translated for experimental studies in animals. The opportunity of monitoring molecular processes non-invasively in the intact organism is highly attractive from a diagnostic point of view but even more so for the drug developer, who can use the techniques for proof-of-mechanism and proof-of-efficacy studies. This review shall elucidate the current status and potential of fluorescence tomography including recent advances in multimodality imaging approaches for preclinical and clinical drug development. PMID:24310495

DEVELOPMENT OF A MOLECULAR METHOD TO IDENTIFY ASTROVIRUS IN WATER

EPA Science Inventory

Astrovirus is a common cause of gastroenteritis that has been determined to be responsible for several outbreaks. Since astrovirus can be waterborne, there is interest in testing environmental water for astrovirus. We have developed a sensitive reverse transcription-polymerase ...

Comparison of Non-Culture-Based Methods for Detection of Systemic Fungal Infections, with an Emphasis on Invasive Candida Infections

PubMed Central

White, P. Lewis; Archer, Alice E.; Barnes, Rosemary A.

2005-01-01

The accepted limitations associated with classic culture techniques for the diagnosis of invasive fungal infections have lead to the emergence of many non-culture-based methods. With superior sensitivities and quicker turnaround times, non-culture-based methods may aid the diagnosis of invasive fungal infections. In this review of the diagnostic service, we assessed the performances of two antigen detection techniques (enzyme-linked immunosorbent assay [ELISA] and latex agglutination) with a molecular method for the detection of invasive Candida infection and invasive aspergillosis. The specificities for all three assays were high (≥97%), although the Candida PCR method had enhanced sensitivity over both ELISA and latex agglutination with values of 95%, 75%, and 25%, respectively. However, calculating significant sensitivity values for the Aspergillus detection methods was not feasible due to a low number of proven/probable cases. Despite enhanced sensitivity, the PCR method failed to detect nucleic acid in a probable case of invasive Candida infection that was detected by ELISA. In conclusion, both PCR and ELISA techniques should be used in unison to aid the detection of invasive fungal infections. PMID:15872239

Molecular analysis of tumor margins by MALDI mass spectrometry in renal carcinoma.

PubMed

Oppenheimer, Stacey R; Mi, Deming; Sanders, Melinda E; Caprioli, Richard M

2010-05-07

The rate of tumor recurrence post resection suggests that there are underlying molecular changes in nearby histologically normal tissue that go undetected by conventional diagnostic methods that utilize contrast agents and immunohistochemistry. MALDI MS is a molecular technology that has the specificity and sensitivity to monitor and identify molecular species indicative of these changes. The current study utilizes this technology to assess molecular distributions within a tumor and adjacent normal tissue in clear cell renal cell carcinoma biopsies. Results indicate that the histologically normal tissue adjacent to the tumor expresses many of the molecular characteristics of the tumor. Proteins of the mitochondrial electron transport system are examples of such distributions. This work demonstrates the utility of MALDI MS for the analysis of tumor tissue in the elucidation of aberrant molecular changes in the tumor microenvironment.

Small business development for molecular diagnostics.

PubMed

Anagostou, Anthanasia; Liotta, Lance A

2012-01-01

Molecular profiling, which is the application of molecular diagnostics technology to tissue and blood -specimens, is an integral element in the new era of molecular medicine and individualized therapy. Molecular diagnostics is a fertile ground for small business development because it can generate products that meet immediate demands in the health-care sector: (a) Detection of disease risk, or early-stage disease, with a higher specificity and sensitivity compared to previous testing methods, and (b) "Companion diagnostics" for stratifying patients to receive a treatment choice optimized to their individual disease. This chapter reviews the promise and challenges of business development in this field. Guidelines are provided for the creation of a business model and the generation of a marketing plan around a candidate molecular diagnostic product. Steps to commercialization are outlined using existing molecular diagnostics companies as learning examples.

Molecular epidemiology for vector research on leishmaniasis.

PubMed

Kato, Hirotomo; Gomez, Eduardo A; Cáceres, Abraham G; Uezato, Hiroshi; Mimori, Tatsuyuki; Hashiguchi, Yoshihisa

2010-03-01

Leishmaniasis is a protozoan disease caused by the genus Leishmania transmitted by female phlebotomine sand flies. Surveillance of the prevalence of Leishmania and responsive vector species in endemic and surrounding areas is important for predicting the risk and expansion of the disease. Molecular biological methods are now widely applied to epidemiological studies of infectious diseases including leishmaniasis. These techniques are used to detect natural infections of sand fly vectors with Leishmania protozoa and are becoming powerful tools due to their sensitivity and specificity. Recently, genetic analyses have been performed on sand fly species and genotyping using PCR-RFLP has been applied to the sand fly taxonomy. In addition, a molecular mass screening method has been established that enables both sand fly species and natural leishmanial infections to be identified simultaneously in hundreds of sand flies with limited effort. This paper reviews recent advances in the study of sand flies, vectors of leishmaniasis, using molecular biological approaches.

Molecular Epidemiology for Vector Research on Leishmaniasis

PubMed Central

Kato, Hirotomo; Gomez, Eduardo A; Cáceres, Abraham G; Uezato, Hiroshi; Mimori, Tatsuyuki; Hashiguchi, Yoshihisa

2010-01-01

Leishmaniasis is a protozoan disease caused by the genus Leishmania transmitted by female phlebotomine sand flies. Surveillance of the prevalence of Leishmania and responsive vector species in endemic and surrounding areas is important for predicting the risk and expansion of the disease. Molecular biological methods are now widely applied to epidemiological studies of infectious diseases including leishmaniasis. These techniques are used to detect natural infections of sand fly vectors with Leishmania protozoa and are becoming powerful tools due to their sensitivity and specificity. Recently, genetic analyses have been performed on sand fly species and genotyping using PCR-RFLP has been applied to the sand fly taxonomy. In addition, a molecular mass screening method has been established that enables both sand fly species and natural leishmanial infections to be identified simultaneously in hundreds of sand flies with limited effort. This paper reviews recent advances in the study of sand flies, vectors of leishmaniasis, using molecular biological approaches. PMID:20617005

Brewster-plate spoiler - A novel method for reducing the amplitude of interference fringes that limit tunable-laser absorption sensitivities

NASA Technical Reports Server (NTRS)

Webster, C. R.

1985-01-01

A simple method is described for substantially reducing the amplitude of interference fringes that limit the sensitivities of tunable-laser high-resolution absorption spectrometers. A lead-salt diode laser operating in the 7-micron region is used with a single Brewster-plate spoiler to reduce the fringe amplitude by a factor of 30 and also to allow the detection of absorptances 0.001 percent in a single laser scan without subtraction techniques, without complex frequency modulation, and without distortion of the molecular line-shape signals. Application to multipass-cell spectrometers is described.

Cells-nano interactions and molecular toxicity after delayed hypersensitivity, in guinea pigs on exposure to hydroxyapatite nanoparticles.

PubMed

Geetha, C S; Remya, N S; Leji, K B; Syama, S; Reshma, S C; Sreekanth, P J; Varma, H K; Mohanan, P V

2013-12-01

The aim of the study was to evaluate the cells-nanoparticle interactions and molecular toxicity after delayed hypersensitivity in Guinea pigs, exposed to hydroxyapatite nanoparticles (HANP). The study focuses on synthesizing and characterizing HANPs and gaining an insight into the cytotoxicity, molecular toxicity, hypersensitivity and oxidative stress caused by them in vitro and in vivo. HANP was synthesized by chemical method and characterized by standard methods. Cytotoxicity was assessed on L929 cells by MTT assay and in vitro studies were carried out on rat liver homogenate. In vivo study was carried out by topical exposure of Guinea pigs with HANP, repeatedly, and evaluating the skin sensitization potential, blood parameters, oxidative stress in liver and brain and DNA damage (8-hydroxyl-2-deoxyguanosine: 8-OHdG) in liver. The results of the study indicated that there was no cytotoxicity (up to 600μg/mL) and oxidative damage (up to 100μg/mL), when exposed to HANPs. It was also evident that, there was no skin sensitization and oxidative damage when HANP were exposed to Guinea pigs. Copyright © 2013 Elsevier B.V. All rights reserved.

Method for efficient storage and transportation of sputum specimens for molecular testing of tuberculosis.

PubMed

Guio, H; Okayama, H; Ashino, Y; Saitoh, H; Xiao, P; Miki, M; Yoshihara, N; Nakanowatari, S; Hattori, T

2006-08-01

The polymerase chain reaction (PCR) is a highly sensitive method for the detection of Mycobacterium tuberculosis and is available in most countries, though to a lesser extent in rural areas. To amplify M. tuberculosis DNA sequences of sputum spotted on FTA cards and compare them with the results of microscopic examination among culture-positive samples. A total of 102 sputum specimens of TB patients in treatment were spotted on FTA cards and stored at room temperature until DNA analysis. We assessed the IS6110 region of M. tuberculosis. The efficacy of the PCR assay for the direct detection of M. tuberculosis was evaluated and compared with the results of cultures (Middlebrook 7H9 broth) and smears of fresh sputum specimens. We were able to detect 10 fg/microl of mycobacterial DNA even after 6 months in storage. The PCR sensitivity and specificity using the FTA card system were 82% and 96%, while microscopic examination showed 41% and 95%, respectively. The FTA card system for the storage of bacterial DNA from sputum samples should be considered for the molecular diagnosis of tuberculosis. Samples can easily be obtained from geographically isolated populations and shipped by mail for accurate molecular diagnosis.

Temperature sensitivity of organic compound destruction in SCWO process.

PubMed

Tan, Yaqin; Shen, Zhemin; Guo, Weimin; Ouyang, Chuang; Jia, Jinping; Jiang, Weili; Zhou, Haiyun

2014-03-01

To study the temperature sensitivity of the destruction of organic compounds in supercritical water oxidation process (SCWO), oxidation effects of twelve chemicals in supercritical water were investigated. The SCWO reaction rates of different compounds improved to varying degrees with the increase of temperature, so the highest slope of the temperature-effect curve (imax) was defined as the maximum ratio of removal ratio to working temperature. It is an important index to stand for the temperature sensitivity effect in SCWO. It was proven that the higher imax is, the more significant the effect of temperature on the SCWO effect is. Since the high-temperature area of SCWO equipment is subject to considerable damage from fatigue, the temperature is of great significance in SCWO equipment operation. Generally, most compounds (imax > 0.25) can be completely oxidized when the reactor temperature reaches 500°C. However, some compounds (imax > 0.25) need a higher temperature for complete oxidation, up to 560°C. To analyze the correlation coefficients between imax and various molecular descriptors, a quantum chemical method was used in this study. The structures of the twelve organic compounds were optimized by the Density Functional Theory B3LYP/6-311G method, as well as their quantum properties. It was shown that six molecular descriptors were negatively correlated to imax while other three descriptors were positively correlated to imax. Among them, dipole moment had the greatest effect on the oxidation thermodynamics of the twelve organic compounds. Once a correlation between molecular descriptors and imax is established, SCWO can be run at an appropriate temperature according to molecular structure. Copyright © 2014 The Research Centre for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V. All rights reserved.

Automated Protein Biomarker Analysis: on-line extraction of clinical samples by Molecularly Imprinted Polymers

NASA Astrophysics Data System (ADS)

Rossetti, Cecilia; Świtnicka-Plak, Magdalena A.; Grønhaug Halvorsen, Trine; Cormack, Peter A. G.; Sellergren, Börje; Reubsaet, Léon

2017-03-01

Robust biomarker quantification is essential for the accurate diagnosis of diseases and is of great value in cancer management. In this paper, an innovative diagnostic platform is presented which provides automated molecularly imprinted solid-phase extraction (MISPE) followed by liquid chromatography-mass spectrometry (LC-MS) for biomarker determination using ProGastrin Releasing Peptide (ProGRP), a highly sensitive biomarker for Small Cell Lung Cancer, as a model. Molecularly imprinted polymer microspheres were synthesized by precipitation polymerization and analytical optimization of the most promising material led to the development of an automated quantification method for ProGRP. The method enabled analysis of patient serum samples with elevated ProGRP levels. Particularly low sample volumes were permitted using the automated extraction within a method which was time-efficient, thereby demonstrating the potential of such a strategy in a clinical setting.

Precision Atomic Beam Laser Spectroscopy

DTIC Science & Technology

1999-02-20

optical efficiency with a new coupled- cavity scheme. We have locked a MISER Nd:YAG laser to a finesse 50,000 cavity with a...sensitivity of optical heterodyne detection is preserved with ZERO sensitivity to small laser / cavity frequency noises. The new method is called Noise-Immune...1996), P. Dube, L.- S. Ma, J. Ye, and J.L.Hall. 9 . "Free-induction decay in molecular iodine measured with an extended - cavity diode laser ,"

Ab initio study of energy transfer rates and impact sensitivities of crystalline explosives.

PubMed

Bernstein, Jonathan

2018-02-28

Impact sensitivities of various crystalline explosives were predicted by means of plane wave-density functional theory calculations. Crystal structures and complete vibrational spectra of TATB, PETN, FOX7, TEX, 14DNI, and β-HMX molecular crystals were calculated. A correlation between the phonon-vibron coupling (which is proportionally related to the energy transfer rate between the phonon manifold and the intramolecular vibrational modes) and impact sensitivities of secondary explosives was found. We propose a method, based on ab initio calculations, for the evaluation of impact sensitivities, which consequently can assist in screening candidates for chemical synthesis of high energetic materials.

Ab initio study of energy transfer rates and impact sensitivities of crystalline explosives

NASA Astrophysics Data System (ADS)

Bernstein, Jonathan

2018-02-01

Impact sensitivities of various crystalline explosives were predicted by means of plane wave-density functional theory calculations. Crystal structures and complete vibrational spectra of TATB, PETN, FOX7, TEX, 14DNI, and β-HMX molecular crystals were calculated. A correlation between the phonon-vibron coupling (which is proportionally related to the energy transfer rate between the phonon manifold and the intramolecular vibrational modes) and impact sensitivities of secondary explosives was found. We propose a method, based on ab initio calculations, for the evaluation of impact sensitivities, which consequently can assist in screening candidates for chemical synthesis of high energetic materials.

Comparison of five assays for detection of Clostridium difficile toxin.

PubMed

Chapin, Kimberle C; Dickenson, Roberta A; Wu, Fongman; Andrea, Sarah B

2011-07-01

Performance characteristics of five assays for detection of Clostridium difficile toxin were compared using fresh stool samples from patients with C. difficile infection (CDI). Assays were performed simultaneously and according to the manufacturers' instructions. Patients were included in the study if they exhibited clinical symptoms consistent with CDI. Nonmolecular assays included glutamate dehydrogenase antigen tests, with positive findings followed by the Premier Toxin A and B Enzyme Immunoassay (GDH/EIA), and the C. Diff Quik Chek Complete test. Molecular assays (PCR) included the BD GeneOhm Cdiff Assay, the Xpert C. difficile test, and the ProGastro Cd assay. Specimens were considered true positive if results were positive in two or more assays. For each method, the Youden index was calculated and cost-effectiveness was analyzed. Of 81 patients evaluated, 26 (32.1%) were positive for CDI. Sensitivity of the BD GeneOhm Cdiff assay, the Xpert C. difficile test, the ProGastro Cd assay, C. Diff Quik Chek Complete test, and two-step GDH/EIA was 96.2%, 96.2%, 88.5%, 61.5%, and 42.3%, respectively. Specificity of the Xpert C. difficile test was 96.4%, and for the other four assays was 100%. Compared with nonmolecular methods, molecular methods detected 34.7% more positive specimens. Assessment of performance characteristics and cost-effectiveness demonstrated that the BD GeneOhm Cdiff assay yielded the best results. While costly, the Xpert C. difficile test required limited processing and yielded rapid results. Because of discordant results, specimen processing, and extraction equipment requirements, the ProGastro Cd assay was the least favored molecular assay. The GDH/EIA method lacked sufficient sensitivity to be recommended. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Ultra-Sensitive Detection of Plasmodium falciparum by Amplification of Multi-Copy Subtelomeric Targets

PubMed Central

Hofmann, Natalie; Mwingira, Felista; Shekalaghe, Seif; Robinson, Leanne J.; Mueller, Ivo; Felger, Ingrid

2015-01-01

Background Planning and evaluating malaria control strategies relies on accurate definition of parasite prevalence in the population. A large proportion of asymptomatic parasite infections can only be identified by surveillance with molecular methods, yet these infections also contribute to onward transmission to mosquitoes. The sensitivity of molecular detection by PCR is limited by the abundance of the target sequence in a DNA sample; thus, detection becomes imperfect at low densities. We aimed to increase PCR diagnostic sensitivity by targeting multi-copy genomic sequences for reliable detection of low-density infections, and investigated the impact of these PCR assays on community prevalence data. Methods and Findings Two quantitative PCR (qPCR) assays were developed for ultra-sensitive detection of Plasmodium falciparum, targeting the high-copy telomere-associated repetitive element 2 (TARE-2, ∼250 copies/genome) and the var gene acidic terminal sequence (varATS, 59 copies/genome). Our assays reached a limit of detection of 0.03 to 0.15 parasites/μl blood and were 10× more sensitive than standard 18S rRNA qPCR. In a population cross-sectional study in Tanzania, 295/498 samples tested positive using ultra-sensitive assays. Light microscopy missed 169 infections (57%). 18S rRNA qPCR failed to identify 48 infections (16%), of which 40% carried gametocytes detected by pfs25 quantitative reverse-transcription PCR. To judge the suitability of the TARE-2 and varATS assays for high-throughput screens, their performance was tested on sample pools. Both ultra-sensitive assays correctly detected all pools containing one low-density P. falciparum–positive sample, which went undetected by 18S rRNA qPCR, among nine negatives. TARE-2 and varATS qPCRs improve estimates of prevalence rates, yet other infections might still remain undetected when absent in the limited blood volume sampled. Conclusions Measured malaria prevalence in communities is largely determined by the sensitivity of the diagnostic tool used. Even when applying standard molecular diagnostics, prevalence in our study population was underestimated by 8% compared to the new assays. Our findings highlight the need for highly sensitive tools such as TARE-2 and varATS qPCR in community surveillance and for monitoring interventions to better describe malaria epidemiology and inform malaria elimination efforts. PMID:25734259

Comparative analysis of detection limits and specificity of molecular diagnostic markers for three pathogens (Microsporidia, Nosema spp.) in the key pollinators Apis mellifera and Bombus terrestris.

PubMed

Erler, Silvio; Lommatzsch, Stefanie; Lattorff, H Michael G

2012-04-01

Global pollinator decline has recently been discussed in the context of honey and bumble bee infections from various pathogens including viruses, bacteria, microsporidia and mites. The microsporidian pathogens Nosema apis, Nosema ceranae and Nosema bombi may in fact be major candidates contributing to this decline. Different molecular and non-molecular detection methods have been developed; however, a comparison, especially of the highly sensitive PCR based methods, is currently lacking. Here, we present the first comparative quantitative real-time PCR study of nine Nosema spp. primers within the framework of primer specificity and sensitivity. With the help of dilution series of defined numbers of spores, we reveal six primer pairs amplifying N. apis, six for N. bombi and four for N. ceranae. All appropriate primer pairs detected an amount of at least 10(4) spores, the majority of which were even as sensitive to detect such low amounts as 10(3) to ten spores. Species specificity of primers was observed for N. apis and N. bombi, but not for N. ceranae. Additionally, we did not find any significant correlation for the amplified fragments with PCR efficiency or the limit of detection. We discuss our findings on the background of false positive and negative results using quantitative real-time PCR. On the basis of these results, future research might be based on appropriate primer selection depending on the experimental needs. Primers may be selected on the basis of specificity or sensitivity. Pathogen species and load may be determined with higher precision enhancing all kinds of diagnostic studies.

Ultrafast, sensitive and large-volume on-chip real-time PCR for the molecular diagnosis of bacterial and viral infections.

PubMed

Houssin, Timothée; Cramer, Jérémy; Grojsman, Rébecca; Bellahsene, Lyes; Colas, Guillaume; Moulet, Hélène; Minnella, Walter; Pannetier, Christophe; Leberre, Maël; Plecis, Adrien; Chen, Yong

2016-04-21

To control future infectious disease outbreaks, like the 2014 Ebola epidemic, it is necessary to develop ultrafast molecular assays enabling rapid and sensitive diagnoses. To that end, several ultrafast real-time PCR systems have been previously developed, but they present issues that hinder their wide adoption, notably regarding their sensitivity and detection volume. An ultrafast, sensitive and large-volume real-time PCR system based on microfluidic thermalization is presented herein. The method is based on the circulation of pre-heated liquids in a microfluidic chip that thermalize the PCR chamber by diffusion and ultrafast flow switches. The system can achieve up to 30 real-time PCR cycles in around 2 minutes, which makes it the fastest PCR thermalization system for regular sample volume to the best of our knowledge. After biochemical optimization, anthrax and Ebola simulating agents could be respectively detected by a real-time PCR in 7 minutes and a reverse transcription real-time PCR in 7.5 minutes. These detections are respectively 6.4 and 7.2 times faster than with an off-the-shelf apparatus, while conserving real-time PCR sample volume, efficiency, selectivity and sensitivity. The high-speed thermalization also enabled us to perform sharp melting curve analyses in only 20 s and to discriminate amplicons of different lengths by rapid real-time PCR. This real-time PCR microfluidic thermalization system is cost-effective, versatile and can be then further developed for point-of-care, multiplexed, ultrafast and highly sensitive molecular diagnoses of bacterial and viral diseases.

NONINVASIVE APPROACHES FOR TOXICOLOICAL RESEARCH OF THE LUNG

EPA Science Inventory

Four presentations are planned to overview this topic: 1) a review of gas chromatographic and mass spectrometric methods and sensitivity of these measures of volatile molecules present in exhaled breath. Molecular species present in breath have been predictive markers in a number...

Predictive and Prognostic Molecular Biomarkers for Response to Neoadjuvant Chemoradiation in Rectal Cancer.

PubMed

Dayde, Delphine; Tanaka, Ichidai; Jain, Rekha; Tai, Mei Chee; Taguchi, Ayumu

2017-03-07

The standard of care in locally advanced rectal cancer is neoadjuvant chemoradiation (nCRT) followed by radical surgery. Response to nCRT varies among patients and pathological complete response is associated with better outcome. However, there is a lack of effective methods to select rectal cancer patients who would or would not have a benefit from nCRT. The utility of clinicopathological and radiological features are limited due to lack of adequate sensitivity and specificity. Molecular biomarkers have the potential to predict response to nCRT at an early time point, but none have currently reached the clinic. Integration of diverse types of biomarkers including clinicopathological and imaging features, identification of mechanistic link to tumor biology, and rigorous validation using samples which represent disease heterogeneity, will allow to develop a sensitive and cost-effective molecular biomarker panel for precision medicine in rectal cancer. Here, we aim to review the recent advance in tissue- and blood-based molecular biomarker research and illustrate their potential in predicting nCRT response in rectal cancer.

Fluorophore-assisted carbohydrate electrophoresis for the determination of molecular mass of heparins and low-molecular-weight (LMW) heparins.

PubMed

Buzzega, Dania; Maccari, Francesca; Volpi, Nicola

2008-11-01

We report the use of fluorophore-assisted carbohydrate electrophoresis (FACE) to determine the molecular mass (M) values of heparins (Heps) and low-molecular-weight (LMW)-Hep derivatives. Hep are labeled with 8-aminonaphthalene-1,3,6-trisulfonic acid and FACE is able to resolve each fraction as a discrete band depending on their M. After densitometric acquisition, the migration distance of each Hep standard is acquired and the third-grade polynomial calibration standard curve is determined by plotting the logarithms of the M values as a function of migration ratio. Purified Hep samples having different properties, pharmaceutical Heps and various LMW-Heps were analyzed by both FACE and conventional high-performance size-exclusion liquid chromatography (HPSEC) methods. The molecular weight value on the top of the chromatographic peak (Mp), the number-average Mn, weight-average Mw and polydispersity (Mw/Mn) were examined by both techniques and found to be similar. This approach offers certain advantages over the HPSEC method. The derivatization process with 8-aminonaphthalene-1,3,6-trisulfonic acid is complete after 4 h so that many samples may be analyzed in a day also considering that multiple samples can be run simultaneously and in parallel and that a single FACE analysis requires approx. 15 min. Furthermore, FACE is a very sensitive method as it requires approx. 5-10 microg of Heps, about 10-100-fold lower than samples and standards used in HPSEC evaluation. Finally, the utilization of mini-gels allows the use of very low amounts of reagents with neither expensive equipment nor any complicated procedures having to be applied. This study demonstrates that FACE analysis is a sensitive method for the determination of the M values of Heps and LMW-Heps with possible utilization in virtually any kind of research and development such as quality control laboratories due to its rapid, parallel analysis of multiple samples by means of common and simple largely used analytical laboratory equipment.

Using molecular recognition of beta-cyclodextrin to determine molecular weights of low-molecular-weight explosives by MALDI-TOF mass spectrometry.

PubMed

Zhang, Min; Shi, Zhen; Bai, Yinjuan; Gao, Yong; Hu, Rongzu; Zhao, Fenqi

2006-02-01

This study presents a novel method for determining the molecular weights of low molecular weight (MW) energetic compounds through their complexes of beta-cyclodextrin (beta-CD) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in a mass range of 500 to 1700 Da, avoiding matrix interference. The MWs of one composite explosive composed of 2,6-DNT, TNT, and RDX, one propellant with unknown components, and 14 single-compound explosives (RDX, HMX, 3,4-DNT, 2,6-DNT, 2,5-DNT, 2,4,6-TNT, TNAZ, DNI, BTTN, NG, TO, NTO, NP, and 662) were measured. The molecular recognition and inclusion behavior of beta-CD to energetic materials (EMs) were investigated. The results show that (1) the established method is sensitive, simple, accurate, and suitable for determining the MWs of low-MW single-compound explosives and energetic components in composite explosives and propellants; and (2) beta-CD has good inclusion and modular recognition abilities to the above EMs.

Molecular beacon-based real-time PCR method for detection of porcine DNA in gelatin and gelatin capsules.

PubMed

Mohamad, Nurhidayatul Asma; Mustafa, Shuhaimi; Khairil Mokhtar, Nur Fadhilah; El Sheikha, Aly Farag

2018-03-05

The pharmaceutical industry has boosted gelatin consumption worldwide. This is supported by the availability of cost-effective gelatin production from porcine by-products. However, cross-contamination of gelatin materials, where porcine gelatin was unintentionally included in the other animal sources of gelatin, has caused significant concerns about halal authenticity. The real-time polymerase chain reaction (PCR) has enabled a highly specific and sensitive animal species detection method in various food products. Hence, such a technique was employed in the present study to detect and quantify porcine DNA in gelatin using a molecular beacon probe, with differences in performance between mitochondrial (cytochrome b gene) and chromosomal DNA-(MPRE42 repetitive element) based porcine-specific PCR assays being compared. A higher sensitivity was observed in chromosomal DNA (MPRE-PCR assay), where this assay allows the detection of gelatin DNA at amounts as as low as 1 pg, whereas mitochondrial DNA (CBH-PCR assay) can only detect at levels down to 10 pg of gelatin DNA. When an analysis with commercial gelatin and gelatin capsule samples was conducted, the same result was observed, with a significantly more sensitive detection being provided by the repetitive element of chromosomal DNA. The present study has established highly sensitive DNA-based porcine detection systems derived from chromosomal DNA that are feasible for highly processed products such as gelatin and gelatin capsules containing a minute amount of DNA. This sensitive detection method can also be implemented to assist the halal authentication process of various food products available on the market. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

A cascaded QSAR model for efficient prediction of overall power conversion efficiency of all-organic dye-sensitized solar cells.

PubMed

Li, Hongzhi; Zhong, Ziyan; Li, Lin; Gao, Rui; Cui, Jingxia; Gao, Ting; Hu, Li Hong; Lu, Yinghua; Su, Zhong-Min; Li, Hui

2015-05-30

A cascaded model is proposed to establish the quantitative structure-activity relationship (QSAR) between the overall power conversion efficiency (PCE) and quantum chemical molecular descriptors of all-organic dye sensitizers. The cascaded model is a two-level network in which the outputs of the first level (JSC, VOC, and FF) are the inputs of the second level, and the ultimate end-point is the overall PCE of dye-sensitized solar cells (DSSCs). The model combines quantum chemical methods and machine learning methods, further including quantum chemical calculations, data division, feature selection, regression, and validation steps. To improve the efficiency of the model and reduce the redundancy and noise of the molecular descriptors, six feature selection methods (multiple linear regression, genetic algorithms, mean impact value, forward selection, backward elimination, and +n-m algorithm) are used with the support vector machine. The best established cascaded model predicts the PCE values of DSSCs with a MAE of 0.57 (%), which is about 10% of the mean value PCE (5.62%). The validation parameters according to the OECD principles are R(2) (0.75), Q(2) (0.77), and Qcv2 (0.76), which demonstrate the great goodness-of-fit, predictivity, and robustness of the model. Additionally, the applicability domain of the cascaded QSAR model is defined for further application. This study demonstrates that the established cascaded model is able to effectively predict the PCE for organic dye sensitizers with very low cost and relatively high accuracy, providing a useful tool for the design of dye sensitizers with high PCE. © 2015 Wiley Periodicals, Inc.

Efficiencies of Dye-Sensitized Solar Cells using Ferritin-Encapsulated Quantum Dots with Various Staining Methods

NASA Astrophysics Data System (ADS)

Perez, Luis

Dye-sensitized solar cells (DSSC) have the potential to replace traditional and cost-inefficient crystalline silicon or ruthenium solar cells. This can only be accomplished by optimizing DSSC's energy efficiency. One of the major components in a dye-sensitized solar cell is the porous layer of titanium dioxide. This layer is coated with a molecular dye that absorbs sunlight. The research conducted for this paper focuses on the different methods used to dye the porous TiO2 layer with ferritin-encapsulated quantum dots. Multiple anodes were dyed using a method known as SILAR which involves deposition through alternate immersion in two different solutions. The efficiencies of DSSCs with ferritin-encapsulated lead sulfide dye deposited using SILAR were subsequently compared against the efficiencies produced by cells using the traditional immersion method. It was concluded that both methods resulted in similar efficiencies (? .074%) however, the SILAR method dyed the TiO2 coating significantly faster than the immersion method. On a related note, our experiments concluded that conducting 2 SILAR cycles yields the highest possible efficiency for this particular binding method. National Science Foundation.

Combined electrophoresis-electrospray interface and method

DOEpatents

Smith, Richard D.; Udseth, Harold R.; Barinaga, Charles J.

1995-01-01

An improvement to the system and method for analyzing molecular constituents of a composition sample that comprises improvements to an electrospray ionization source for interfacing to mass spectrometers and other detection devices. The improvement consists of establishing a unique electrical circuit pattern and nozzle configuration, a metallic coated and conical shaped capillary outlet, coupled with sizing of the capillary to obtain maximum sensitivity.

Identification of Pseudallescheria and Scedosporium species by three molecular methods.

PubMed

Lu, Qiaoyun; Gerrits van den Ende, A H G; Bakkers, J M J E; Sun, Jiufeng; Lackner, M; Najafzadeh, M J; Melchers, W J G; Li, Ruoyu; de Hoog, G S

2011-03-01

The major clinically relevant species in Scedosporium (teleomorph Pseudallescheria) are Pseudallescheria boydii, Scedosporium aurantiacum, Scedosporium apiospermum, and Scedosporium prolificans, while Pseudallescheria minutispora, Petriellopsis desertorum, and Scedosporium dehoogii are exceptional agents of disease. Three molecular methods targeting the partial β-tubulin gene were developed and evaluated to identify six closely related species of the S. apiospermum complex using quantitative real-time PCR (qPCR), PCR-based reverse line blot (PCR-RLB), and loop-mediated isothermal amplification (LAMP). qPCR was not specific enough for the identification of all species but had the highest sensitivity. The PCR-RLB assay was efficient for the identification of five species. LAMP distinguished all six species unambiguously. The analytical sensitivities of qPCR, PCR-RLB, and LAMP combined with MagNAPure, CTAB (cetyltrimethylammonium bromide), and FTA filter (Whatman) extraction were 50, 5 × 10(3), and 5 × 10(2) cells/μl, respectively. When LAMP was combined with a simplified DNA extraction method using an FTA filter, identification to the species level was achieved within 2 h, including DNA extraction. The FTA-LAMP assay is therefore recommended as a cost-effective, simple, and rapid method for the identification of Scedosporium species.

Novel real-time polymerase chain reaction assay for simultaneous detection of recurrent fusion genes in acute myeloid leukemia.

PubMed

Dolz, Sandra; Barragán, Eva; Fuster, Óscar; Llop, Marta; Cervera, José; Such, Esperanza; De Juan, Inmaculada; Palanca, Sarai; Murria, Rosa; Bolufer, Pascual; Luna, Irene; Gómez, Inés; López, María; Ibáñez, Mariam; Sanz, Miguel A

2013-09-01

The recent World Health Organization classification recognizes different subtypes of acute myeloid leukemia (AML) according to the presence of several recurrent genetic abnormalities. Detection of these abnormalities and other molecular changes is of increasing interest because it contributes to a refined diagnosis and prognostic assessment in AML and enables monitoring of minimal residual disease. These genetic abnormalities can be detected using single RT-PCR, although the screening is still labor intensive and costly. We have developed a novel real-time RT-PCR assay to simultaneously detect 15 AML-associated rearrangements that is a simple and easily applicable method for use in clinical diagnostic laboratories. This method showed 100% specificity and sensitivity (95% confidence interval, 91% to 100% and 92% to 100%, respectively). The procedure was validated in a series of 105 patients with AML. The method confirmed all translocations detected using standard cytogenetics and fluorescence in situ hybridization and some additional undetected rearrangements. Two patients demonstrated two molecular rearrangements simultaneously, with BCR-ABL1 implicated in both, in addition to RUNX1-MECOM in one patient and PML-RARA in another. In conclusion, this novel real-time RT-PCR assay for simultaneous detection of multiple AML-associated fusion genes is a versatile and sensitive method for reliable screening of recurrent rearrangements in AML. Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Diagnostic Performance of a Molecular Test versus Clinician Assessment of Vaginitis

PubMed Central

Gaydos, Charlotte A.; Nyirjesy, Paul; Paradis, Sonia; Kodsi, Salma; Cooper, Charles K.

2018-01-01

ABSTRACT Vaginitis is a common complaint, diagnosed either empirically or using Amsel's criteria and wet mount microscopy. This study sought to determine characteristics of an investigational test (a molecular test for vaginitis), compared to reference, for detection of bacterial vaginosis, Candida spp., and Trichomonas vaginalis. Vaginal specimens from a cross-sectional study were obtained from 1,740 women (≥18 years old), with vaginitis symptoms, during routine clinic visits (across 10 sites in the United States). Specimens were analyzed using a commercial PCR/fluorogenic probe-based investigational test that detects bacterial vaginosis, Candida spp., and Trichomonas vaginalis. Clinician diagnosis and in-clinic testing (Amsel's test, potassium hydroxide preparation, and wet mount) were also employed to detect the three vaginitis causes. All testing methods were compared to the respective reference methods (Nugent Gram stain for bacterial vaginosis, detection of the Candida gene its2, and Trichomonas vaginalis culture). The investigational test, clinician diagnosis, and in-clinic testing were compared to reference methods for bacterial vaginosis, Candida spp., and Trichomonas vaginalis. The investigational test resulted in significantly higher sensitivity and negative predictive value than clinician diagnosis or in-clinic testing. In addition, the investigational test showed a statistically higher overall percent agreement with each of the three reference methods than did clinician diagnosis or in-clinic testing. The investigational test showed significantly higher sensitivity for detecting vaginitis, involving more than one cause, than did clinician diagnosis. Taken together, these results suggest that a molecular investigational test can facilitate accurate detection of vaginitis. PMID:29643195

A fluorescence high throughput screening method for the detection of reactive electrophiles as potential skin sensitizers.

PubMed

Avonto, Cristina; Chittiboyina, Amar G; Rua, Diego; Khan, Ikhlas A

2015-12-01

Skin sensitization is an important toxicological end-point in the risk assessment of chemical allergens. Because of the complexity of the biological mechanisms associated with skin sensitization, integrated approaches combining different chemical, biological and in silico methods are recommended to replace conventional animal tests. Chemical methods are intended to characterize the potential of a sensitizer to induce earlier molecular initiating events. The presence of an electrophilic mechanistic domain is considered one of the essential chemical features to covalently bind to the biological target and induce further haptenation processes. Current in chemico assays rely on the quantification of unreacted model nucleophiles after incubation with the candidate sensitizer. In the current study, a new fluorescence-based method, 'HTS-DCYA assay', is proposed. The assay aims at the identification of reactive electrophiles based on their chemical reactivity toward a model fluorescent thiol. The reaction workflow enabled the development of a High Throughput Screening (HTS) method to directly quantify the reaction adducts. The reaction conditions have been optimized to minimize solubility issues, oxidative side reactions and increase the throughput of the assay while minimizing the reaction time, which are common issues with existing methods. Thirty-six chemicals previously classified with LLNA, DPRA or KeratinoSens™ were tested as a proof of concept. Preliminary results gave an estimated 82% accuracy, 78% sensitivity, 90% specificity, comparable to other in chemico methods such as Cys-DPRA. In addition to validated chemicals, six natural products were analyzed and a prediction of their sensitization potential is presented for the first time. Copyright © 2015 Elsevier Inc. All rights reserved.

A pM leveled photoelectrochemical sensor for microcystin-LR based on surface molecularly imprinted TiO2@CNTs nanostructure.

PubMed

Liu, Meichuan; Ding, Xue; Yang, Qiwei; Wang, Yu; Zhao, Guohua; Yang, Nianjun

2017-06-05

A simple and highly sensitive photoelectrochemical (PEC) sensor towards Microcystin-LR (MC-LR), a kind of typical cyanobacterial toxin in water samples, was developed on a surface molecular imprinted TiO 2 coated multiwalled carbon nanotubes (MI-TiO 2 @CNTs) hybrid nanostructure. It was synthesized using a feasible two-step sol-gel method combining with in situ surface molecular imprinting technique (MIT). With a controllable core-shell tube casing structure, the resultant MI-TiO 2 @CNTs are enhanced greatly in visible-light driven response capacity. In comparison with the traditional TiO 2 (P25) and non-imprinted (NI-)TiO 2 @CNTs, the MI-TiO 2 @CNTs based PEC sensor showed a much higher photoelectric oxidation capacity towards MC-LR. Using this sensor, the determination of MC-LR was doable in a wide linear range from 1.0pM to 3.0nM with a high photocurrent response sensitivity. An outstanding selectivity towards MC-LR was further achieved with this sensor, proven by simultaneously monitoring 100-fold potential co-existing interferences. The superiority of the obtained MC-LR sensor in sensitivity and selectivity is mainly attributed to the high specific surface area and excellent photoelectric activity of TiO 2 @CNTs heterojunction structure, as well as the abundant active recognition sites on its functionalized molecular imprinting surface. A promising PEC analysis platform with high sensitivity and selectivity for MC-LR has thus been provided. Copyright © 2017 Elsevier B.V. All rights reserved.

Iron Oxide Nanoparticle-Micelles (ION-Micelles) for Sensitive (Molecular) Magnetic Particle Imaging and Magnetic Resonance Imaging

PubMed Central

Starmans, Lucas W. E.; Burdinski, Dirk; Haex, Nicole P. M.; Moonen, Rik P. M.; Strijkers, Gustav J.; Nicolay, Klaas; Grüll, Holger

2013-01-01

Background Iron oxide nanoparticles (IONs) are a promising nanoplatform for contrast-enhanced MRI. Recently, magnetic particle imaging (MPI) was introduced as a new imaging modality, which is able to directly visualize magnetic particles and could serve as a more sensitive and quantitative alternative to MRI. However, MPI requires magnetic particles with specific magnetic properties for optimal use. Current commercially available iron oxide formulations perform suboptimal in MPI, which is triggering research into optimized synthesis strategies. Most synthesis procedures aim at size control of iron oxide nanoparticles rather than control over the magnetic properties. In this study, we report on the synthesis, characterization and application of a novel ION platform for sensitive MPI and MRI. Methods and Results IONs were synthesized using a thermal-decomposition method and subsequently phase-transferred by encapsulation into lipidic micelles (ION-Micelles). Next, the material and magnetic properties of the ION-Micelles were analyzed. Most notably, vibrating sample magnetometry measurements showed that the effective magnetic core size of the IONs is 16 nm. In addition, magnetic particle spectrometry (MPS) measurements were performed. MPS is essentially zero-dimensional MPI and therefore allows to probe the potential of iron oxide formulations for MPI. ION-Micelles induced up to 200 times higher signal in MPS measurements than commercially available iron oxide formulations (Endorem, Resovist and Sinerem) and thus likely allow for significantly more sensitive MPI. In addition, the potential of the ION-Micelle platform for molecular MPI and MRI was showcased by MPS and MRI measurements of fibrin-binding peptide functionalized ION-Micelles (FibPep-ION-Micelles) bound to blood clots. Conclusions The presented data underlines the potential of the ION-Micelle nanoplatform for sensitive (molecular) MPI and warrants further investigation of the FibPep-ION-Micelle platform for in vivo, non-invasive imaging of fibrin in preclinical disease models of thrombus-related pathologies and atherosclerosis. PMID:23437371

Quantitative experimental monitoring of molecular diffusion in clay with positron emission tomography

NASA Astrophysics Data System (ADS)

Kulenkampff, Johannes; Zakhnini, Abdelhamid; Gründig, Marion; Lippmann-Pipke, Johanna

2016-08-01

Clay plays a prominent role as barrier material in the geosphere. The small particle sizes cause extremely small pore sizes and induce low permeability and high sorption capacity. Transport of dissolved species by molecular diffusion, driven only by a concentration gradient, is less sensitive to the pore size. Heterogeneous structures on the centimetre scale could cause heterogeneous effects, like preferential transport zones, which are difficult to assess. Laboratory measurements with diffusion cells yield limited information on heterogeneity, and pore space imaging methods have to consider scale effects. We established positron emission tomography (PET), applying a high-resolution PET scanner as a spatially resolved quantitative method for direct laboratory observation of the molecular diffusion process of a PET tracer on the prominent scale of 1-100 mm. Although PET is rather insensitive to bulk effects, quantification required significant improvements of the image reconstruction procedure with respect to Compton scatter and attenuation. The experiments were conducted with 22Na and 124I over periods of 100 and 25 days, respectively. From the images we derived trustable anisotropic diffusion coefficients and, in addition, we identified indications of preferential transport zones. We thus demonstrated the unique potential of the PET imaging modality for geoscientific process monitoring under conditions where other methods fail, taking advantage of the extremely high detection sensitivity that is typical of radiotracer applications.

Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting.

PubMed

Fu, Glenn K; Wilhelmy, Julie; Stern, David; Fan, H Christina; Fodor, Stephen P A

2014-03-18

We present a new approach for the sensitive detection and accurate quantitation of messenger ribonucleic acid (mRNA) gene transcripts in single cells. First, the entire population of mRNAs is encoded with molecular barcodes during reverse transcription. After amplification of the gene targets of interest, molecular barcodes are counted by sequencing or scored on a simple hybridization detector to reveal the number of molecules in the starting sample. Since absolute quantities are measured, calibration to standards is unnecessary, and many of the relative quantitation challenges such as polymerase chain reaction (PCR) bias are avoided. We apply the method to gene expression analysis of minute sample quantities and demonstrate precise measurements with sensitivity down to sub single-cell levels. The method is an easy, single-tube, end point assay utilizing standard thermal cyclers and PCR reagents. Accurate and precise measurements are obtained without any need for cycle-to-cycle intensity-based real-time monitoring or physical partitioning into multiple reactions (e.g., digital PCR). Further, since all mRNA molecules are encoded with molecular barcodes, amplification can be used to generate more material for multiple measurements and technical replicates can be carried out on limited samples. The method is particularly useful for small sample quantities, such as single-cell experiments. Digital encoding of cellular content preserves true abundance levels and overcomes distortions introduced by amplification.

Estimating the Triple-Point Isotope Effect and the Corresponding Uncertainties for Cryogenic Fixed Points

NASA Astrophysics Data System (ADS)

Tew, W. L.

2008-02-01

The sensitivities of melting temperatures to isotopic variations in monatomic and diatomic atmospheric gases using both theoretical and semi-empirical methods are estimated. The current state of knowledge of the vapor-pressure isotope effects (VPIE) and triple-point isotope effects (TPIE) is briefly summarized for the noble gases (except He), and for selected diatomic molecules including oxygen. An approximate expression is derived to estimate the relative shift in the melting temperature with isotopic substitution. In general, the magnitude of the effects diminishes with increasing molecular mass and increasing temperature. Knowledge of the VPIE, molar volumes, and heat of fusion are sufficient to estimate the temperature shift or isotopic sensitivity coefficient via the derived expression. The usefulness of this approach is demonstrated in the estimation of isotopic sensitivities and uncertainties for triple points of xenon and molecular oxygen for which few documented estimates were previously available. The calculated sensitivities from this study are considerably higher than previous estimates for Xe, and lower than other estimates in the case of oxygen. In both these cases, the predicted sensitivities are small and the resulting variations in triple point temperatures due to mass fractionation effects are less than 20 μK.

DEVELOPMENT OF A MOLECULAR METHOD TO DETECT ASTROVIRUS

EPA Science Inventory

Astrovirus is a common cause of gastroenteritis that has been determined to be responsible for several outbreaks. Since astrovirus can be waterborne, there is interest in testing environmental water for astrovirus and we have developed a sensitive RT-PCR assay that is designed t...

Selective chromogenic detection of thiol-containing biomolecules using carbonaceous nanospheres loaded with silver nanoparticles as carrier.

PubMed

Hu, Bo; Zhao, Yang; Zhu, Hai-Zhou; Yu, Shu-Hong

2011-04-26

Thiol-containing biomolecules show strong affinity with noble metal nanostructures and could not only stably protect them but also control the self-assembly process of these special nanostructures. A highly selective and sensitive chromogenic detection method has been designed for the low and high molecular weight thiol-containing biomolecules, including cysteine, glutathione, dithiothreitol, and bovine serum albumin, using a new type of carbonaceous nanospheres loaded with silver nanoparticles (Ag NPs) as carrier. This strategy relies upon the place-exchange process between the reporter dyes on the surface of Ag NPs and the thiol groups of thiol-containing biomolecules. The concentration of biomolecules can be determined by monitoring with the fluorescence intensity of reporter dyes dispersed in solution. This new chromogenic assay method could selectively detect these biomolecules in the presence of various other amino acids and monosaccharides and even sensitively detect the thiol-containing biomolecules with different molecular weight, even including proteins.

Liquid chromatography coupled to molecular fluorescence with postcolumn UV sensitization for thimerosal and derivative compounds monitoring in environmental samples.

PubMed

Acosta, Gimena; Torres, Sabier; Kaplan, Marcos; Fernández, Liliana P; Pacheco, Pablo H; Gil, Raúl A

2016-10-01

A HPLC coupled with molecular fluorescence (MF) spectrometry method for determination of thimerosal (THM, sodium ethylmercurythiosalicylate, C 9 H 9 HgNaO 2 S), and derivatives is proposed. A sensitization of MF was provoked by UV irradiation of analytes in a home-made photoreactor that served as interface between the LC column and MF spectrometer. This method is applied to determination of THM, ethyl mercury, and thiosalicylic acid in samples of pharmaceutical industry effluents, and waters of La Carolina and Jáchal rivers situated in the center-west side of San Luis city and in the east of San Juan city (Middle West, Argentine) where the effluents are dumped. The LODs calculated on basis of 3σ criterion were 1.8, 5, and 0.05 μmol/L for THM, ethyl mercury, and for thiosalicylic acid, respectively. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Surface Plasmon Resonance-Based Optical Fiber Probe Fabricated with Electropolymerized Molecular Imprinting Film for Melamine Detection

PubMed Central

Zheng, Yongping; Zhang, Tingwei; Wu, Songjie; Zhang, Jue; Fang, Jing

2018-01-01

Molecularly imprinted polymer (MIP) films prepared by bulk polymerization suffer from numerous deficiencies, including poor mass transfer ability and difficulty in controlling reaction rate and film thickness, which usually result in poor repeatability. However, polymer film synthesized by electropolymerization methods benefit from high reproducibility, simplicity and rapidity of preparation. In the present study, an Au film served as the refractive index-sensitive metal film to couple with the light leaked out from optical fiber core and the electrode for electropolymerizing MIP film simultaneously. The manufactured probe exhibited satisfactory sensitivity and specificity. Furthermore, the surface morphology and functional groups of the synthesized MIP film were characterized by Atomic Force Microscopy (AFM) and Fourier transform infrared microspectroscopy (FTIR) for further insights into the adsorption and desorption processes. Given the low cost, label-free test, simple preparation process and fast response, this method has a potential application to monitor substances in complicated real samples for out-of-lab test in the future. PMID:29522472

Biomimetic ELISA detection of malachite green based on molecularly imprinted polymer film.

PubMed

Li, Lu; Peng, Ai-Hong; Lin, Zheng-Zhong; Zhong, Hui-Ping; Chen, Xiao-Mei; Huang, Zhi-Yong

2017-08-15

A highly selective and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the detection of malachite green (MG) using a molecularly imprinted polymer (MIP) film as bionic antibody. The MIP film, based on the self-polymerization of dopamine, was fabricated on the surfaces of a 96-well microplate. It showed specific recognition for MG in aqueous solution. A direct competitive ELISA method was established with the sensitivity reaching 10.31μgL -1 and the detection limit being 0.3μgL -1 . The cross-reactivity of two structural analogues to MG was less than 10%. The average recovery tested by MG standard spiking was 88.8% for bass and 90.4% for water, and the relative standard deviations were less than 3.6%. All the above results indicated that the developed method could be used to detect MG in fish and water samples rapidly, specifically and accurately. Copyright © 2017 Elsevier Ltd. All rights reserved.

Simple Molecular Methods for Early Detection of Chloroquine Drug Resistance in Plasmodium vivax and Plasmodium falciparum

PubMed Central

Singh, Raksha; Urhehar, Anant Dattatraya

2016-01-01

Introduction Malaria is a human disease of which causes high morbidity and mortality. In Plasmodium falciparum malaria, the resistance to antimalarial drugs, especially chloroquine (CQ) is one of the paramount factors contributing to the global increase in morbidity and mortality, due to malaria. Hence, there is a need for detection of chloroquine drug resistance genes i.e., pfcrt-o (Plasmodium falciparum chloroquine resistance transporter-o) and pfmdr-1 (Plasmodium falciparum multidrug resistance-1) of P. falciparum and pvcrt-o (Plasmodium vivax chloroquine resistance transporter-o) and pvmdr-1 (Plasmodium vivax multidrug resistance-1) of P. vivax by using molecular methods to prevent mortality in malarial cases. Aim To standardize chloroquine drug sensitivity testing by molecular method so as to provide reports of chloroquine within 6-8 hours to physicians for better treatment. Materials and Methods This study was conducted over a period of one year from January to December 2014. A Total of 300 blood samples were collected from malaria suspected patient attending MGM Hospital, Kamothe, Navi Mumbai, India. Out of 300 blood samples, 44 were malaria positive as assessed by Thick and Thin blood smear stained, by Leishman’s method and examination with light microscope. Chloroquine drug sensitivity testing was performed using WHO III plate method (micro test). Nested PCR was done for detection of pfcrt-o and pfmdr-1 for P. falciparum and pvcrt-o, pvmdr-1 genes for P. vivax. Results Total 44 samples were included in this study, out of which 22 samples confirmed for Plasmodium falciparum and 22 samples confirmed for Plasmodium vivax. Out of 22 P. falciparum 15 (68.18%) samples were chloroquine resistant. P. vivax showed chloroquine resistance to 5 samples (22.73%) by method similar to WHO III plate method (micro test) and nested PCR. Conclusion Drug resistance testing by molecular methods is useful for early detection of antimalarial drug resistance. pfmdr-1 along with pfcrt-o can be used as biomarker for chloroquine drug resistance in P. falciparum and pvmdr-1 along with pvcrt-o for P. vivax. PMID:27630842

Infectious helminth ova in wastewater and sludge: A review on public health issues and current quantification practices.

PubMed

Gyawali, P

2018-02-01

Raw and partially treated wastewater has been widely used to maintain the global water demand. Presence of viable helminth ova and larvae in the wastewater raised significant public health concern especially when used for agriculture and aquaculture. Depending on the prevalence of helminth infections in communities, up to 1.0 × 10 3 ova/larvae can be presented per litre of wastewater and 4 gm (dry weight) of sludge. Multi-barrier approaches including pathogen reduction, risk assessment, and exposure reduction have been suggested by health regulators to minimise the potential health risk. However, with a lack of a sensitive and specific method for the quantitative detection of viable helminth ova from wastewater, an accurate health risk assessment is difficult to achieve. As a result, helminth infections are difficult to control from the communities despite two decades of global effort (mass drug administration). Molecular methods can be more sensitive and specific than currently adapted culture-based and vital stain methods. The molecular methods, however, required more and thorough investigation for its ability with accurate quantification of viable helminth ova/larvae from wastewater and sludge samples. Understanding different cell stages and corresponding gene copy numbers is pivotal for accurate quantification of helminth ova/larvae in wastewater samples. Identifying specific genetic markers including protein, lipid, and metabolites using multiomics approach could be utilized for cheap, rapid, sensitive, specific and point of care detection tools for helminth ova and larva in the wastewater.

Rapid detection of Shigella and enteroinvasive Escherichia coli in produce enrichments by a conventional multiplex PCR assay.

PubMed

Binet, Rachel; Deer, Deanne M; Uhlfelder, Samantha J

2014-06-01

Faster detection of contaminated foods can prevent adulterated foods from being consumed and minimize the risk of an outbreak of foodborne illness. A sensitive molecular detection method is especially important for Shigella because ingestion of as few as 10 of these bacterial pathogens can cause disease. The objectives of this study were to compare the ability of four DNA extraction methods to detect Shigella in six types of produce, post-enrichment, and to evaluate a new and rapid conventional multiplex assay that targets the Shigella ipaH, virB and mxiC virulence genes. This assay can detect less than two Shigella cells in pure culture, even when the pathogen is mixed with background microflora, and it can also differentiate natural Shigella strains from a control strain and eliminate false positive results due to accidental laboratory contamination. The four DNA extraction methods (boiling, PrepMan Ultra [Applied Biosystems], InstaGene Matrix [Bio-Rad], DNeasy Tissue kit [Qiagen]) detected 1.6 × 10(3)Shigella CFU/ml post-enrichment, requiring ∼18 doublings to one cell in 25 g of produce pre-enrichment. Lower sensitivity was obtained, depending on produce type and extraction method. The InstaGene Matrix was the most consistent and sensitive and the multiplex assay accurately detected Shigella in less than 90 min, outperforming, to the best of our knowledge, molecular assays currently in place for this pathogen. Published by Elsevier Ltd.

Specific temperature-induced perturbations of secondary mRNA structures are associated with the cold-adapted temperature-sensitive phenotype of influenza A virus.

PubMed

Chursov, Andrey; Kopetzky, Sebastian J; Leshchiner, Ignaty; Kondofersky, Ivan; Theis, Fabian J; Frishman, Dmitrij; Shneider, Alexander

2012-10-01

For decades, cold-adapted, temperature-sensitive (ca/ts) strains of influenza A virus have been used as live attenuated vaccines. Due to their great public health importance it is crucial to understand the molecular mechanism(s) of cold adaptation and temperature sensitivity that are currently unknown. For instance, secondary RNA structures play important roles in influenza biology. Thus, we hypothesized that a relatively minor change in temperature (32-39°C) can lead to perturbations in influenza RNA structures and, that these structural perturbations may be different for mRNAs of the wild type (wt) and ca/ts strains. To test this hypothesis, we developed a novel in silico method that enables assessing whether two related RNA molecules would undergo (dis)similar structural perturbations upon temperature change. The proposed method allows identifying those areas within an RNA chain where dissimilarities of RNA secondary structures at two different temperatures are particularly pronounced, without knowing particular RNA shapes at either temperature. We identified such areas in the NS2, PA, PB2 and NP mRNAs. However, these areas are not identical for the wt and ca/ts mutants. Differences in temperature-induced structural changes of wt and ca/ts mRNA structures may constitute a yet unappreciated molecular mechanism of the cold adaptation/temperature sensitivity phenomena.

Probing the brain with molecular fMRI.

PubMed

Ghosh, Souparno; Harvey, Peter; Simon, Jacob C; Jasanoff, Alan

2018-06-01

One of the greatest challenges of modern neuroscience is to incorporate our growing knowledge of molecular and cellular-scale physiology into integrated, organismic-scale models of brain function in behavior and cognition. Molecular-level functional magnetic resonance imaging (molecular fMRI) is a new technology that can help bridge these scales by mapping defined microscopic phenomena over large, optically inaccessible regions of the living brain. In this review, we explain how MRI-detectable imaging probes can be used to sensitize noninvasive imaging to mechanistically significant components of neural processing. We discuss how a combination of innovative probe design, advanced imaging methods, and strategies for brain delivery can make molecular fMRI an increasingly successful approach for spatiotemporally resolved studies of diverse neural phenomena, perhaps eventually in people. Copyright © 2018 Elsevier Ltd. All rights reserved.

Pushing the sensitivity envelope of lanthanide-based magnetic resonance imaging (MRI) contrast agents for molecular imaging applications.

PubMed

Aime, Silvio; Castelli, Daniela Delli; Crich, Simonetta Geninatti; Gianolio, Eliana; Terreno, Enzo

2009-07-21

Contrast in magnetic resonance imaging (MRI) arises from changes in the intensity of the proton signal of water between voxels (essentially, the 3D counterpart of pixels). Differences in intervoxel intensity can be significantly enhanced with chemicals that alter the nuclear magnetic resonance (NMR) intensity of the imaged spins; this alteration can occur by various mechanisms. Paramagnetic lanthanide(III) complexes are used in two major classes of MRI contrast agent: the well-established class of Gd-based agents and the emerging class of chemical exchange saturation transfer (CEST) agents. A Gd-based complex increases water signal by enhancing the longitudinal relaxation rate of water protons, whereas CEST agents decrease water signal as a consequence of the transfer of saturated magnetization from the exchangeable protons of the agent. In this Account, we survey recent progress in both areas, focusing on how MRI is becoming a more competitive choice among the various molecular imaging methods. Compared with other imaging modalities, MRI is set apart by its superb anatomical resolution; however, its success in molecular imaging suffers because of its intrinsic insensitivity. A relatively high concentration of molecular agents (0.01-0.1 mM) is necessary to produce a local alteration in the water signal intensity. Unfortunately, the most desirable molecules for visualization in molecular imaging are present at much lower concentrations, in the nano- or picomolar range. Therefore, augmenting the sensitivity of MRI agents is key to the development of MR-based molecular imaging applications. In principle, this task can be tackled either by increasing the sensitivity of the reporting units, through the optimization of their structural and dynamic properties, or by setting up proper amplification strategies that allow the accumulation of a huge number of imaging reporters at the site of interest. For Gd-based agents, high sensitivities can be attained by exploiting a range of nanosized carriers (micelles, liposomes, microemulsions, and the like, as well as biological structures such as apoferritin and lipoproteins) properly loaded with Gd-based chelates. Furthermore, the sensitivity of Gd-based agents can be markedly affected either by their interactions with biological structures or by their cellular localization. For CEST agents, a huge sensitivity enhancement has been obtained by using the water molecules contained in the inner cavity of liposomes as the exchangeable source of protons for magnetization transfer. Several "tricks" (for example, the use of multimeric lanthanide(III) shift reagents, changes in the shape of the liposome container, and so forth) have been devised to improve the chemical shift separation between the intraliposomal water and the "bulk" water resonances. Overall, excellent sensitivity enhancements have been obtained for both classes of agents, enabling their use in MR molecular imaging applications.

Determination of the molecular weight of poly(ethylene glycol) in biological samples by reversed-phase LC-MS with in-source fragmentation.

PubMed

Warrack, Bethanne M; Redding, Brian P; Chen, Guodong; Bolgar, Mark S

2013-05-01

PEGylation has been widely used to improve the biopharmaceutical properties of therapeutic proteins and peptides. Previous studies have used multiple analytical techniques to determine the fate of both the therapeutic molecule and unconjugated poly(ethylene glycol) (PEG) after drug administration. A straightforward strategy utilizing liquid chromatography-mass spectrometry (LC-MS) to characterize high-molecular weight PEG in biologic matrices without a need for complex sample preparation is presented. The method is capable of determining whether high-MW PEG is cleaved in vivo to lower-molecular weight PEG species. Reversed-phase chromatographic separation is used to take advantage of the retention principles of polymeric materials whereby elution order correlates with PEG molecular weight. In-source collision-induced dissociation (CID) combined with selected reaction monitoring (SRM) or selected ion monitoring (SIM) mass spectrometry (MS) is then used to monitor characteristic PEG fragment ions in biological samples. MS provides high sensitivity and specificity for PEG and the observed retention times in reversed-phase LC enable estimation of molecular weight. This method was successfully used to characterize PEG molecular weight in mouse serum samples. No change in molecular weight was observed for 48 h after dosing.

Thermal and chemical treatment of polymer optical fiber Bragg grating sensors for enhanced mechanical sensitivity

NASA Astrophysics Data System (ADS)

Pospori, A.; Marques, C. A. F.; Sáez-Rodríguez, D.; Nielsen, K.; Bang, O.; Webb, D. J.

2017-07-01

An investigation of the thermal annealing effects on the strain, stress, and force sensitivities of polymer optical fiber Bragg grating sensors is performed. We demonstrate for the first time that the fiber annealing can enhance both stress and force sensitivities of Bragg grating sensors, with the possible cause being the molecular relaxation of the polymer when fiber is raised above the β -transition temperature. A simple, cost-effective, but well controlled method for fiber annealing is also presented in this work. In addition, the effects of chemical etching on the strain, stress, and force sensitivities have been investigated. Results show that fiber etching too can increase the force sensitivity, and it can also affect the strain and stress sensitivities of the Bragg grating sensors.

Absolute molecular weight determination of hypromellose acetate succinate by size exclusion chromatography: use of a multi angle laser light scattering detector and a mixed solvent.

PubMed

Chen, Raymond; Ilasi, Nicholas; Sekulic, Sonja S

2011-12-05

Molecular weight distribution is an important quality attribute for hypromellose acetate succinate (HPMCAS), a pharmaceutical excipient used in spray-dried dispersions. Our previous study showed that neither relative nor universal calibration method of size exclusion chromatography (SEC) works for HPMCAS polymers. We here report our effort to develop a SEC method using a mass sensitive multi angle laser light scattering detector (MALLS) to determine molecular weight distributions of HPMCAS polymers. A solvent screen study reveals that a mixed solvent (60:40%, v/v 50mM NaH(2)PO(4) with 0.1M NaNO(3) buffer: acetonitrile, pH* 8.0) is the best for HPMCAS-LF and MF sub-classes. Use of a mixed solvent creates a challenging condition for the method that uses refractive index detector. Therefore, we thoroughly evaluated the method performance and robustness. The mean weight average molecular weight of a polyethylene oxide standard has a 95% confidence interval of (28,443-28,793) g/mol vs. 28,700g/mol from the Certificate of Analysis. The relative standard deviations of average molecular weights for all polymers are 3-6%. These results and the Design of Experiments study demonstrate that the method is accurate and robust. Copyright © 2011 Elsevier B.V. All rights reserved.

[MALDI-TOF mass spectrometry in the investigation of large high-molecular biological compounds].

PubMed

Porubl'ova, L V; Rebriiev, A V; Hromovyĭ, T Iu; Minia, I I; Obolens'ka, M Iu

2009-01-01

MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) mass spectrometry has become, in the recent years, a tool of choice for analyses of biological polymers. The wide mass range, high accuracy, informativity and sensitivity make it a superior method for analysis of all kinds of high-molecular biological compounds including proteins, nucleic acids and lipids. MALDI-TOF-MS is particularly suitable for the identification of proteins by mass fingerprint or microsequencing. Therefore it has become an important technique of proteomics. Furthermore, the method allows making a detailed analysis of post-translational protein modifications, protein-protein and protein-nucleic acid interactions. Recently, the method was also successfully applied to nucleic acid sequencing as well as screening for mutations.

Next Generation LOCAD-PTS Cartridge Development

NASA Technical Reports Server (NTRS)

Morris, H.; Nutter, D.; Weite, E.; Wells, M.; Maule, J.; Damon, M.; Monaco, L.; Steele, A.; Wainwright, N.

2008-01-01

Future astrobiology exploration missions will require rapid, point-of-use techniques for surface science experiments and contamination monitoring. The Lab-On-a-Chip Application Development (LOCAD) team is developing operational instruments that advance spaceflight technologies to molecular-based methods. Currently, LOCAD-Portable Test System (PTS) is quantifying levels of the bacterial molecule endotoxin onboard the Internatioal Space Station. Future research and development will focus on more sensitive molecular techniques that expand the number of compounds detected to include beta-glucan from fungal cell walls.

Multicentric Comparative Analytical Performance Study for Molecular Detection of Low Amounts of Toxoplasma gondii from Simulated Specimens▿ †

PubMed Central

Sterkers, Yvon; Varlet-Marie, Emmanuelle; Cassaing, Sophie; Brenier-Pinchart, Marie-Pierre; Brun, Sophie; Dalle, Frédéric; Delhaes, Laurence; Filisetti, Denis; Pelloux, Hervé; Yera, Hélène; Bastien, Patrick

2010-01-01

Although screening for maternal toxoplasmic seroconversion during pregnancy is based on immunodiagnostic assays, the diagnosis of clinically relevant toxoplasmosis greatly relies upon molecular methods. A problem is that this molecular diagnosis is subject to variation of performances, mainly due to a large diversity of PCR methods and primers and the lack of standardization. The present multicentric prospective study, involving eight laboratories proficient in the molecular prenatal diagnosis of toxoplasmosis, was a first step toward the harmonization of this diagnosis among university hospitals in France. Its aim was to compare the analytical performances of different PCR protocols used for Toxoplasma detection. Each center extracted the same concentrated Toxoplasma gondii suspension and tested serial dilutions of the DNA using its own assays. Differences in analytical sensitivities were observed between assays, particularly at low parasite concentrations (≤2 T. gondii genomes per reaction tube), with “performance scores” differing by a 20-fold factor among laboratories. Our data stress the fact that differences do exist in the performances of molecular assays in spite of expertise in the matter; we propose that laboratories work toward a detection threshold defined for a best sensitivity of this diagnosis. Moreover, on the one hand, intralaboratory comparisons confirmed previous studies showing that rep529 is a more adequate DNA target for this diagnosis than the widely used B1 gene. But, on the other hand, interlaboratory comparisons showed differences that appear independent of the target, primers, or technology and that hence rely essentially on proficiency and care in the optimization of PCR conditions. PMID:20610670

Detection of Entamoeba histolytica by Recombinase Polymerase Amplification

PubMed Central

Nair, Gayatri; Rebolledo, Mauricio; White, A. Clinton; Crannell, Zachary; Richards-Kortum, R. Rebecca; Pinilla, A. Elizabeth; Ramírez, Juan David; López, M. Consuelo; Castellanos-Gonzalez, Alejandro

2015-01-01

Amebiasis is an important cause of diarrheal disease worldwide and has been associated with childhood malnutrition. Traditional microscopy approaches are neither sensitive nor specific for Entamoeba histolytica. Antigen assays are more specific, but many cases are missed unless tested by molecular methods. Although polymerase chain reaction (PCR) is effective, the need for sophisticated, expensive equipment, infrastructure, and trained personnel limits its usefulness, especially in the resource-limited, endemic areas. Here, we report development of a recombinase polymerase amplification (RPA) method to detect E. histolytica specifically. Using visual detection by lateral flow (LF), the test was highly sensitive and specific and could be performed without additional equipment. The availability of this inexpensive, sensitive, and field-applicable diagnostic test could facilitate rapid diagnosis and treatment of amebiasis in endemic regions. PMID:26123960

Imaging-based molecular barcoding with pixelated dielectric metasurfaces.

PubMed

Tittl, Andreas; Leitis, Aleksandrs; Liu, Mingkai; Yesilkoy, Filiz; Choi, Duk-Yong; Neshev, Dragomir N; Kivshar, Yuri S; Altug, Hatice

2018-06-08

Metasurfaces provide opportunities for wavefront control, flat optics, and subwavelength light focusing. We developed an imaging-based nanophotonic method for detecting mid-infrared molecular fingerprints and implemented it for the chemical identification and compositional analysis of surface-bound analytes. Our technique features a two-dimensional pixelated dielectric metasurface with a range of ultrasharp resonances, each tuned to a discrete frequency; this enables molecular absorption signatures to be read out at multiple spectral points, and the resulting information is then translated into a barcode-like spatial absorption map for imaging. The signatures of biological, polymer, and pesticide molecules can be detected with high sensitivity, covering applications such as biosensing and environmental monitoring. Our chemically specific technique can resolve absorption fingerprints without the need for spectrometry, frequency scanning, or moving mechanical parts, thereby paving the way toward sensitive and versatile miniaturized mid-infrared spectroscopy devices. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

A new instrument of VUV laser desorption/ionization mass spectrometry imaging with micrometer spatial resolution and low level of molecular fragmentation.

PubMed

Wang, Jia; Liu, Feng; Mo, Yuxiang; Wang, Zhaoying; Zhang, Sichun; Zhang, Xinrong

2017-11-01

Mass spectrometry imaging (MSI) has important applications in material research, biology, and medicine. The MSI method based on UV laser desorption/ionization (UVLDI) can obtain images of intact samples, but has a high level of molecular fragmentation. In this work, we report a new MSI instrument that uses a VUV laser (125.3 nm) as a desorption/ionization source to exploit its advantages of high single photon energy and small focus size. The new instrument was tested by the mass spectra of Nile red and FGB (Fibrinogen beta chain) samples and mass spectrometric images of a fly brain section. For the tested samples, the VUVDI method offers lower levels of molecular fragmentations and higher sensitivities than those of the UVLDI method and second ion mass spectrometry imaging method using a Bi 3 + beam. The ablation crater produced by the focused VUV laser on a quartz plate has an area of 10 μm 2 . The VUV laser is prepared based on the four-wave mixing method using three collimated laser beams and a heated Hg cell.

A new instrument of VUV laser desorption/ionization mass spectrometry imaging with micrometer spatial resolution and low level of molecular fragmentation

NASA Astrophysics Data System (ADS)

Wang, Jia; Liu, Feng; Mo, Yuxiang; Wang, Zhaoying; Zhang, Sichun; Zhang, Xinrong

2017-11-01

Mass spectrometry imaging (MSI) has important applications in material research, biology, and medicine. The MSI method based on UV laser desorption/ionization (UVLDI) can obtain images of intact samples, but has a high level of molecular fragmentation. In this work, we report a new MSI instrument that uses a VUV laser (125.3 nm) as a desorption/ionization source to exploit its advantages of high single photon energy and small focus size. The new instrument was tested by the mass spectra of Nile red and FGB (Fibrinogen beta chain) samples and mass spectrometric images of a fly brain section. For the tested samples, the VUVDI method offers lower levels of molecular fragmentations and higher sensitivities than those of the UVLDI method and second ion mass spectrometry imaging method using a Bi3+ beam. The ablation crater produced by the focused VUV laser on a quartz plate has an area of 10 μm2. The VUV laser is prepared based on the four-wave mixing method using three collimated laser beams and a heated Hg cell.

A multiplex PCR method for detection of Aspergillus spp. and Mycobacterium tuberculosis in BAL specimens.

PubMed

Amini, F; Kachuei, R; Noorbakhsh, F; Imani Fooladi, A A

2015-06-01

The aim of this study was the detection of Aspergillus species and Mycobacterium tuberculosis together in bronchoalveolar lavage (BAL) using of multiplex PCR. In this study, from September 2012 until June 2013, 100 bronchoalveolar lavage (BAL) specimens were collected from patients suspected of tuberculosis (TB). After the direct and culture test, multiplex PCR were utilized in order to diagnose Aspergillus species and M. tuberculosis. Phenol-chloroform manual method was used in order to extract DNA from these microorganisms. Aspergillus specific primers, M. tuberculosis designed primers and beta actin primers were used for multiplex PCR. In this study, by multiplex PCR method, Aspergillus species were identified in 12 samples (12%), positive samples in direct and culture test were respectively 11% and 10%. Sensitivity and specificity of this method in comparison to direct test were respectively 100% and 98.8%, also sensitivity and specificity of this method in comparison to culture test were respectively 100% and 97.7%. In this assay, M. tuberculosis was identified in 8 samples (8%). Mycobacterium-positive samples in molecular method, direct and culture test were respectively 6%, 5% and 7%. Sensitivity and specificity of PCR method in comparison to direct test were 80% and 97.8% also sensitivity and specificity of this method in comparison to culture test was 71.4% and 98.9%. In the present study, multiplex PCR method had higher sensitivity than direct and culture test in order to identify and detect Aspergillus, also this method had lower sensitivity for identification of M. tuberculosis, suggesting that the method of DNA extraction was not suitable. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Gold nanoparticles: enhanced optical trapping and sensitivity coupled with significant heating.

PubMed

Seol, Yeonee; Carpenter, Amanda E; Perkins, Thomas T

2006-08-15

Gold nanoparticles appear to be superior handles in optical trapping assays. We demonstrate that relatively large gold particles (R(b)=50 nm) indeed yield a sixfold enhancement in trapping efficiency and detection sensitivity as compared to similar-sized polystyrene particles. However, optical absorption by gold at the most common trapping wavelength (1064 nm) induces dramatic heating (266 degrees C/W). We determined this heating by comparing trap stiffness from three different methods in conjunction with detailed modeling. Due to this heating, gold nanoparticles are not useful for temperature-sensitive optical-trapping experiments, but may serve as local molecular heaters. Also, such particles, with their increased detection sensitivity, make excellent probes for certain zero-force biophysical assays.

DEVELOPMENT OF A MOLECULAR METHOD TO IDENTIFY ASTROVIRUS IN WATER.

EPA Science Inventory

Astrovirus is a common cause of gastroenteritis that has been determined to be responsible for several outbreaks. Since astrovirus can be waterborne, there is interest in testing environmental water for astrovirus and we have developed a sensitive RT-PCR assay that is designed t...

Instrumentation in molecular imaging.

PubMed

Wells, R Glenn

2016-12-01

In vivo molecular imaging is a challenging task and no single type of imaging system provides an ideal solution. Nuclear medicine techniques like SPECT and PET provide excellent sensitivity but have poor spatial resolution. Optical imaging has excellent sensitivity and spatial resolution, but light photons interact strongly with tissues and so only small animals and targets near the surface can be accurately visualized. CT and MRI have exquisite spatial resolution, but greatly reduced sensitivity. To overcome the limitations of individual modalities, molecular imaging systems often combine individual cameras together, for example, merging nuclear medicine cameras with CT or MRI to allow the visualization of molecular processes with both high sensitivity and high spatial resolution.

Study of complex molecular systems by probe vibrational spectroscopy method

NASA Astrophysics Data System (ADS)

Boldeskul, A. E.; Zatsepin, V. M.; Atakhodjaev, A. K.; Shermatov, A. N.; Ashburiev, R.

1984-03-01

Experimental study of benzonitril as a probe in aqueous solution of sodium lauril sulphate /SDS/ by Raman spectroscopy technique showed integral moments of √ /C X N/ line to be extremely sensitive to the structural transitions in micellar systems. The central part of the experimental contour was used to determine integral moments with the help of line shape approximant received by Mori method

Combined electrophoresis-electrospray interface and method

DOEpatents

Smith, R.D.; Udseth, H.R.; Barinaga, C.J.

1995-06-13

An improvement to the system and method is disclosed for analyzing molecular constituents of a composition sample that comprises improvements to an electrospray ionization source for interfacing to mass spectrometers and other detection devices. The improvement consists of establishing a unique electrical circuit pattern and nozzle configuration, a metallic coated and conical shaped capillary outlet, coupled with sizing of the capillary to obtain maximum sensitivity. 10 figs.

PCR/LDR/universal array platforms for the diagnosis of infectious disease.

PubMed

Pingle, Maneesh; Rundell, Mark; Das, Sanchita; Golightly, Linnie M; Barany, Francis

2010-01-01

Infectious diseases account for between 14 and 17 million deaths worldwide each year. Accurate and rapid diagnosis of bacterial, fungal, viral, and parasitic infections is therefore essential to reduce the morbidity and mortality associated with these diseases. Classical microbiological and serological methods have long served as the gold standard for diagnosis but are increasingly being replaced by molecular diagnostic methods that demonstrate increased sensitivity and specificity and provide an identification of the etiologic agent in a shorter period of time. PCR/LDR coupled with universal array detection provides a highly sensitive and specific platform for the detection and identification of bacterial and viral infections.

PCR/LDR/Universal Array Platforms for the Diagnosis of Infectious Disease

PubMed Central

Pingle, Maneesh; Rundell, Mark; Das, Sanchita; Golightly, Linnie M.; Barany, Francis

2015-01-01

Infectious diseases account for between 14 and 17 million deaths worldwide each year. Accurate and rapid diagnosis of bacterial, fungal, viral, and parasitic infections is therefore essential to reduce the morbidity and mortality associated with these diseases. Classical microbiological and serological methods have long served as the gold standard for diagnosis but are increasingly being replaced by molecular diagnostic methods that demonstrate increased sensitivity and specificity and provide an identification of the etiologic agent in a shorter period of time. PCR/LDR coupled with universal array detection provides a highly sensitive and specific platform for the detection and identification of bacterial and viral infections. PMID:20217576

New perspectives in tracing vector-borne interaction networks.

PubMed

Gómez-Díaz, Elena; Figuerola, Jordi

2010-10-01

Disentangling trophic interaction networks in vector-borne systems has important implications in epidemiological and evolutionary studies. Molecular methods based on bloodmeal typing in vectors have been increasingly used to identify hosts. Although most molecular approaches benefit from good specificity and sensitivity, their temporal resolution is limited by the often rapid digestion of blood, and mixed bloodmeals still remain a challenge for bloodmeal identification in multi-host vector systems. Stable isotope analyses represent a novel complementary tool that can overcome some of these problems. The utility of these methods using examples from different vector-borne systems are discussed and the extents to which they are complementary and versatile are highlighted. There are excellent opportunities for progress in the study of vector-borne transmission networks resulting from the integration of both molecular and stable isotope approaches. Copyright © 2010 Elsevier Ltd. All rights reserved.

Development and Testing of Molecular Adsorber Coatings

NASA Technical Reports Server (NTRS)

Abraham, Nithin; Hasegawa, Mark; Straka, Sharon

2012-01-01

The effect of on-orbit molecular contamination has the potential to degrade the performance of spaceflight hardware and diminish the lifetime of the spacecraft. For example, sensitive surfaces, such as optical surfaces, electronics, detectors, and thermal control surfaces, are vulnerable to the damaging effects of contamination from outgassed materials. The current solution to protect these surfaces is through the use of zeolite coated ceramic adsorber pucks. However, these pucks and its additional complex mounting hardware requirements result in several disadvantages, such as size, weight, and cost related concerns, that impact the spacecraft design and the integration and test schedule. As a result, a new innovative molecular adsorber coating was developed as a sprayable alternative to mitigate the risk of on-orbit molecular contamination. In this study, the formulation for molecular adsorber coatings was optimized using various binders, pigment treatment methods, binder to pigment ratios, thicknesses, and spray application techniques. The formulations that passed coating adhesion and vacuum thermal cycling tests were further tested for its adsorptive capacity. Accelerated molecular capacitance tests were performed in an innovatively designed multi-unit system containing idealized contaminant sources. This novel system significantly increased the productivity of the testing phase for the various formulations that were developed. Work performed during the development and testing phases has demonstrated successful application of molecular adsorber coatings onto metallic substrates, as well as, very promising results for the adhesion performance and the molecular capacitance of the coating. Continued testing will assist in the qualification of molecular adsorber coatings for use on future contamination sensitive spaceflight missions.

Quantification of trans-1,4-polyisoprene in Eucommia ulmoides by fourier transform infrared spectroscopy and pyrolysis-gas chromatography/mass spectrometry.

PubMed

Takeno, Shinya; Bamba, Takeshi; Nakazawa, Yoshihisa; Fukusaki, Eiichiro; Okazawa, Atsushi; Kobayashi, Akio

2008-04-01

Commercial development of trans-1,4-polyisoprene from Eucommia ulmoides Oliver (EU-rubber) requires specific knowledge on selection of high-rubber-content lines and establishment of agronomic cultivation methods for achieving maximum EU-rubber yield. The development can be facilitated by high-throughput and highly sensitive analytical techniques for EU-rubber extraction and quantification. In this paper, we described an efficient EU-rubber extraction method, and validated that the accuracy was equivalent to that of the conventional Soxhlet extraction method. We also described a highly sensitive quantification method for EU-rubber by Fourier transform infrared spectroscopy (FT-IR) and pyrolysis-gas chromatography/mass spectrometry (PyGC/MS). We successfully applied the extraction/quantification method for study of seasonal changes in EU-rubber content and molecular weight distribution.

A rapid method to visualize von willebrand factor multimers by using agarose gel electrophoresis, immunolocalization and luminographic detection.

PubMed

Krizek, D R; Rick, M E

2000-03-15

A highly sensitive and rapid clinical method for the visualization of the multimeric structure of von Willebrand Factor in plasma and platelets is described. The method utilizes submerged horizontal agarose gel electrophoresis, followed by transfer of the von Willebrand Factor onto a polyvinylidine fluoride membrane, and immunolocalization and luminographic visualization of the von Willebrand Factor multimeric pattern. This method distinguishes type 1 from types 2A and 2B von Willebrand disease, allowing timely evaluation and classification of von Willebrand Factor in patient plasma. It also allows visualization of the unusually high molecular weight multimers present in platelets. There are several major advantages to this method including rapid processing, simplicity of gel preparation, high sensitivity to low concentrations of von Willebrand Factor, and elimination of radioactivity.

Parasites under the Spotlight: Applications of Vibrational Spectroscopy to Malaria Research.

PubMed

Perez-Guaita, David; Marzec, Katarzyna M; Hudson, Andrew; Evans, Corey; Chernenko, Tatyana; Matthäus, Christian; Miljkovic, Milos; Diem, Max; Heraud, Philip; Richards, Jack S; Andrew, Dean; Anderson, David A; Doerig, Christian; Garcia-Bustos, Jose; McNaughton, Don; Wood, Bayden R

2018-04-20

New technologies to diagnose malaria at high sensitivity and specificity are urgently needed in the developing world where the disease continues to pose a huge burden on society. Infrared and Raman spectroscopy-based diagnostic methods have a number of advantages compared with other diagnostic tests currently on the market. These include high sensitivity and specificity for detecting low levels of parasitemia along with ease of use and portability. Here, we review the application of vibrational spectroscopic techniques for monitoring and detecting malaria infection. We discuss the role of vibrational (infrared and Raman) spectroscopy in understanding the processes of parasite biology and its application to the study of interactions with antimalarial drugs. The distinct molecular phenotype that characterizes malaria infection and the high sensitivity enabling detection of low parasite densities provides a genuine opportunity for vibrational spectroscopy to become a front-line tool in the elimination of this deadly disease and provide molecular insights into the chemistry of this unique organism.

Study of translational dynamics in molten polymer by variation of gradient pulse-width of PGSE.

PubMed

Stepišnik, Janez; Lahajnar, Gojmir; Zupančič, Ivan; Mohorič, Aleš

2013-11-01

Pulsed gradient spin echo is a method of measuring molecular translation. Changing Δ makes it sensitive to diffusion spectrum. Spin translation effects the buildup of phase structure during the application of gradient pulses as well. The time scale of the self-diffusion measurement shortens if this is taken into account. The method of diffusion spectrometry with variable δ is also less sensitive to artifacts caused by spin relaxation and internal gradient fields. Here the method is demonstrated in the case of diffusion spectrometry of molten polyethylene. The results confirm a model of constraint release in a system of entangled polymer chains as a sort of tube Rouse motion. Copyright © 2013 Elsevier Inc. All rights reserved.

Sensitivity, Specificity, PPV, and NPV for Predictive Biomarkers

PubMed Central

2015-01-01

Molecularly targeted cancer drugs are often developed with companion diagnostics that attempt to identify which patients will have better outcome on the new drug than the control regimen. Such predictive biomarkers are playing an increasingly important role in precision oncology. For diagnostic tests, sensitivity, specificity, positive predictive value, and negative predictive are usually used as performance measures. This paper discusses these indices for predictive biomarkers, provides methods for their calculation with survival or response endpoints, and describes assumptions involved in their use. PMID:26109105

Semi-Automated Curation Allows Causal Network Model Building for the Quantification of Age-Dependent Plaque Progression in ApoE-/- Mouse.

PubMed

Szostak, Justyna; Martin, Florian; Talikka, Marja; Peitsch, Manuel C; Hoeng, Julia

2016-01-01

The cellular and molecular mechanisms behind the process of atherosclerotic plaque destabilization are complex, and molecular data from aortic plaques are difficult to interpret. Biological network models may overcome these difficulties and precisely quantify the molecular mechanisms impacted during disease progression. The atherosclerosis plaque destabilization biological network model was constructed with the semiautomated curation pipeline, BELIEF. Cellular and molecular mechanisms promoting plaque destabilization or rupture were captured in the network model. Public transcriptomic data sets were used to demonstrate the specificity of the network model and to capture the different mechanisms that were impacted in ApoE -/- mouse aorta at 6 and 32 weeks. We concluded that network models combined with the network perturbation amplitude algorithm provide a sensitive, quantitative method to follow disease progression at the molecular level. This approach can be used to investigate and quantify molecular mechanisms during plaque progression.

Method and apparatus for molecular imaging using x-rays at resonance wavelengths

DOEpatents

Chapline, G.F. Jr.

Holographic x-ray images are produced representing the molecular structure of a microscopic object, such as a living cell, by directing a beam of coherent x-rays upon the object to produce scattering of the x-rays by the object, producing interference on a recording medium between the scattered x-rays from the object and unscattered coherent x-rays and thereby producing holograms on the recording surface, and establishing the wavelength of the coherent x-rays to correspond with a molecular resonance of a constituent of such object and thereby greatly improving the contrast, sensitivity and resolution of the holograms as representations of molecular structures involving such constituent. For example, the coherent x-rays may be adjusted to the molecular resonant absorption line of nitrogen at about 401.3 eV to produce holographic images featuring molecular structures involving nitrogen.

Method and apparatus for molecular imaging using X-rays at resonance wavelengths

DOEpatents

Chapline, Jr., George F.

1985-01-01

Holographic X-ray images are produced representing the molecular structure of a microscopic object, such as a living cell, by directing a beam of coherent X-rays upon the object to produce scattering of the X-rays by the object, producing interference on a recording medium between the scattered X-rays from the object and unscattered coherent X-rays and thereby producing holograms on the recording surface, and establishing the wavelength of the coherent X-rays to correspond with a molecular resonance of a constituent of such object and thereby greatly improving the contrast, sensitivity and resolution of the holograms as representations of molecular structures involving such constituent. For example, the coherent X-rays may be adjusted to the molecular resonant absorption line of nitrogen at about 401.3 eV to produce holographic images featuring molecular structures involving nitrogen.

Predicting honey bee sensitivity based on the conservation of the pesticide molecular initiating event

EPA Science Inventory

Concern surrounding the potential adverse impacts of pesticides to honey bee colonies has led to the need for rapid/cost efficient methods for aiding decision making relative to the protection of this important pollinator species. Neonicotinoids represent a class of pesticides th...

Molecular Cloning of the Human Genes(s) Directing the Synthesis of Nervous System Cholinesterases.

DTIC Science & Technology

1985-12-01

anesthesia (1) This clinical om~pli- cation ould be diagnosed by a rapid method to detect such deficiencies or preventeod by injecting, as a scavenger...the enzyme could hence be employed to develop a method for the clinical diagnosis of this dims. Thus both for basic research and multiple clinical...translation, crossed imunoelec- trophoresis, and atoiradiogar t. This approach gives a method of high sensitivity and low back . It has the advantage over

The diagnosis of chronic endometritis in infertile asymptomatic women: a comparative study of histology, microbial cultures, hysteroscopy, and molecular microbiology.

PubMed

Moreno, Inmaculada; Cicinelli, Ettore; Garcia-Grau, Iolanda; Gonzalez-Monfort, Marta; Bau, Davide; Vilella, Felipe; De Ziegler, Dominique; Resta, Leonardo; Valbuena, Diana; Simon, Carlos

2018-06-01

Chronic endometritis is a persistent inflammation of the endometrial mucosa caused by bacterial pathogens such as Enterobacteriaceae, Enterococcus, Streptococcus, Staphylococcus, Mycoplasma, and Ureaplasma. Although chronic endometritis can be asymptomatic, it is found in up to 40% of infertile patients and is responsible for repeated implantation failure and recurrent miscarriage. Diagnosis of chronic endometritis is based on hysteroscopy of the uterine cavity, endometrial biopsy with plasma cells being identified histologically, while specific treatment is determined based on microbial culture. However, not all microorganisms implicated are easily or readily culturable needing a turnaround time of up to 1 week. We sought to develop a molecular diagnostic tool for chronic endometritis based on real-time polymerase chain reaction equivalent to using the 3 classic methods together, overcoming the bias of using any of them alone. Endometrial samples from patients assessed for chronic endometritis (n = 113) using at least 1 or several conventional diagnostic methods namely histology, hysteroscopy, and/or microbial culture, were blindly evaluated by real-time polymerase chain reaction for the presence of 9 chronic endometritis pathogens: Chlamydia trachomatis, Enterococcus, Escherichia coli, Gardnerella vaginalis, Klebsiella pneumoniae, Mycoplasma hominis, Neisseria gonorrhoeae, Staphylococcus, and Streptococcus. The sensitivity and specificity of the molecular analysis vs the classic diagnostic techniques were compared in the 65 patients assessed by all 3 recognized classic methods. The molecular method showed concordant results with histological diagnosis in 30 samples (14 double positive and 16 double negative) with a matching accuracy of 46.15%. Concordance of molecular and hysteroscopic diagnosis was observed in 38 samples (37 double positive and 1 double negative), with an accuracy of 58.46%. When the molecular method was compared to microbial culture, concordance was present in 37 samples (22 double positive and 15 double negative), a matching rate of 56.92%. When cases of potential contamination and/or noncultivable bacteria were considered, the accuracy increased to 66.15%. Of these 65 patients, only 27 patients had consistent histological + hysteroscopic diagnosis, revealing 58.64% of nonconcordant results. Only 13 of 65 patients (20%) had consistent histology + hysteroscopy + microbial culture results. In these cases, the molecular microbiology matched in 10 cases showing a diagnostic accuracy of 76.92%. Interestingly, the molecular microbiology confirmed over half of the isolated pathogens and provided additional detection of nonculturable microorganisms. These results were confirmed by the microbiome assessed by next-generation sequencing. In the endometrial samples with concordant histology + hysteroscopy + microbial culture results, the molecular microbiology diagnosis demonstrates 75% sensitivity, 100% specificity, 100% positive and 25% negative predictive values, and 0% false-positive and 25% false-negative rates. The molecular microbiology method describe herein is a fast and inexpensive diagnostic tool that allows for the identification of culturable and nonculturable endometrial pathogens associated with chronic endometritis. The results obtained were similar to all 3 classic diagnostic methods together with a degree of concordance of 76.92% providing an opportunity to improve the clinical management of infertile patients with a risk of experiencing this ghost endometrial pathology. Copyright © 2018 Elsevier Inc. All rights reserved.

Advancing Cell Biology Through Proteomics in Space and Time (PROSPECTS)*

PubMed Central

Lamond, Angus I.; Uhlen, Mathias; Horning, Stevan; Makarov, Alexander; Robinson, Carol V.; Serrano, Luis; Hartl, F. Ulrich; Baumeister, Wolfgang; Werenskiold, Anne Katrin; Andersen, Jens S.; Vorm, Ole; Linial, Michal; Aebersold, Ruedi; Mann, Matthias

2012-01-01

The term “proteomics” encompasses the large-scale detection and analysis of proteins and their post-translational modifications. Driven by major improvements in mass spectrometric instrumentation, methodology, and data analysis, the proteomics field has burgeoned in recent years. It now provides a range of sensitive and quantitative approaches for measuring protein structures and dynamics that promise to revolutionize our understanding of cell biology and molecular mechanisms in both human cells and model organisms. The Proteomics Specification in Time and Space (PROSPECTS) Network is a unique EU-funded project that brings together leading European research groups, spanning from instrumentation to biomedicine, in a collaborative five year initiative to develop new methods and applications for the functional analysis of cellular proteins. This special issue of Molecular and Cellular Proteomics presents 16 research papers reporting major recent progress by the PROSPECTS groups, including improvements to the resolution and sensitivity of the Orbitrap family of mass spectrometers, systematic detection of proteins using highly characterized antibody collections, and new methods for absolute as well as relative quantification of protein levels. Manuscripts in this issue exemplify approaches for performing quantitative measurements of cell proteomes and for studying their dynamic responses to perturbation, both during normal cellular responses and in disease mechanisms. Here we present a perspective on how the proteomics field is moving beyond simply identifying proteins with high sensitivity toward providing a powerful and versatile set of assay systems for characterizing proteome dynamics and thereby creating a new “third generation” proteomics strategy that offers an indispensible tool for cell biology and molecular medicine. PMID:22311636

A fluorescence high throughput screening method for the detection of reactive electrophiles as potential skin sensitizers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Avonto, Cristina; Chittiboyina, Amar G.; Rua, Diego

2015-12-01

Skin sensitization is an important toxicological end-point in the risk assessment of chemical allergens. Because of the complexity of the biological mechanisms associated with skin sensitization, integrated approaches combining different chemical, biological and in silico methods are recommended to replace conventional animal tests. Chemical methods are intended to characterize the potential of a sensitizer to induce earlier molecular initiating events. The presence of an electrophilic mechanistic domain is considered one of the essential chemical features to covalently bind to the biological target and induce further haptenation processes. Current in chemico assays rely on the quantification of unreacted model nucleophiles aftermore » incubation with the candidate sensitizer. In the current study, a new fluorescence-based method, ‘HTS-DCYA assay’, is proposed. The assay aims at the identification of reactive electrophiles based on their chemical reactivity toward a model fluorescent thiol. The reaction workflow enabled the development of a High Throughput Screening (HTS) method to directly quantify the reaction adducts. The reaction conditions have been optimized to minimize solubility issues, oxidative side reactions and increase the throughput of the assay while minimizing the reaction time, which are common issues with existing methods. Thirty-six chemicals previously classified with LLNA, DPRA or KeratinoSens™ were tested as a proof of concept. Preliminary results gave an estimated 82% accuracy, 78% sensitivity, 90% specificity, comparable to other in chemico methods such as Cys-DPRA. In addition to validated chemicals, six natural products were analyzed and a prediction of their sensitization potential is presented for the first time. - Highlights: • A novel fluorescence-based method to detect electrophilic sensitizers is proposed. • A model fluorescent thiol was used to directly quantify the reaction products. • A discussion of the reaction workflow and critical parameters is presented. • The method could provide a useful tool to complement existing chemical assays.« less

Potentiometric Sensors Based on Surface Molecular Imprinting: Detection of Cancer Biomarkers and Viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Y.; Zhang, Z; Jain, V

2010-01-01

The continuing discovery of cancer biomarkers necessitates improved methods for their detection. Molecular imprinting using artificial materials provides an alternative to the detection of a wide range of substances. We applied surface molecular imprinting using self-assembled monolayers to design sensing elements for the detection of cancer biomarkers and other proteins. These elements consist of a gold-coated silicon chip onto which hydroxyl-terminated alkanethiol molecules and template biomolecule are co-adsorbed, where the thiol molecules are chemically bound to the metal substrate and self-assembled into highly ordered monolayers, the biomolecules can be removed, creating the foot-print cavities in the monolayer matrix for thismore » kind of template molecules. Re-adsorption of the biomolecules to the sensing chip changes its potential, which can be measured potentiometrically. We applied this method to the detection of carcinoembryonic antigen (CEA) in both solutions of purified CEA and in the culture medium of a CEA-producing human colon cancer cell line. The CEA assay, validated also against a standard immunoassay, was both sensitive (detection range 2.5-250 ng/mL) and specific (no cross-reactivity with hemoglobin; no response by a non-imprinted sensor). Similar results were obtained for human amylase. In addition, we detected virions of poliovirus in a specific manner (no cross-reactivity to adenovirus, no response by a non-imprinted sensor). Our findings demonstrate the application of the principles of molecular imprinting to the development of a new method for the detection of protein cancer biomarkers and to protein-based macromolecular structures such as the capsid of a virion. This approach has the potential of generating a general assay methodology that could be highly sensitive, specific, simple and likely inexpensive.« less

Combining vibrational biomolecular spectroscopy with chemometric techniques for the study of response and sensitivity of molecular structures/functional groups mainly related to lipid biopolymer to various processing applications.

PubMed

Yu, Gloria Qingyu; Yu, Peiqiang

2015-09-01

The objectives of this project were to (1) combine vibrational spectroscopy with chemometric multivariate techniques to determine the effect of processing applications on molecular structural changes of lipid biopolymer that mainly related to functional groups in green- and yellow-type Crop Development Centre (CDC) pea varieties [CDC strike (green-type) vs. CDC meadow (yellow-type)] that occurred during various processing applications; (2) relatively quantify the effect of processing applications on the antisymmetric CH3 ("CH3as") and CH2 ("CH2as") (ca. 2960 and 2923 cm(-1), respectively), symmetric CH3 ("CH3s") and CH2 ("CH2s") (ca. 2873 and 2954 cm(-1), respectively) functional groups and carbonyl C=O ester (ca. 1745 cm(-1)) spectral intensities as well as their ratios of antisymmetric CH3 to antisymmetric CH2 (ratio of CH3as to CH2as), ratios of symmetric CH3 to symmetric CH2 (ratio of CH3s to CH2s), and ratios of carbonyl C=O ester peak area to total CH peak area (ratio of C=O ester to CH); and (3) illustrate non-invasive techniques to detect the sensitivity of individual molecular functional group to the various processing applications in the recently developed different types of pea varieties. The hypothesis of this research was that processing applications modified the molecular structure profiles in the processed products as opposed to original unprocessed pea seeds. The results showed that the different processing methods had different impacts on lipid molecular functional groups. Different lipid functional groups had different sensitivity to various heat processing applications. These changes were detected by advanced molecular spectroscopy with chemometric techniques which may be highly related to lipid utilization and availability. The multivariate molecular spectral analyses, cluster analysis, and principal component analysis of original spectra (without spectral parameterization) are unable to fully distinguish the structural differences in the antisymmetric and symmetric CH3 and CH2 spectral region (ca. 3001-2799 cm(-1)) and carbonyl C=O ester band region (ca. 1771-1714 cm(-1)). This result indicated that the sensitivity to detect treatment difference by multivariate analysis of cluster analysis (CLA) and principal components analysis (PCA) might be lower compared with univariate molecular spectral analysis. In the future, other more sensitive techniques such as "discriminant analysis" could be considered for discriminating and classifying structural differences. Molecular spectroscopy can be used as non-invasive technique to study processing-induced structural changes that are related to lipid compound in legume seeds.

Diagnostic Performance of a Molecular Test versus Clinician Assessment of Vaginitis.

PubMed

Schwebke, Jane R; Gaydos, Charlotte A; Nyirjesy, Paul; Paradis, Sonia; Kodsi, Salma; Cooper, Charles K

2018-06-01

Vaginitis is a common complaint, diagnosed either empirically or using Amsel's criteria and wet mount microscopy. This study sought to determine characteristics of an investigational test (a molecular test for vaginitis), compared to reference, for detection of bacterial vaginosis, Candida spp., and Trichomonas vaginalis Vaginal specimens from a cross-sectional study were obtained from 1,740 women (≥18 years old), with vaginitis symptoms, during routine clinic visits (across 10 sites in the United States). Specimens were analyzed using a commercial PCR/fluorogenic probe-based investigational test that detects bacterial vaginosis, Candida spp., and Trichomonas vaginalis Clinician diagnosis and in-clinic testing (Amsel's test, potassium hydroxide preparation, and wet mount) were also employed to detect the three vaginitis causes. All testing methods were compared to the respective reference methods (Nugent Gram stain for bacterial vaginosis, detection of the Candida gene its2 , and Trichomonas vaginalis culture). The investigational test, clinician diagnosis, and in-clinic testing were compared to reference methods for bacterial vaginosis, Candida spp., and Trichomonas vaginalis The investigational test resulted in significantly higher sensitivity and negative predictive value than clinician diagnosis or in-clinic testing. In addition, the investigational test showed a statistically higher overall percent agreement with each of the three reference methods than did clinician diagnosis or in-clinic testing. The investigational test showed significantly higher sensitivity for detecting vaginitis, involving more than one cause, than did clinician diagnosis. Taken together, these results suggest that a molecular investigational test can facilitate accurate detection of vaginitis. Copyright © 2018 Schwebke et al.

Application of the adverse outcome pathway (AOP) concept to structure the available in vivo and in vitro mechanistic data for allergic sensitization to food proteins.

PubMed

van Bilsen, Jolanda H M; Sienkiewicz-Szłapka, Edyta; Lozano-Ojalvo, Daniel; Willemsen, Linette E M; Antunes, Celia M; Molina, Elena; Smit, Joost J; Wróblewska, Barbara; Wichers, Harry J; Knol, Edward F; Ladics, Gregory S; Pieters, Raymond H H; Denery-Papini, Sandra; Vissers, Yvonne M; Bavaro, Simona L; Larré, Colette; Verhoeckx, Kitty C M; Roggen, Erwin L

2017-01-01

The introduction of whole new foods in a population may lead to sensitization and food allergy. This constitutes a potential public health problem and a challenge to risk assessors and managers as the existing understanding of the pathophysiological processes and the currently available biological tools for prediction of the risk for food allergy development and the severity of the reaction are not sufficient. There is a substantial body of in vivo and in vitro data describing molecular and cellular events potentially involved in food sensitization. However, these events have not been organized in a sequence of related events that is plausible to result in sensitization, and useful to challenge current hypotheses. The aim of this manuscript was to collect and structure the current mechanistic understanding of sensitization induction to food proteins by applying the concept of adverse outcome pathway (AOP). The proposed AOP for food sensitization is based on information on molecular and cellular mechanisms and pathways evidenced to be involved in sensitization by food and food proteins and uses the AOPs for chemical skin sensitization and respiratory sensitization induction as templates. Available mechanistic data on protein respiratory sensitization were included to fill out gaps in the understanding of how proteins may affect cells, cell-cell interactions and tissue homeostasis. Analysis revealed several key events (KE) and biomarkers that may have potential use in testing and assessment of proteins for their sensitizing potential. The application of the AOP concept to structure mechanistic in vivo and in vitro knowledge has made it possible to identify a number of methods, each addressing a specific KE, that provide information about the food allergenic potential of new proteins. When applied in the context of an integrated strategy these methods may reduce, if not replace, current animal testing approaches. The proposed AOP will be shared at the www.aopwiki.org platform to expand the mechanistic data, improve the confidence in each of the proposed KE and key event relations (KERs), and allow for the identification of new, or refinement of established KE and KERs.

Evolution of microbiological analytical methods for dairy industry needs

PubMed Central

Sohier, Danièle; Pavan, Sonia; Riou, Armelle; Combrisson, Jérôme; Postollec, Florence

2014-01-01

Traditionally, culture-based methods have been used to enumerate microbial populations in dairy products. Recent developments in molecular methods now enable faster and more sensitive analyses than classical microbiology procedures. These molecular tools allow a detailed characterization of cell physiological states and bacterial fitness and thus, offer new perspectives to integration of microbial physiology monitoring to improve industrial processes. This review summarizes the methods described to enumerate and characterize physiological states of technological microbiota in dairy products, and discusses the current deficiencies in relation to the industry’s needs. Recent studies show that Polymerase chain reaction-based methods can successfully be applied to quantify fermenting microbes and probiotics in dairy products. Flow cytometry and omics technologies also show interesting analytical potentialities. However, they still suffer from a lack of validation and standardization for quality control analyses, as reflected by the absence of performance studies and official international standards. PMID:24570675

Evolution of microbiological analytical methods for dairy industry needs.

PubMed

Sohier, Danièle; Pavan, Sonia; Riou, Armelle; Combrisson, Jérôme; Postollec, Florence

2014-01-01

Traditionally, culture-based methods have been used to enumerate microbial populations in dairy products. Recent developments in molecular methods now enable faster and more sensitive analyses than classical microbiology procedures. These molecular tools allow a detailed characterization of cell physiological states and bacterial fitness and thus, offer new perspectives to integration of microbial physiology monitoring to improve industrial processes. This review summarizes the methods described to enumerate and characterize physiological states of technological microbiota in dairy products, and discusses the current deficiencies in relation to the industry's needs. Recent studies show that Polymerase chain reaction-based methods can successfully be applied to quantify fermenting microbes and probiotics in dairy products. Flow cytometry and omics technologies also show interesting analytical potentialities. However, they still suffer from a lack of validation and standardization for quality control analyses, as reflected by the absence of performance studies and official international standards.

Bio-Mimetic Sensors Based on Molecularly Imprinted Membranes

PubMed Central

Algieri, Catia; Drioli, Enrico; Guzzo, Laura; Donato, Laura

2014-01-01

An important challenge for scientific research is the production of artificial systems able to mimic the recognition mechanisms occurring at the molecular level in living systems. A valid contribution in this direction resulted from the development of molecular imprinting. By means of this technology, selective molecular recognition sites are introduced in a polymer, thus conferring it bio-mimetic properties. The potential applications of these systems include affinity separations, medical diagnostics, drug delivery, catalysis, etc. Recently, bio-sensing systems using molecularly imprinted membranes, a special form of imprinted polymers, have received the attention of scientists in various fields. In these systems imprinted membranes are used as bio-mimetic recognition elements which are integrated with a transducer component. The direct and rapid determination of an interaction between the recognition element and the target analyte (template) was an encouraging factor for the development of such systems as alternatives to traditional bio-assay methods. Due to their high stability, sensitivity and specificity, bio-mimetic sensors-based membranes are used for environmental, food, and clinical uses. This review deals with the development of molecularly imprinted polymers and their different preparation methods. Referring to the last decades, the application of these membranes as bio-mimetic sensor devices will be also reported. PMID:25196110

Trifunctional molecular beacon-mediated quadratic amplification for highly sensitive and rapid detection of mercury(II) ion with tunable dynamic range.

PubMed

Zhao, Yue; Liu, Huaqing; Chen, Feng; Bai, Min; Zhao, Junwu; Zhao, Yongxi

2016-12-15

Analyses of target with low abundance or concentration varying over many orders of magnitude are severe challenges faced by numerous assay methods due to their modest sensitivity and limited dynamic range. Here, we introduce a homogeneous and rapid quadratic polynomial amplification strategy through rational design of a trifunctional molecular beacon, which serves as not only a reporter molecule but also a bridge to couple two stage amplification modules without adding any reaction components or process other than basic linear amplification. As a test bed for our studies, we took mercury(II) ion as an example and obtained a high sensitivity with detection limit down to 200 pM within 30min. In order to create a tunable dynamic range, homotropic allostery is employed to modulate the target specific binding. When the number of metal binding site varies from 1 to 3, signal response is programmed accordingly with useful dynamic range spanning 50, 25 and 10 folds, respectively. Furthermore, the applicability of the proposed method in river water and biological samples are successfully verified with good recovery and reproducibility, indicating considerable potential for its practicality in complex real samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Application of graphene-ionic liquid-chitosan composite-modified carbon molecular wire electrode for the sensitive determination of adenosine-5'-monophosphate.

PubMed

Shi, Fan; Gong, Shixing; Xu, Li; Zhu, Huanhuan; Sun, Zhenfan; Sun, Wei

2013-12-01

In this paper, a graphene (GR) ionic liquid (IL) 1-octyl-3-methylimidazolium hexafluorophosphate and chitosan composite-modified carbon molecular wire electrode (CMWE) was fabricated by a drop-casting method and further applied to the sensitive electrochemical detection of adenosine-5'-monophosphate (AMP). CMWE was prepared with diphenylacetylene (DPA) as the modifier and the binder. The properties of modified electrode were examined by scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. Electrochemical behaviors of AMP was carefully investigated with enhanced responses appeared, which was due to the presence of GR-IL composite on the electrode surface with excellent electrocatalytic ability. A well-defined oxidation peak of AMP appeared at 1.314 V and the electrochemical parameters were calculated by electrochemical methods. Under the selected conditions, the oxidation peak current of AMP was proportional to its concentration in the range from 0.01 μM to 80.0 μM with the detection limit as 3.42 nM (3σ) by differential pulse voltammetry. The proposed method exhibited good selectivity and was applied to the detection of vidarabine monophosphate injection samples with satisfactory results. © 2013.

Identification of Pseudallescheria and Scedosporium Species by Three Molecular Methods▿

PubMed Central

Lu, Qiaoyun; Gerrits van den Ende, A. H. G.; Bakkers, J. M. J. E.; Sun, Jiufeng; Lackner, M.; Najafzadeh, M. J.; Melchers, W. J. G.; Li, Ruoyu; de Hoog, G. S.

2011-01-01

The major clinically relevant species in Scedosporium (teleomorph Pseudallescheria) are Pseudallescheria boydii, Scedosporium aurantiacum, Scedosporium apiospermum, and Scedosporium prolificans, while Pseudallescheria minutispora, Petriellopsis desertorum, and Scedosporium dehoogii are exceptional agents of disease. Three molecular methods targeting the partial β-tubulin gene were developed and evaluated to identify six closely related species of the S. apiospermum complex using quantitative real-time PCR (qPCR), PCR-based reverse line blot (PCR-RLB), and loop-mediated isothermal amplification (LAMP). qPCR was not specific enough for the identification of all species but had the highest sensitivity. The PCR-RLB assay was efficient for the identification of five species. LAMP distinguished all six species unambiguously. The analytical sensitivities of qPCR, PCR-RLB, and LAMP combined with MagNAPure, CTAB (cetyltrimethylammonium bromide), and FTA filter (Whatman) extraction were 50, 5 × 103, and 5 × 102 cells/μl, respectively. When LAMP was combined with a simplified DNA extraction method using an FTA filter, identification to the species level was achieved within 2 h, including DNA extraction. The FTA-LAMP assay is therefore recommended as a cost-effective, simple, and rapid method for the identification of Scedosporium species. PMID:21177887

Hybrid optimal descriptors as a tool to predict skin sensitization in accordance to OECD principles.

PubMed

Toropova, Alla P; Toropov, Andrey A

2017-06-05

Skin sensitization (allergic contact dermatitis) is a widespread problem arising from the contact of chemicals with the skin. The detection of molecular features with undesired effect for skin is complex task owing to unclear biochemical mechanisms and unclearness of conditions of action of chemicals to skin. The development of computational methods for estimation of this endpoint in order to reduce animal testing is recommended (Cosmetics Directive EC regulation 1907/2006; EU Regulation, Regulation, 1223/2009). The CORAL software (http://www.insilico.eu/coral) gives good predictive models for the skin sensitization. Simplified molecular input-line entry system (SMILES) together with molecular graph are used to represent the molecular structure for these models. So-called hybrid optimal descriptors are used to establish quantitative structure-activity relationships (QSARs). The aim of this study is the estimation of the predictive potential of the hybrid descriptors. Three different distributions into the training (≈70%), calibration (≈15%), and validation (≈15%) sets are studied. QSAR for these three distributions are built up with using the Monte Carlo technique. The statistical characteristics of these models for external validation set are used as a measure of predictive potential of these models. The best model, according to the above criterion, is characterized by n validation =29, r 2 validation =0.8596, RMSE validation =0.489. Mechanistic interpretation and domain of applicability for these models are defined. Copyright © 2017 Elsevier B.V. All rights reserved.

A molecular-beacon-based asymmetric PCR assay for easy visualization of amplicons in the diagnosis of trichomoniasis.

PubMed

Sonkar, Subash C; Sachdev, Divya; Mishra, Prashant K; Kumar, Anita; Mittal, Pratima; Saluja, Daman

2016-12-15

The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecularly imprinted electrochemical biosensor based on Fe@Au nanoparticles involved in 2-aminoethanethiol functionalized multi-walled carbon nanotubes for sensitive determination of cefexime in human plasma.

PubMed

Yola, Mehmet Lütfi; Eren, Tanju; Atar, Necip

2014-10-15

The molecular imprinting technique depends on the molecular recognition. It is a polymerization method around the target molecule. Hence, this technique creates specific cavities in the cross-linked polymeric matrices. In present study, a sensitive imprinted electrochemical biosensor based on Fe@Au nanoparticles (Fe@AuNPs) involved in 2-aminoethanethiol (2-AET) functionalized multi-walled carbon nanotubes (f-MWCNs) modified glassy carbon (GC) electrode was developed for determination of cefexime (CEF). The results of X-ray photoelectron spectroscopy (XPS) and reflection-absorption infrared spectroscopy (RAIRS) confirmed the formation of the developed surfaces. CEF imprinted film was constructed by cyclic voltammetry (CV) for 9 cycles in the presence of 80 mM pyrrole in phosphate buffer solution (pH 6.0) containing 20mM CEF. The developed electrochemical biosensor was validated according to the International Conference on Harmonisation (ICH) guideline and found to be linear, sensitive, selective, precise and accurate. The linearity range and the detection limit were obtained as 1.0 × 10(-10)-1.0 × 10(-8)M and 2.2 × 10(-11)M, respectively. The developed CEF imprinted sensor was successfully applied to real samples such as human plasma. In addition, the stability and reproducibility of the prepared molecular imprinted electrode were investigated. The excellent long-term stability and reproducibility of the prepared CEF imprinted electrodes make them attractive in electrochemical sensors. Copyright © 2014 Elsevier B.V. All rights reserved.

Highly sensitive and selective sugar detection by terahertz nano-antennas

NASA Astrophysics Data System (ADS)

Lee, Dong-Kyu; Kang, Ji-Hun; Lee, Jun-Seok; Kim, Hyo-Seok; Kim, Chulki; Hun Kim, Jae; Lee, Taikjin; Son, Joo-Hiuk; Park, Q.-Han; Seo, Minah

2015-10-01

Molecular recognition and discrimination of carbohydrates are important because carbohydrates perform essential roles in most living organisms for energy metabolism and cell-to-cell communication. Nevertheless, it is difficult to identify or distinguish various carbohydrate molecules owing to the lack of a significant distinction in the physical or chemical characteristics. Although there has been considerable effort to develop a sensing platform for individual carbohydrates selectively using chemical receptors or an ensemble array, their detection and discrimination limits have been as high in the millimolar concentration range. Here we show a highly sensitive and selective detection method for the discrimination of carbohydrate molecules using nano-slot-antenna array-based sensing chips which operate in the terahertz (THz) frequency range (0.5-2.5 THz). This THz metamaterial sensing tool recognizes various types of carbohydrate molecules over a wide range of molecular concentrations. Strongly localized and enhanced terahertz transmission by nano-antennas can effectively increase the molecular absorption cross sections, thereby enabling the detection of these molecules even at low concentrations. We verified the performance of nano-antenna sensing chip by both THz spectra and images of transmittance. Screening and identification of various carbohydrates can be applied to test even real market beverages with a high sensitivity and selectivity.

Highly sensitive and selective sugar detection by terahertz nano-antennas

PubMed Central

Lee, Dong-Kyu; Kang, Ji-Hun; Lee, Jun-Seok; Kim, Hyo-Seok; Kim, Chulki; Hun Kim, Jae; Lee, Taikjin; Son, Joo-Hiuk; Park, Q-Han; Seo, Minah

2015-01-01

Molecular recognition and discrimination of carbohydrates are important because carbohydrates perform essential roles in most living organisms for energy metabolism and cell-to-cell communication. Nevertheless, it is difficult to identify or distinguish various carbohydrate molecules owing to the lack of a significant distinction in the physical or chemical characteristics. Although there has been considerable effort to develop a sensing platform for individual carbohydrates selectively using chemical receptors or an ensemble array, their detection and discrimination limits have been as high in the millimolar concentration range. Here we show a highly sensitive and selective detection method for the discrimination of carbohydrate molecules using nano-slot-antenna array-based sensing chips which operate in the terahertz (THz) frequency range (0.5–2.5 THz). This THz metamaterial sensing tool recognizes various types of carbohydrate molecules over a wide range of molecular concentrations. Strongly localized and enhanced terahertz transmission by nano-antennas can effectively increase the molecular absorption cross sections, thereby enabling the detection of these molecules even at low concentrations. We verified the performance of nano-antenna sensing chip by both THz spectra and images of transmittance. Screening and identification of various carbohydrates can be applied to test even real market beverages with a high sensitivity and selectivity. PMID:26494203

Rapid and sensitive detection of mink circovirus by recombinase polymerase amplification.

PubMed

Ge, Junwei; Shi, Yunjia; Cui, Xingyang; Gu, Shanshan; Zhao, Lili; Chen, Hongyan

2018-06-01

To date, the pathogenic role of mink circovirus (MiCV) remains unclear, and its prevalence and economic importance are unknown. Therefore, a rapid and sensitive molecular diagnosis is necessary for disease management and epidemiological surveillance. However, only PCR methods can identify MiCV infection at present. In this study, we developed a nested PCR and established a novel recombinase polymerase amplification (RPA) assay for MiCV detection. Sensitivity analysis showed that the detection limit of nested PCR and RPA assay was 10 1 copies/reaction, and these methods were more sensitive than conventional PCR, which has a detection limit of 10 5 copies/reaction. The RPA assay had no cross-reactivity with other related viral pathogens, and amplification was completed in less than 20 min with a simple device. Further assessment of clinical samples showed that the two assays were accurate in identifying positive and negative conventional PCR samples. The detection rate of MiCV by the RPA assay in clinical samples was 38.09%, which was 97% consistent with that by the nested PCR. The developed nested PCR is a highly sensitive tool for practical use, and the RPA assay is a simple, sensitive, and potential alternative method for rapid and accurate MiCV diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

Detection of Entamoeba histolytica by Recombinase Polymerase Amplification.

PubMed

Nair, Gayatri; Rebolledo, Mauricio; White, A Clinton; Crannell, Zachary; Richards-Kortum, R Rebecca; Pinilla, A Elizabeth; Ramírez, Juan David; López, M Consuelo; Castellanos-Gonzalez, Alejandro

2015-09-01

Amebiasis is an important cause of diarrheal disease worldwide and has been associated with childhood malnutrition. Traditional microscopy approaches are neither sensitive nor specific for Entamoeba histolytica. Antigen assays are more specific, but many cases are missed unless tested by molecular methods. Although polymerase chain reaction (PCR) is effective, the need for sophisticated, expensive equipment, infrastructure, and trained personnel limits its usefulness, especially in the resource-limited, endemic areas. Here, we report development of a recombinase polymerase amplification (RPA) method to detect E. histolytica specifically. Using visual detection by lateral flow (LF), the test was highly sensitive and specific and could be performed without additional equipment. The availability of this inexpensive, sensitive, and field-applicable diagnostic test could facilitate rapid diagnosis and treatment of amebiasis in endemic regions. © The American Society of Tropical Medicine and Hygiene.

Improved detection of endoparasite DNA in soil sample PCR by the use of anti-inhibitory substances.

PubMed

Krämer, F; Vollrath, T; Schnieder, T; Epe, C

2002-09-26

Although there have been numerous microbial examinations of soil for the presence of human pathogenic developmental parasite stages of Ancylostoma caninum and Toxocara canis, molecular techniques (e.g. DNA extraction, purification and subsequent PCR) have scarcely been applied. Here, DNA preparations of soil samples artificially contaminated with genomic DNA or parasite eggs were examined by PCR. A. caninum and T. canis-specific primers based on the ITS-2 sequence were used for amplification. After the sheer DNA preparation a high content of PCR-interfering substances was still detectable. Subsequently, two different inhibitors of PCR-interfering agents (GeneReleaser, Bioventures Inc. and Maximator, Connex GmbH) were compared in PCR. Both substances increased PCR sensitivity greatly. However, comparison of the increase in sensitivity achieved with the two compounds demonstrated the superiority of Maximator, which enhanced sensitivity to the point of permitting positive detection of a single A. caninum egg and three T. canis eggs in a soil sample. This degree of sensitivity could not be achieved with GeneReleaser for either parasite Furthermore, Maximator not only increased sensitivity; it also cost less, required less time and had a lower risk of contamination. Future applications of molecular methods in epidemiological examinations of soil samples are discussed/elaborated.

Photooxidation of 3-substituted pyrroles:  a postcolumn reaction detection system for singlet molecular oxygen in HPLC.

PubMed

Denham, K; Milofsky, R E

1998-10-01

A postcolumn photochemical reaction detection scheme, based on the reaction of 3-substituted pyrroles with singlet molecular oxygen ((1)O(2)), has been developed. The method is selective and sensitive for the determination of a class of organic compounds called (1)O(2)-sensitizers and is readily coupled to HPLC. Following separation by HPLC, analytes ((1)O(2)-sensitizers) are excited by a Hg pen-ray lamp. Analytes that are efficient (1)O(2)-sensitizers promote ground-state O(2) ((3)Σ(g)(-)) to an excited state ((1)Σ(g)(+) or (1)Δ(g)), which reacts rapidly with tert-butyl-3,4,5-trimethylpyrrolecarboxylate (BTMPC) or N-benzyl-3-methoxypyrrole-2-tert-carboxylate (BMPC), which is added to the mobile phase. Detection is based on the loss of pyrrole (BTMPC or BMPC). The reaction is catalytic in nature since one analyte molecule may absorb light many times, producing large amounts of (1)O(2). Detection limits for several (1)O(2)-sensitizers were improved by 1-2 orders of magnitude over optimized UV-absorbance detection. This paper discusses the optimization of the reaction conditions for this photochemical reaction detection scheme and its application to the detection of PCBs, nitrogen heterocycles, nitro and chloro aromatics, and other substituted aromatic compounds.

Molecular typing of Sarcocystis neurona: current status and future trends.

PubMed

Elsheikha, Hany M; Mansfield, Linda S

2007-10-21

Sarcocystis neurona is an important protozoal pathogen because it causes the serious neurological disease equine protozoal myeloencephalitis (EPM). The capacity of this organism to cause a wide spectrum of neurological signs in horses and the broad geographic distribution of observed cases in the Americas drive the need for sensitive, reliable and rapid typing methods to characterize strains. Various molecular methods have been developed and used to diagnose EPM due to S. neurona, to identify S. neurona isolates and to determine the heterogeneity and evolutionary relatedness within this species and related Sarcocystis spp. These methods included sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immuno-fluorescent assay (IFA), slide agglutination test (SAT), SnSAG-specific ELISA, random amplified polymorphic DNA (RAPD), PCR-based restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP) fingerprinting, and sequence analysis of surface protein genes, ribosomal genes, microsatellite alleles and other molecular markers. Here, the utility of these molecular methods is reviewed and evaluated with respect to the need for molecular approaches that utilize well-characterized polymorphic, simple, independent, and stable genetic markers. These tools have the potential to add to knowledge of the genetic population structure of S. neurona and to provide new insights into the pathogenesis of EPM and S. neurona epidemiology. In particular, these methods provide new tools to address the hypothesis that particular genetic variants are associated with adverse clinical outcomes (severe pathotypes). The ultimate goal is to utilize them in future studies to improve treatment and prevention strategies.

Toward a Droplet-Based Single-Cell Radiometric Assay.

PubMed

Gallina, Maria Elena; Kim, Tae Jin; Shelor, Mark; Vasquez, Jaime; Mongersun, Amy; Kim, Minkyu; Tang, Sindy K Y; Abbyad, Paul; Pratx, Guillem

2017-06-20

Radiotracers are widely used to track molecular processes, both in vitro and in vivo, with high sensitivity and specificity. However, most radionuclide detection methods have spatial resolution inadequate for single-cell analysis. A few existing methods can extract single-cell information from radioactive decays, but the stochastic nature of the process precludes high-throughput measurement (and sorting) of single cells. In this work, we introduce a new concept for translating radioactive decays occurring stochastically within radiolabeled single-cells into an integrated, long-lasting fluorescence signal. Single cells are encapsulated in radiofluorogenic droplets containing molecular probes sensitive to byproducts of ionizing radiation (primarily reactive oxygen species, or ROS). Different probes were examined in bulk solutions, and dihydrorhodamine 123 (DHRh 123) was selected as the lead candidate due to its sensitivity and reproducibility. Fluorescence intensity of DHRh 123 in bulk increased at a rate of 54% per Gy of X-ray radiation and 15% per MBq/ml of 2-deoxy-2-[ 18 F]-fluoro-d-glucose ([ 18 F]FDG). Fluorescence imaging of microfluidic droplets showed the same linear response, but droplets were less sensitive overall than the bulk ROS sensor (detection limit of 3 Gy per droplet). Finally, droplets encapsulating radiolabeled cancer cells allowed, for the first time, the detection of [ 18 F]FDG radiotracer uptake in single cells through fluorescence activation. With further improvements, we expect this technology to enable quantitative measurement and selective sorting of single cells based on the uptake of radiolabeled small molecules.

Type-specific identification of anogenital herpes simplex virus infections by use of a commercially available nucleic acid amplification test.

PubMed

Van Der Pol, Barbara; Warren, Terri; Taylor, Stephanie N; Martens, Mark; Jerome, Keith R; Mena, Leandro; Lebed, Joel; Ginde, Savita; Fine, Paul; Hook, Edward W

2012-11-01

Herpes infections are among the most common sexually transmitted infections (STI), but diagnostic methods for genital herpes have not kept pace with the movement toward molecular testing. Here, we describe an FDA-approved molecular assay that identifies and types herpes simplex virus (HSV) infections for use in routine clinical settings. Paired samples from anogenital lesions were tested using the BD ProbeTec HSV Q(x) (HSVQ(x)) system, HSV culture and, a laboratory-developed PCR assay. Family planning, obstetrics/gynecology (OB/GYN), or sexually transmitted disease (STD) clinics in the United States served as recruitment sites. Sensitivity and specificity estimates, head-to-head comparisons, measures of agreement, and latent-class analyses were performed to provide robust estimates of performance. A total of 508 participants (174 men and 334 women) with anogenital lesions were included; 260 HSV-2 and 73 HSV-1 infections were identified. No differences in test performance based on gender, clinic type, location of the lesion, or type of lesion were observed. The sensitivity of HSV-2 detection ranged from 98.4 to 100% depending on the analytical approach, while the specificity ranged from 80.6%, compared to the less sensitive culture method, to 97.0%, compared to PCR. For HSV-1, the sensitivity and specificity ranges were 96.7 to 100% and 95.1 to 99.4%, respectively. This assay may improve our ability to accurately diagnose anogenital lesions due to herpes infection.

Clustering the Orion B giant molecular cloud based on its molecular emission

PubMed Central

Bron, Emeric; Daudon, Chloé; Pety, Jérôme; Levrier, François; Gerin, Maryvonne; Gratier, Pierre; Orkisz, Jan H.; Guzman, Viviana; Bardeau, Sébastien; Goicoechea, Javier R.; Liszt, Harvey; Öberg, Karin; Peretto, Nicolas; Sievers, Albrecht; Tremblin, Pascal

2017-01-01

Context Previous attempts at segmenting molecular line maps of molecular clouds have focused on using position-position-velocity data cubes of a single molecular line to separate the spatial components of the cloud. In contrast, wide field spectral imaging over a large spectral bandwidth in the (sub)mm domain now allows one to combine multiple molecular tracers to understand the different physical and chemical phases that constitute giant molecular clouds (GMCs). Aims We aim at using multiple tracers (sensitive to different physical processes and conditions) to segment a molecular cloud into physically/chemically similar regions (rather than spatially connected components), thus disentangling the different physical/chemical phases present in the cloud. Methods We use a machine learning clustering method, namely the Meanshift algorithm, to cluster pixels with similar molecular emission, ignoring spatial information. Clusters are defined around each maximum of the multidimensional Probability Density Function (PDF) of the line integrated intensities. Simple radiative transfer models were used to interpret the astrophysical information uncovered by the clustering analysis. Results A clustering analysis based only on the J = 1 – 0 lines of three isotopologues of CO proves suffcient to reveal distinct density/column density regimes (nH ~ 100 cm−3, ~ 500 cm−3, and > 1000 cm−3), closely related to the usual definitions of diffuse, translucent and high-column-density regions. Adding two UV-sensitive tracers, the J = 1 − 0 line of HCO+ and the N = 1 − 0 line of CN, allows us to distinguish two clearly distinct chemical regimes, characteristic of UV-illuminated and UV-shielded gas. The UV-illuminated regime shows overbright HCO+ and CN emission, which we relate to a photochemical enrichment effect. We also find a tail of high CN/HCO+ intensity ratio in UV-illuminated regions. Finer distinctions in density classes (nH ~ 7 × 103 cm−3 ~ 4 × 104 cm−3) for the densest regions are also identified, likely related to the higher critical density of the CN and HCO+ (1 – 0) lines. These distinctions are only possible because the high-density regions are spatially resolved. Conclusions Molecules are versatile tracers of GMCs because their line intensities bear the signature of the physics and chemistry at play in the gas. The association of simultaneous multi-line, wide-field mapping and powerful machine learning methods such as the Meanshift clustering algorithm reveals how to decode the complex information available in these molecular tracers. PMID:29456256

DFT/TD-semiempirical study on the structural and electronic properties and absorption spectra of supramolecular fullerene-porphyrine-metalloporphyrine triads based dye-sensitized solar cells.

PubMed

Rezvani, M; Darvish Ganji, M; Jameh-Bozorghi, S; Niazi, A

2018-04-05

In the present work density functional theory (DFT) and time-dependent semiempirical ZNIDO/S (TD-ZNIDO/S) methods have been used to investigate the ground state geometries, electronic structures and excited state properties of triad systems. The influences of the type of metal in the porphyrin ring, change in bridge position and porphyrine-ZnP duplicate on the energies of frontier molecular orbital and UV-Vis spectra has been studied. Geometry optimization, the energy levels and electron density of the Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO), chemical hardness (η), electrophilicity index (ω), electron accepting power (ω + ) were calculated using ZINDO/S method to predict which molecule is the most efficient with a great capability to be used as a triad molecule in solar industry. Moreover the light harvesting efficiency (LHE) was calculated by means of the oscillator strengths which are obtained by TD-ZINDO/S calculation. Theoretical studies of the electronic spectra by ZINDO/S method were helpful in interpreting the observed electronic transitions. This aspect was systematically explored in a series of C 60 -Porphyrine-Metalloporphyrine (C 60 -P-Mp) triad system with M being Fe, Co, Ni, Ti, and Zn. Generally, transition metal coordination compounds are used as effective sensitizers, due to their intense charge-transfer absorption over the whole visible range and highly efficient metal-to-ligand charge transfer. We aim to optimize the performance of the title solar cells by altering the frontier orbital energy gaps. The results reveal that cell efficiency can be enhanced by metal functionalization of the free base porphyrin. Ti-porphyrin was found to be the most efficient dye sensitizer for dye sensitized solar cells (DSSCs) based on C 60 -P-Mptriad system due to C 60 -Por-TiP complex has lower chemical hardness, gap energy and chemical potential as well as higher electron accepting power among other complexes. In addition, the performance of solar cells favors better with doubly and increasing the π conjugated of the bridge. Copyright © 2018 Elsevier B.V. All rights reserved.

DFT/TD-semiempirical study on the structural and electronic properties and absorption spectra of supramolecular fullerene-porphyrine-metalloporphyrine triads based dye-sensitized solar cells

NASA Astrophysics Data System (ADS)

Rezvani, M.; Darvish Ganji, M.; Jameh-Bozorghi, S.; Niazi, A.

2018-04-01

In the present work density functional theory (DFT) and time-dependent semiempirical ZNIDO/S (TD-ZNIDO/S) methods have been used to investigate the ground state geometries, electronic structures and excited state properties of triad systems. The influences of the type of metal in the porphyrin ring, change in bridge position and porphyrine-ZnP duplicate on the energies of frontier molecular orbital and UV-Vis spectra has been studied. Geometry optimization, the energy levels and electron density of the Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO), chemical hardness (η), electrophilicity index (ω), electron accepting power (ω+) were calculated using ZINDO/S method to predict which molecule is the most efficient with a great capability to be used as a triad molecule in solar industry. Moreover the light harvesting efficiency (LHE) was calculated by means of the oscillator strengths which are obtained by TD-ZINDO/S calculation. Theoretical studies of the electronic spectra by ZINDO/S method were helpful in interpreting the observed electronic transitions. This aspect was systematically explored in a series of C60-Porphyrine-Metalloporphyrine (C60-P-Mp) triad system with M being Fe, Co, Ni, Ti, and Zn. Generally, transition metal coordination compounds are used as effective sensitizers, due to their intense charge-transfer absorption over the whole visible range and highly efficient metal-to-ligand charge transfer. We aim to optimize the performance of the title solar cells by altering the frontier orbital energy gaps. The results reveal that cell efficiency can be enhanced by metal functionalization of the free base porphyrin. Ti-porphyrin was found to be the most efficient dye sensitizer for dye sensitized solar cells (DSSCs) based on C60-P-Mptriad system due to C60-Por-TiP complex has lower chemical hardness, gap energy and chemical potential as well as higher electron accepting power among other complexes. In addition, the performance of solar cells favors better with doubly and increasing the π conjugated of the bridge.

A novel approach for the quantitation of carbohydrates in mash, wort, and beer with RP-HPLC using 1-naphthylamine for precolumn derivatization.

PubMed

Rakete, Stefan; Glomb, Marcus A

2013-04-24

A novel universal method for the determination of reducing mono-, di-, and oligosaccharides in complex matrices on RP-HPLC using 1-naphthylamine for precolumn derivatization with sodium cyanoborhydride was established to study changes in the carbohydrate profile during beer brewing. Fluorescence and mass spectrometric detection enabled very sensitive analyses of beer-relevant carbohydrates. Mass spectrometry additionally allowed the identification of the molecular weight and thereby the degree of polymerization of unknown carbohydrates. Thus, carbohydrates with up to 16 glucose units were detected. Comparison demonstrated that the novel method was superior to fluorophore-assisted carbohydrate electrophoresis (FACE). The results proved the HPLC method clearly to be more powerful in regard to sensitivity and resolution. Analogous to FACE, this method was designated fluorophore-assisted carbohydrate HPLC (FAC-HPLC).

The LLNA: A Brief Review of Recent Advances and Limitations

PubMed Central

Anderson, Stacey E.; Siegel, Paul D.; Meade, B. J.

2011-01-01

Allergic contact dermatitis is the second most commonly reported occupational illness, accounting for 10% to 15% of all occupational diseases. This highlights the importance of developing rapid and sensitive methods for hazard identification of chemical sensitizers. The murine local lymph node assay (LLNA) was developed and validated for the identification of low molecular weight sensitizing chemicals. It provides several benefits over other tests for sensitization because it provides a quantitative endpoint, dose-responsive data, and allows for prediction of potency. However, there are also several concerns with this assay including: levels of false positive responses, variability due to vehicle, and predictivity. This report serves as a concise review which briefly summarizes the progress, advances and limitations of the assay over the last decade. PMID:21747867

Theoretical studies on a carbonaceous molecular bearing: association thermodynamics and dual-mode rolling dynamics† †Electronic supplementary information (ESI) available: Supplementary figures, tables and atomic coordinates of representative geometries. See DOI: 10.1039/c5sc00335k

PubMed Central

Nakamura, Kosuke; Hitosugi, Shunpei; Sato, Sota; Tokoyama, Hiroaki; Yamakado, Hideo; Ohno, Koichi

2015-01-01

The thermodynamics and dynamics of a carbonaceous molecular bearing comprising a belt-persistent tubular molecule and a fullerene molecule have been investigated using density functional theory (DFT). Among ten representative methods, two DFT methods afforded an association energy that reasonably reproduced the experimental enthalpy of –12.5 kcal mol–1 at the unique curved π-interface. The dynamics of the molecular bearing, which was assembled solely with van der Waals interactions, exhibited small energy barriers with maximum values of 2–3 kcal mol–1 for the rolling motions. The dynamic motions responded sensitively to the steric environment and resulted in two distinct motions, precession and spin, which explained the unique NMR observations that were not clarified in previous experimental studies. PMID:29142679

Whole Genome Sequencing Increases Molecular Diagnostic Yield Compared with Current Diagnostic Testing for Inherited Retinal Disease.

PubMed

Ellingford, Jamie M; Barton, Stephanie; Bhaskar, Sanjeev; Williams, Simon G; Sergouniotis, Panagiotis I; O'Sullivan, James; Lamb, Janine A; Perveen, Rahat; Hall, Georgina; Newman, William G; Bishop, Paul N; Roberts, Stephen A; Leach, Rick; Tearle, Rick; Bayliss, Stuart; Ramsden, Simon C; Nemeth, Andrea H; Black, Graeme C M

2016-05-01

To compare the efficacy of whole genome sequencing (WGS) with targeted next-generation sequencing (NGS) in the diagnosis of inherited retinal disease (IRD). Case series. A total of 562 patients diagnosed with IRD. We performed a direct comparative analysis of current molecular diagnostics with WGS. We retrospectively reviewed the findings from a diagnostic NGS DNA test for 562 patients with IRD. A subset of 46 of 562 patients (encompassing potential clinical outcomes of diagnostic analysis) also underwent WGS, and we compared mutation detection rates and molecular diagnostic yields. In addition, we compared the sensitivity and specificity of the 2 techniques to identify known single nucleotide variants (SNVs) using 6 control samples with publically available genotype data. Diagnostic yield of genomic testing. Across known disease-causing genes, targeted NGS and WGS achieved similar levels of sensitivity and specificity for SNV detection. However, WGS also identified 14 clinically relevant genetic variants through WGS that had not been identified by NGS diagnostic testing for the 46 individuals with IRD. These variants included large deletions and variants in noncoding regions of the genome. Identification of these variants confirmed a molecular diagnosis of IRD for 11 of the 33 individuals referred for WGS who had not obtained a molecular diagnosis through targeted NGS testing. Weighted estimates, accounting for population structure, suggest that WGS methods could result in an overall 29% (95% confidence interval, 15-45) uplift in diagnostic yield. We show that WGS methods can detect disease-causing genetic variants missed by current NGS diagnostic methodologies for IRD and thereby demonstrate the clinical utility and additional value of WGS. Copyright © 2016 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

An empirical assessment of validation practices for molecular classifiers

PubMed Central

Castaldi, Peter J.; Dahabreh, Issa J.

2011-01-01

Proposed molecular classifiers may be overfit to idiosyncrasies of noisy genomic and proteomic data. Cross-validation methods are often used to obtain estimates of classification accuracy, but both simulations and case studies suggest that, when inappropriate methods are used, bias may ensue. Bias can be bypassed and generalizability can be tested by external (independent) validation. We evaluated 35 studies that have reported on external validation of a molecular classifier. We extracted information on study design and methodological features, and compared the performance of molecular classifiers in internal cross-validation versus external validation for 28 studies where both had been performed. We demonstrate that the majority of studies pursued cross-validation practices that are likely to overestimate classifier performance. Most studies were markedly underpowered to detect a 20% decrease in sensitivity or specificity between internal cross-validation and external validation [median power was 36% (IQR, 21–61%) and 29% (IQR, 15–65%), respectively]. The median reported classification performance for sensitivity and specificity was 94% and 98%, respectively, in cross-validation and 88% and 81% for independent validation. The relative diagnostic odds ratio was 3.26 (95% CI 2.04–5.21) for cross-validation versus independent validation. Finally, we reviewed all studies (n = 758) which cited those in our study sample, and identified only one instance of additional subsequent independent validation of these classifiers. In conclusion, these results document that many cross-validation practices employed in the literature are potentially biased and genuine progress in this field will require adoption of routine external validation of molecular classifiers, preferably in much larger studies than in current practice. PMID:21300697

Highly sensitive fluorescence quantitative detection of specific DNA sequences with molecular beacons and nucleic acid dye SYBR Green I.

PubMed

Xiang, Dongshan; Zhai, Kun; Xiang, Wenjun; Wang, Lianzhi

2014-11-01

A highly sensitive fluorescence method of quantitative detection for specific DNA sequence is developed based on molecular beacon (MB) and nucleic acid dye SYBR Green I by synchronous fluorescence analysis. It is demonstrated by an oligonucleotide sequence of wild-type HBV (target DNA) as a model system. In this strategy, the fluorophore of MB is designed to be 6-carboxyfluorescein group (FAM), and the maximum excitation wavelength and maximum emission wavelength are both very close to that of SYBR Green I. In the presence of targets DNA, the MBs hybridize with the targets DNA and form double-strand DNA (dsDNA), the fluorophore FAM is separated from the quencher BHQ-1, thus the fluorophore emit fluorescence. At the same time, SYBR Green I binds to dsDNA, the fluorescence intensity of SYBR Green I is significantly enhanced. When targets DNA are detected by synchronous fluorescence analysis, the fluorescence peaks of FAM and SYBR Green I overlap completely, so the fluorescence signal of system will be significantly enhanced. Thus, highly sensitive fluorescence quantitative detection for DNA can be realized. Under the optimum conditions, the total fluorescence intensity of FAM and SYBR Green I exhibits good linear dependence on concentration of targets DNA in the range from 2×10(-11) to 2.5×10(-9)M. The detection limit of target DNA is estimated to be 9×10(-12)M (3σ). Compared with previously reported methods of detection DNA with MB, the proposed method can significantly enhance the detection sensitivity. Copyright © 2014 Elsevier B.V. All rights reserved.

Novel biotechnology approaches in colorectal cancer diagnosis and therapy.

PubMed

Kavousipour, Soudabeh; Khademi, Fathemeh; Zamani, Mozhdeh; Vakili, Bahareh; Mokarram, Pooneh

2017-06-01

With ever-increasing molecular information about colorectal cancer (CRC), there is an expectation to detect more sensitive and specific molecular markers for new advanced diagnostic methods that can surpass the limitations of current screening tests. Moreover, enhanced molecular pathology knowledge about cancer has led to the development of targeted therapies, designed to interfere with specific aberrant biological pathways in cancer. Furthermore, biotechnology has opened a new window in CRC diagnosis and treatment by introducing different application of antibodies, antibody fragments, non-Ig scaffold proteins, and aptamers in targeted therapy and drug delivery. This review summarizes the molecular diagnostic and therapeutic approaches in CRC with a focus on genetic and epigenetic alterations, protein and metabolite markers as well as targeted therapy and drug delivery by Ig-scaffold proteins, non-Ig scaffold proteins, nanobodies, and aptamers.

Molecular diagnosis of canine visceral leishmaniasis: a comparative study of three methods using skin and spleen from dogs with natural Leishmania infantum infection.

PubMed

Reis, Levi Eduardo Soares; Coura-Vital, Wendel; Roatt, Bruno Mendes; Bouillet, Leoneide Érica Maduro; Ker, Henrique Gama; Fortes de Brito, Rory Cristiane; Resende, Daniela de Melo; Carneiro, Mariângela; Giunchetti, Rodolfo Cordeiro; Marques, Marcos José; Carneiro, Cláudia Martins; Reis, Alexandre Barbosa

2013-11-08

Polymerase chain reaction (PCR) and its variations represent highly sensitive and specific methods for Leishmania DNA detection and subsequent canine visceral leishmaniasis (CVL) diagnosis. The aim of this work was to compare three different molecular diagnosis techniques (conventional PCR [cPCR], seminested PCR [snPCR], and quantitative PCR [qPCR]) in samples of skin and spleen from 60 seropositive dogs by immunofluorescence antibody test and enzyme-linked immunosorbent assay. Parasitological analysis was conducted by culture of bone marrow aspirate and optical microscopic assessment of ear skin and spleen samples stained with Giemsa, the standard tests for CVL diagnosis. The primers L150/L152 and LINR4/LIN17/LIN19 were used to amplify the conserved region of the Leishmania kDNA minicircle in the cPCR, and snPCR and qPCR were performed using the DNA polymerase gene (DNA pol α) primers from Leishmania infantum. The parasitological analysis revealed parasites in 61.7% of the samples. Sensitivities were 89.2%, 86.5%, and 97.3% in the skin and 81.1%, 94.6%, and 100.0% in spleen samples used for cPCR, snPCR, and qPCR, respectively. We demonstrated that the qPCR method was the best technique to detect L. infantum in both skin and spleen samples. However, we recommend the use of skin due to the high sensitivity and sampling being less invasive. Copyright © 2013 Elsevier B.V. All rights reserved.

CarcinoPred-EL: Novel models for predicting the carcinogenicity of chemicals using molecular fingerprints and ensemble learning methods.

PubMed

Zhang, Li; Ai, Haixin; Chen, Wen; Yin, Zimo; Hu, Huan; Zhu, Junfeng; Zhao, Jian; Zhao, Qi; Liu, Hongsheng

2017-05-18

Carcinogenicity refers to a highly toxic end point of certain chemicals, and has become an important issue in the drug development process. In this study, three novel ensemble classification models, namely Ensemble SVM, Ensemble RF, and Ensemble XGBoost, were developed to predict carcinogenicity of chemicals using seven types of molecular fingerprints and three machine learning methods based on a dataset containing 1003 diverse compounds with rat carcinogenicity. Among these three models, Ensemble XGBoost is found to be the best, giving an average accuracy of 70.1 ± 2.9%, sensitivity of 67.0 ± 5.0%, and specificity of 73.1 ± 4.4% in five-fold cross-validation and an accuracy of 70.0%, sensitivity of 65.2%, and specificity of 76.5% in external validation. In comparison with some recent methods, the ensemble models outperform some machine learning-based approaches and yield equal accuracy and higher specificity but lower sensitivity than rule-based expert systems. It is also found that the ensemble models could be further improved if more data were available. As an application, the ensemble models are employed to discover potential carcinogens in the DrugBank database. The results indicate that the proposed models are helpful in predicting the carcinogenicity of chemicals. A web server called CarcinoPred-EL has been built for these models ( http://ccsipb.lnu.edu.cn/toxicity/CarcinoPred-EL/ ).

A simplified protocol for molecular identification of Eimeria species in field samples.

PubMed

Haug, Anita; Thebo, Per; Mattsson, Jens G

2007-05-15

This study aimed to find a fast, sensitive and efficient protocol for molecular identification of chicken Eimeria spp. in field samples. Various methods for each of the three steps of the protocol were evaluated: oocyst wall rupturing methods, DNA extraction methods, and identification of species-specific DNA sequences by PCR. We then compared and evaluated five complete protocols. Three series of oocyst suspensions of known number of oocysts from Eimeria mitis, Eimeria praecox, Eimeria maxima and Eimeria tenella were prepared and ground using glass beads or mini-pestle. DNA was extracted from ruptured oocysts using commercial systems (GeneReleaser, Qiagen Stoolkit and Prepman) or phenol-chloroform DNA extraction, followed by identification of species-specific ITS-1 sequences by optimised single species PCR assays. The Stoolkit and Prepman protocols showed insufficient repeatability, and the former was also expensive and relatively time-consuming. In contrast, both the GeneReleaser protocol and phenol-chloroform protocols were robust and sensitive, detecting less than 0.4 oocysts of each species per PCR. Finally, we evaluated our new protocol on 68 coccidia positive field samples. Our data suggests that rupturing the oocysts by mini-pestle grinding, preparing the DNA with GeneReleaser, followed by optimised single species PCR assays, makes a robust and sensitive procedure for identifying chicken Eimeria species in field samples. Importantly, it also provides minimal hands-on-time in the pre-PCR process, lower contamination risk and no handling of toxic chemicals.

First International External Quality Assessment Study on Molecular and Serological Methods for Yellow Fever Diagnosis

PubMed Central

Domingo, Cristina; Escadafal, Camille; Rumer, Leonid; Méndez, Jairo A.; García, Paquita; Sall, Amadou A.; Teichmann, Anette; Donoso-Mantke, Oliver; Niedrig, Matthias

2012-01-01

Objective We describe an external quality assurance (EQA) study designed to assess the efficiency and accurateness of molecular and serological methods used by expert laboratories performing YF diagnosis. Study Design For molecular diagnosis evaluation, a panel was prepared of 14 human plasma samples containing specific RNA of different YFV strains (YFV-17D, YFV South American strain [Brazil], YFV IvoryC1999 strain), and specificity samples containing other flaviviruses and negative controls. For the serological panel, 13 human plasma samples with anti-YFV-specific antibodies against different strains of YFV (YFV-17D strain, YFV IvoryC1999 strain, and YFV Brazilian strain), as well as specificity and negative controls, were included. Results Thirty-six laboratories from Europe, the Americas, Middle East, and Africa participated in these EQA activities. Only 16% of the analyses reported met all evaluation criteria with optimal performance. Serial dilutions of YFV-17D showed that in general the methodologies reported provided a suitable sensitivity. Failures were mainly due to the inability to detect wild-type strains or the presence of false positives. Performance in the serological diagnosis varied, mainly depending on the methodology used. Anti-YFV IgM detection was not performed in 16% of the reports using IIF or ELISA techniques, although it is preferable for the diagnosis of YFV acute infections. A good sensitivity profile was achieved in general; however, in the detection of IgM antibodies a lack of sensitivity of anti-YFV antibodies against the vaccine strain 17D was observed, and of the anti-YFV IgG antibodies against a West African strain. Neutralization assays showed a very good performance; however, the unexpected presence of false positives underlined the need of improving the running protocols. Conclusion This EQA provides information on each laboratory's efficacy of RT-PCR and serological YFV diagnosis techniques. The results indicate the need for improving serological and molecular diagnosis techniques and provide a follow-up of the diagnostic profiles. PMID:22570700

First international external quality assessment study on molecular and serological methods for yellow fever diagnosis.

PubMed

Domingo, Cristina; Escadafal, Camille; Rumer, Leonid; Méndez, Jairo A; García, Paquita; Sall, Amadou A; Teichmann, Anette; Donoso-Mantke, Oliver; Niedrig, Matthias

2012-01-01

We describe an external quality assurance (EQA) study designed to assess the efficiency and accurateness of molecular and serological methods used by expert laboratories performing YF diagnosis. For molecular diagnosis evaluation, a panel was prepared of 14 human plasma samples containing specific RNA of different YFV strains (YFV-17D, YFV South American strain [Brazil], YFV IvoryC1999 strain), and specificity samples containing other flaviviruses and negative controls. For the serological panel, 13 human plasma samples with anti-YFV-specific antibodies against different strains of YFV (YFV-17D strain, YFV IvoryC1999 strain, and YFV Brazilian strain), as well as specificity and negative controls, were included. Thirty-six laboratories from Europe, the Americas, Middle East, and Africa participated in these EQA activities. Only 16% of the analyses reported met all evaluation criteria with optimal performance. Serial dilutions of YFV-17D showed that in general the methodologies reported provided a suitable sensitivity. Failures were mainly due to the inability to detect wild-type strains or the presence of false positives. Performance in the serological diagnosis varied, mainly depending on the methodology used. Anti-YFV IgM detection was not performed in 16% of the reports using IIF or ELISA techniques, although it is preferable for the diagnosis of YFV acute infections. A good sensitivity profile was achieved in general; however, in the detection of IgM antibodies a lack of sensitivity of anti-YFV antibodies against the vaccine strain 17D was observed, and of the anti-YFV IgG antibodies against a West African strain. Neutralization assays showed a very good performance; however, the unexpected presence of false positives underlined the need of improving the running protocols. This EQA provides information on each laboratory's efficacy of RT-PCR and serological YFV diagnosis techniques. The results indicate the need for improving serological and molecular diagnosis techniques and provide a follow-up of the diagnostic profiles.

A theoretical investigation into the strength of N-NO2 bonds, ring strain and electrostatic potential upon formation of intermolecular H-bonds between HF and the nitro group in nitrogen heterocyclic rings C n H2n N-NO2 (n = 2-5), RDX and HMX.

PubMed

Wang, Bao-Guo; Ren, Fu-de; Shi, Wen-Jing

2015-11-01

Changes in N-NO2 bond strength, ring strain energy and electrostatic potential upon formation of intermolecular H-bonds between HF and the nitro group in nitrogen heterocyclic rings C n H2n N-NO2 (n = 2-5), RDX and HMX were investigated using DFT-B3LYP and MP2(full) methods with the 6-311++G(2df,2p) and aug-cc-pVTZ basis sets. Analysis of electron density shifts was also carried out. The results indicate that H-bonding energy correlates well with the increment of ring strain energy. Upon complex formation, the strength of the N-NO2 trigger-bond is enhanced, suggesting reduced sensitivity, while judged by the increased ring strain energy, sensitivity is increased. However, some features of the molecular surface electrostatic potential, such as a local maximum above the N-NO2 bond and ring, σ + (2) and electrostatic balance parameter ν, remain essentially unchanged upon complex formation, and only a small change in the impact sensitivity h 50 is suggested. It is not sufficient to determine sensitivity solely on the basis of trigger bond or ring strain; as a global feature of a molecule, the molecular surface electrostatic potential is available to help judge the change of sensitivity in H-bonded complexes. Graphical Abstract The strengthened N-NO2 bond suggests reduced sensitivity, while it is reverse by theincreased ring strain energy upon the complex formation. However, the molecular surfaceelectrostatic potential (V S) shows the little change of h 50. The V S should be taken into accountin the analysis of explosive sensitivity in the H-bonded complex.

Molecular dynamics of oligofluorenes: A dielectric spectroscopy investigation

NASA Astrophysics Data System (ADS)

Papadopoulos, P.; Floudas, G.; Chi, C.; Wegner, G.

2004-02-01

The molecular dynamics were investigated in a series of "defect-free" oligofluorenes up to the polymer by dielectric spectroscopy (DS). The method is very sensitive to the presence of keto "defects" that when incorporated on the backbone give rise to poor optical and electronic properties. Two dielectrically active processes were found (β and α process). The latter process (α) displays strongly temperature dependent relaxation times and temperature- and molecular weight-dependent spectral broadening associated with intramolecular correlations. The glass temperature (Tg) obeys the Fox-Flory equation and the polymer Tg is obtained by DS at 332 K. The effective dipole moment associated with the α process is 0.27±0.03 D.

Molecular orbital imaging via above-threshold ionization with circularly polarized pulses.

PubMed

Zhu, Xiaosong; Zhang, Qingbin; Hong, Weiyi; Lu, Peixiang; Xu, Zhizhan

2011-07-18

Above-threshold ionization (ATI) for aligned or orientated linear molecules by circularly polarized laser pulsed is investigated. It is found that the all-round structural information of the molecular orbital is extracted with only one shot by the circularly polarized probe pulse rather than with multi-shot detections in a linearly polarized case. The obtained photoelectron momentum spectrum directly depicts the symmetry and electron distribution of the occupied molecular orbital, which results from the strong sensitivity of the ionization probability to these structural features. Our investigation indicates that the circularly polarized probe scheme would present a simple method to study the angle-dependent ionization and image the occupied electronic orbital.

Molecular Detection of Chilo infuscatellus

PubMed Central

Wang, Weizhong; Wang, Rong; Zheng, Haotian; Gao, Sanji

2017-01-01

Abstract The Chilo infuscatellus (Snellen; Lepidoptera: Pyralidae) is the main pest of sugarcane in China. The shortage of easily distinguishable morphological characters especially in early stage make it challenging to diagnosed and promptly take steps for pest management. In the present study, we described a PCR method for the molecular identification of based on barcode region of COI sequences between C. infuscatellus and four other sugarcane borer species. A 285bp fragment was successfully amplified from all life stages and different geographical populations. Sensitivity tests revealed that diagnostic bands were generated as low as the DNA template concentration of 5 ng/µl. Our work demonstrated a rapid and accurate way for the molecular diagnosis of C. infuscatellus. PMID:29117377

Cavity-Enhanced Spectroscopy of Molecular Ions in the Mid-Infrared with Up-Conversion Detection and Brewster-Plate Spoilers

NASA Astrophysics Data System (ADS)

Markus, Charles R.; McCollum, Jefferson E.; Hodges, James Neil; Perry, Adam J.; McCall, Benjamin J.

2017-06-01

Molecular ions are challenging to study with conventional spectroscopic methods. Laboratory discharges produce ions in trace quantities which can be obscured by the abundant neutral molecules present. The technique Noise Immune Cavity Enhanced Optical Heterodyne Velocity Modulation Spectroscopy (NICE-OHVMS) overcomes these challenges by combining the ion-neutral discrimination of velocity modulation spectroscopy with the sensitivity of Noise-Immune Cavity-Enhanced Optical Heterodyne Molecular Spectroscopy (NICE-OHMS), and has been able to determine transition frequencies of molecular ions in the mid-infrared (mid-IR) with sub-MHz uncertainties when calibrated with an optical frequency comb. However, the extent of these studies was limited by the presence of fringes due to parasitic etalons and the speed and noise characteristics of mid-IR detectors. Recently, we have overcome these limitations by implementing up-conversion detection and dithered optics. We performed up-conversion using periodically poled lithium niobate to convert light from the mid-IR to the visible to be within the coverage of sensitive and fast silicon detectors while maintaining our heterodyne and velocity modulation signals. The parasitic etalons were removed by rapidly rotating CaF_2 windows with galvanometers, which is known as a Brewster-plate spoiler, which averaged out the fringes in detection. Together, these improved the sensitivity by more than an order of magnitude and have enabled extended spectroscopic surveys of molecular ions in the mid-IR. J. N. Hodges, A. J. Perry, P. A. Jenkins II, B. M. Siller, and B. J. McCall, J. Chem. Phys. (2013), 139, 164201. C. R. Webster, J. Opt. Soc. Am. B (1985), 2, 1464. C. R. Markus, A. J. Perry, J. N. Hodges, and B. J. McCall, Opt. Express (2017), 25, 3709-3721.

Quenching of cascade reaction between triplet and photochrome probes with nitroxide radicals. A novel labeling method in study of membranes and surface systems.

PubMed

Papper, V; Medvedeva, N; Fishov, I; Likhtenshtein, G I

2000-01-01

We proposed a new method for the study of molecular dynamics and fluidity of the living and model biomembranes and surface systems. The method is based on the measurements of the sensitized photoisomerization kinetics of a photochrome probe. The cascade triplet cis-trans photoisomerization of the excited stilbene derivative sensitized with the excited triplet Erythrosin B has been studied in a model liposome membrane. The photoisomerization reaction is depressed with nitroxide radicals quenching the excited triplet state of the sensitizer. The enhanced fluorescence polarization of the stilbene probe incorporated into liposome membranes indicates that the stilbene molecules are squeezed in a relatively viscous media of the phospholipids. Calibration of the "triple" cascade system is based on a previously proposed method that allows the measurement of the product of the quenching rate constant and the sensitizer's triplet lifetime, as well as the quantitative detection of the nitroxide radicals in the vicinity of the membrane surface. The experiment was conducted using the constant-illumination fluorescence technique. Sensitivity of the method using a standard commercial spectrofluorimeter is about 10(-12) mol of fluorescence molecules per sample and can be improved using an advanced fluorescence technique. The minimal local concentration of nitroxide radicals or any other quenchers being detected is about 10(-5) M. This method enables the investigation of any chemical and biological surface processes of microscopic scale when the minimal volume is about 10(-3) microL or less.

Evaluation of Molecular Methods for Identification of Salmonella Serovars

PubMed Central

Gurnik, Simone; Ahmad, Aaminah; Blimkie, Travis; Murphy, Stephanie A.; Kropinski, Andrew M.; Nash, John H. E.

2016-01-01

Classification by serotyping is the essential first step in the characterization of Salmonella isolates and is important for surveillance, source tracking, and outbreak detection. To improve detection and reduce the burden of salmonellosis, several rapid and high-throughput molecular Salmonella serotyping methods have been developed. The aim of this study was to compare three commercial kits, Salm SeroGen (Salm Sero-Genotyping AS-1 kit), Check&Trace (Check-Points), and xMAP (xMAP Salmonella serotyping assay), to the Salmonella genoserotyping array (SGSA) developed by our laboratory. They were assessed using a panel of 321 isolates that represent commonly reported serovars from human and nonhuman sources globally. The four methods correctly identified 73.8% to 94.7% of the isolates tested. The methods correctly identified 85% and 98% of the clinically important Salmonella serovars Enteritidis and Typhimurium, respectively. The methods correctly identified 75% to 100% of the nontyphoidal, broad host range Salmonella serovars, including Heidelberg, Hadar, Infantis, Kentucky, Montevideo, Newport, and Virchow. The sensitivity and specificity of Salmonella serovars Typhimurium and Enteritidis ranged from 85% to 100% and 99% to 100%, respectively. It is anticipated that whole-genome sequencing will replace serotyping in public health laboratories in the future. However, at present, it is approximately three times more expensive than molecular methods. Until consistent standards and methodologies are deployed for whole-genome sequencing, data analysis and interlaboratory comparability remain a challenge. The use of molecular serotyping will provide a valuable high-throughput alternative to traditional serotyping. This comprehensive analysis provides a detailed comparison of commercial kits available for the molecular serotyping of Salmonella. PMID:27194688

Determination of Diffusion Parameters of CO2 Through Microporous PTFE Using a Potentiometric Method

NASA Astrophysics Data System (ADS)

Tarsiche, I.; Ciurchea, D.

Dk values at the diffusion of CO2 through microporous PTFE of 1 to 7 × 10- 7 cm2 s- 1 in the concentration range from 4 × 10- 4 to 0.22 g/l CO2 are determined using a simple, fast and reliable potentiometric method. The method is based on the least-squares fitting of the potential versus time response of a self made CO2 sensitive Severinghaus type sensor with PTFE as a gas-permeable membrane. The obtained results are in good agreement with other reported literature data, both experimental or calculated ones using molecular dynamics simulations. The proposed technique is very sensitive especially at low concentrations of gas and may be used for the study of other polymeric membranes too.

Molecularly imprinted solid-phase extraction combined with electrochemical oxidation fluorimetry for the determination of methotrexate in human serum and urine

NASA Astrophysics Data System (ADS)

Chen, Suming; Zhang, Zhujun

2008-06-01

The method of synthesis and evaluation of molecularly imprinted polymers was reported. As a selective solid-phase extraction sorbent, the polymers were coupled with electrochemical fluorimetry detection for the efficient determination of methotrexate in serum and urine. Methotrexate was preconcentrated in the molecularly imprinted solid-phase extraction microcolumn packed with molecularly imprinted polymers, and then eluted. The eluate was detected by fluorescence spectrophotometer after electrochemical oxidation. The conditions of preconcentration, elution, electrochemical oxidation and determination were carefully studied. Under the selected experimental conditions, the calibration graph of the fluorescence intensity versus methotrexate concentration was linear from 4 × 10 -9 g mL -1 to 5 × 10 -7 g mL -1, and the detection limit was 8.2 × 10 -10 g mL -1 (3 σ). The relative standard deviation was 3.92% ( n = 7) for 1 × 10 -7 g mL -1 methotrexate. The experiments showed that the selectivity and sensitivity of fluorimetry could be greatly improved by the proposed method. This method has been successfully applied to the determination of methotrexate. At the same time, the binding characteristics of the polymers to the methotrexate were evaluated by batch and dynamic methods.

Peptide kinetics from picoseconds to microseconds using boxed molecular dynamics: Power law rate coefficients in cyclisation reactions

NASA Astrophysics Data System (ADS)

Shalashilin, Dmitrii V.; Beddard, Godfrey S.; Paci, Emanuele; Glowacki, David R.

2012-10-01

Molecular dynamics (MD) methods are increasingly widespread, but simulation of rare events in complex molecular systems remains a challenge. We recently introduced the boxed molecular dynamics (BXD) method, which accelerates rare events, and simultaneously provides both kinetic and thermodynamic information. We illustrate how the BXD method may be used to obtain high-resolution kinetic data from explicit MD simulations, spanning picoseconds to microseconds. The method is applied to investigate the loop formation dynamics and kinetics of cyclisation for a range of polypeptides, and recovers a power law dependence of the instantaneous rate coefficient over six orders of magnitude in time, in good agreement with experimental observations. Analysis of our BXD results shows that this power law behaviour arises when there is a broad and nearly uniform spectrum of reaction rate coefficients. For the systems investigated in this work, where the free energy surfaces have relatively small barriers, the kinetics is very sensitive to the initial conditions: strongly non-equilibrium conditions give rise to power law kinetics, while equilibrium initial conditions result in a rate coefficient with only a weak dependence on time. These results suggest that BXD may offer us a powerful and general algorithm for describing kinetics and thermodynamics in chemical and biochemical systems.

Peptidylation for the determination of low-molecular-weight compounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Tang, Feng; Cen, Si-Ying; He, Huan; Liu, Yi; Yuan, Bi-Feng; Feng, Yu-Qi

2016-05-23

Determination of low-molecular-weight compounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been a great challenge in the analytical research field. Here we developed a universal peptide-based derivatization (peptidylation) strategy for the sensitive analysis of low-molecular-weight compounds by MALDI-TOF-MS. Upon peptidylation, the molecular weights of target analytes increase, thus avoiding serious matrix ion interference in the low-molecular-weight region in MALDI-TOF-MS. Since peptides typically exhibit good signal response during MALDI-TOF-MS analysis, peptidylation endows high detection sensitivities of low-molecular-weight analytes. As a proof-of-concept, we analyzed low-molecular-weight compounds of aldehydes and thiols by the developed peptidylation strategy. Our results showed that aldehydes and thiols can be readily determined upon peptidylation, thus realizing the sensitive and efficient determination of low-molecular-weight compounds by MALDI-TOF-MS. Moreover, target analytes also can be unambiguously detected in biological samples using the peptidylation strategy. The established peptidylation strategy is a universal strategy and can be extended to the sensitive analysis of various low-molecular-weight compounds by MALDI-TOF-MS, which may be potentially used in areas such as metabolomics.

Improved HF183 reverse primer and probe for greater analytical sensitivity of human Bacteroides in the environment

EPA Science Inventory

Background: Numerous indicators have been used to assess the presence of fecal pollution, many relying on molecular methods such as qPCR. One of the targets frequently used, the human-associated Bacteroides 16s rRNA region, has several assays in current usage. These assays vary...

Complex molecular assemblies at hand via interactive simulations.

PubMed

Delalande, Olivier; Férey, Nicolas; Grasseau, Gilles; Baaden, Marc

2009-11-30

Studying complex molecular assemblies interactively is becoming an increasingly appealing approach to molecular modeling. Here we focus on interactive molecular dynamics (IMD) as a textbook example for interactive simulation methods. Such simulations can be useful in exploring and generating hypotheses about the structural and mechanical aspects of biomolecular interactions. For the first time, we carry out low-resolution coarse-grain IMD simulations. Such simplified modeling methods currently appear to be more suitable for interactive experiments and represent a well-balanced compromise between an important gain in computational speed versus a moderate loss in modeling accuracy compared to higher resolution all-atom simulations. This is particularly useful for initial exploration and hypothesis development for rare molecular interaction events. We evaluate which applications are currently feasible using molecular assemblies from 1900 to over 300,000 particles. Three biochemical systems are discussed: the guanylate kinase (GK) enzyme, the outer membrane protease T and the soluble N-ethylmaleimide-sensitive factor attachment protein receptors complex involved in membrane fusion. We induce large conformational changes, carry out interactive docking experiments, probe lipid-protein interactions and are able to sense the mechanical properties of a molecular model. Furthermore, such interactive simulations facilitate exploration of modeling parameters for method improvement. For the purpose of these simulations, we have developed a freely available software library called MDDriver. It uses the IMD protocol from NAMD and facilitates the implementation and application of interactive simulations. With MDDriver it becomes very easy to render any particle-based molecular simulation engine interactive. Here we use its implementation in the Gromacs software as an example. Copyright 2009 Wiley Periodicals, Inc.

Optically stimulated slowing of polar heavy-atom molecules with a constant beat phase

NASA Astrophysics Data System (ADS)

Yin, Yanning; Xu, Supeng; Xia, Meng; Xia, Yong; Yin, Jianping

2018-04-01

Polar heavy-atom molecules have been well recognized as promising candidates for precision measurements and tests of fundamental physics. A much slower molecular beam to increase the interaction time should lead to a more sensitive measurement. Here we theoretically demonstrate the possibility of the stimulated longitudinal slowing of heavy-atom molecules by the coherent optical bichromatic force with a constant beat phase. Taking the YbF meolecule as an example, we show that a rapid and short-distance deceleration of heavy molecules by a phase-compensation method is feasible with moderate conditions. A molecular beam of YbF with a forward velocity of 120 m/s can be decelerated below 10 m/s within a distance of 3.5 cm and with a laser irradiance for each traveling wave of 107.2 W/cm 2 . Our proposed slowing method could be a promising approach to break through the space constraint or the limited capture efficiency of molecules loadable into a magneto-optical trap in traditional deceleration schemes, opening the possibility for a significant improvement of the precision measurement sensitivity.

Mesoporous structured MIPs@CDs fluorescence sensor for highly sensitive detection of TNT.

PubMed

Xu, Shoufang; Lu, Hongzhi

2016-11-15

A facile strategy was developed to prepare mesoporous structured molecularly imprinted polymers capped carbon dots (M-MIPs@CDs) fluorescence sensor for highly sensitive and selective determination of TNT. The strategy using amino-CDs directly as "functional monomer" for imprinting simplify the imprinting process and provide well recognition sites accessibility. The as-prepared M-MIPs@CDs sensor, using periodic mesoporous silica as imprinting matrix, and amino-CDs directly as "functional monomer", exhibited excellent selectivity and sensitivity toward TNT with detection limit of 17nM. The recycling process was sustainable for 10 times without obvious efficiency decrease. The feasibility of the developed method in real samples was successfully evaluated through the analysis of TNT in soil and water samples with satisfactory recoveries of 88.6-95.7%. The method proposed in this work was proved to be a convenient and practical way to prepare high sensitive and selective fluorescence MIPs@CDs sensors. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular sensitivity threshold of wet mount and an immunochromatographic assay evaluated by quantitative real-time PCR for diagnosis of Trichomonas vaginalis infection in a low-risk population of childbearing women.

PubMed

Leli, Christian; Castronari, Roberto; Levorato, Lucia; Luciano, Eugenio; Pistoni, Eleonora; Perito, Stefano; Bozza, Silvia; Mencacci, Antonella

2016-06-01

Vaginal trichomoniasis is a sexually transmitted infection caused by Trichomonas vaginalis, a flagellated protozoan. Diagnosis of T. vaginalis infection is mainly performed by wet mount microscopy, with a sensitivity ranging from 38% to 82%, compared to culture, still considered the gold standard. Commercial immunochromatographic tests for monoclonal-antibody-based detection have been introduced as alternative methods for diagnosis of T. vaginalis infection and have been reported in some studies to be more sensitive than wet mount. Real-time PCR methods have been recently developed, with optimal sensitivity and specificity. The aim of this study was to evaluate whether there is a molecular sensitivity threshold for both wet mount and imunochromatographic assays. To this aim, a total of 1487 low-risk childbearing women (median age 32 years, interquartile range 27-37) were included in the study, and underwent vaginal swab for T. vaginalis detection by means of a quantitative real-time PCR assay, wet mount and an immunochromatographic test. Upon comparing the results, prevalence values observed were 1.3% for real-time PCR, 0.5% for microscopic examination, and 0.8% for the immunochromatographic test. Compared to real-time PCR, wet mount sensitivity was 40% (95% confidence interval 19.1% to 63.9%) and specificity was 100% (95% CI 99.7% to 100%). The sensitivity and specificity of the immunochromatographic assay were 57.9% (95% CI 33.5% to 79.8%) and 99.9% (95% CI 99.6% to 100%), respectively. Evaluation of the wet mount results and those of immunochromatographic assay detection in relation to the number of T. vaginalis DNA copies detected in vaginal samples showed that the lower identification threshold for both wet mount (chi-square 6.1; P = 0.016) and the immunochromatographic assay (chi-square 10.7; P = 0.002) was ≥100 copies of T. vaginalis DNA/5 mcl of eluted DNA.

BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks.

PubMed

Yan, Winston X; Mirzazadeh, Reza; Garnerone, Silvano; Scott, David; Schneider, Martin W; Kallas, Tomasz; Custodio, Joaquin; Wernersson, Erik; Li, Yinqing; Gao, Linyi; Federova, Yana; Zetsche, Bernd; Zhang, Feng; Bienko, Magda; Crosetto, Nicola

2017-05-12

Precisely measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality. Here we present Breaks Labeling In Situ and Sequencing (BLISS), featuring the following: (1) direct labelling of DSBs in fixed cells or tissue sections on a solid surface; (2) low-input requirement by linear amplification of tagged DSBs by in vitro transcription; (3) quantification of DSBs through unique molecular identifiers; and (4) easy scalability and multiplexing. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells and liver tissue. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. Our results establish BLISS as a versatile, sensitive and efficient method for genome-wide DSB mapping in many applications.

Comparison of real-time PCR methods for the detection of Naegleria fowleri in surface water and sediment.

PubMed

Streby, Ashleigh; Mull, Bonnie J; Levy, Karen; Hill, Vincent R

2015-05-01

Naegleria fowleri is a thermophilic free-living ameba found in freshwater environments worldwide. It is the cause of a rare but potentially fatal disease in humans known as primary amebic meningoencephalitis. Established N. fowleri detection methods rely on conventional culture techniques and morphological examination followed by molecular testing. Multiple alternative real-time PCR assays have been published for rapid detection of Naegleria spp. and N. fowleri. Foursuch assays were evaluated for the detection of N. fowleri from surface water and sediment. The assays were compared for thermodynamic stability, analytical sensitivity and specificity, detection limits, humic acid inhibition effects, and performance with seeded environmental matrices. Twenty-one ameba isolates were included in the DNA panel used for analytical sensitivity and specificity analyses. N. fowleri genotypes I and III were used for method performance testing. Two of the real-time PCR assays were determined to yield similar performance data for specificity and sensitivity for detecting N. fowleri in environmental matrices.

Comparison of real-time PCR methods for the detection of Naegleria fowleri in surface water and sediment

PubMed Central

Streby, Ashleigh; Mull, Bonnie J.; Levy, Karen

2015-01-01

Naegleria fowleri is a thermophilic free-living ameba found in freshwater environments worldwide. It is the cause of a rare but potentially fatal disease in humans known as primary amebic meningoencephalitis. Established N. fowleri detection methods rely on conventional culture techniques and morphological examination followed by molecular testing. Multiple alternative real-time PCR assays have been published for rapid detection of Naegleria spp. and N. fowleri. Four such assays were evaluated for the detection of N. fowleri from surface water and sediment. The assays were compared for thermodynamic stability, analytical sensitivity and specificity, detection limits, humic acid inhibition effects, and performance with seeded environmental matrices. Twenty-one ameba isolates were included in the DNA panel used for analytical sensitivity and specificity analyses. N. fowleri genotypes I and III were used for method performance testing. Two of the real-time PCR assays were determined to yield similar performance data for specificity and sensitivity for detecting N. fowleri in environmental matrices. PMID:25855343

Determination of low molecular weight alcohols including fusel oil in various samples by diethyl ether extraction and capillary gas chromatography.

PubMed

Woo, Kang-Lyung

2005-01-01

Low molecular weight alcohols including fusel oil were determined using diethyl ether extraction and capillary gas chromatography. Twelve kinds of alcohols were successfully resolved on the HP-FFAP (polyethylene glycol) capillary column. The diethyl ether extraction method was very useful for the analysis of alcohols in alcoholic beverages and biological samples with excellent cleanliness of the resulting chromatograms and high sensitivity compared to the direct injection method. Calibration graphs for all standard alcohols showed good linearity in the concentration range used, 0.001-2% (w/v) for all alcohols. Salting out effects were significant (p < 0.01) for the low molecular weight alcohols methanol, isopropanol, propanol, 2-butanol, n-butanol and ethanol, but not for the relatively high molecular weight alcohols amyl alcohol, isoamyl alcohol, and heptanol. The coefficients of variation of the relative molar responses were less than 5% for all of the alcohols. The limits of detection and quantitation were 1-5 and 10-60 microg/L for the diethyl ether extraction method, and 10-50 and 100-350 microg/L for the direct injection method, respectively. The retention times and relative retention times of standard alcohols were significantly shifted in the direct injection method when the injection volumes were changed, even with the same analysis conditions, but they were not influenced in the diethyl ether extraction method. The recoveries by the diethyl ether extraction method were greater than 95% for all samples and greater than 97% for biological samples.

Evaluation of the sensitizing potential of antibiotics in vitro using the human cell lines THP-1 and MUTZ-LC and primary monocyte‐derived dendritic cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sebastian, Katrin, E-mail: ksebastian@ukaachen.de; Ott, Hagen; Zwadlo-Klarwasser, Gabriele

Since the 7th amendment to the EU cosmetics directive foresees a complete ban on animal testing, alternative in vitro methods have been established to evaluate the sensitizing potential of small molecular weight compounds. To find out whether these novel in vitro assays are also capable to predict the sensitizing potential of small molecular weight drugs, model compounds such as beta-lactams and sulfonamides – which are the most frequent cause of adverse drug reactions – were co-incubated with THP-1, MUTZ-LC, or primary monocyte‐derived dendritic cells for 48 h and subsequent expression of selected marker genes (IL-8, IL-1β, CES1, NQO1, GCLM, PIRmore » and TRIM16) was studied by real time PCR. Benzylpenicillin and phenoxymethylpenicillin were recognized as sensitizing compounds because they are capable to induce the mRNA expression of these genes in moDCs and, except for IL-8, in THP-1 cells but not in MUTZ-LC. Ampicillin stimulated the expression of some marker genes in moDCs and THP-1 cells. SMX did not affect the expression of these genes in THP-1, however, in moDCs, at least PIR was enhanced and there was an increase of the release of IL-8. These data reveal that novel in vitro DC based assays might play a role in the evaluation of the allergenic potential of novel drug compounds, but these systems seem to lack the ability to detect the sensitizing potential of prohaptens that require metabolic activation prior to sensitization and moDCs seem to be superior with regard to the sensitivity compared with THP-1 and MUTZ-3 cell lines. -- Highlights: ► We tested the sensitizing potential of small molecular weight drugs in vitro. ► In vitro assays were performed with moDCs and THP-1 cells. ► Beta-lactam antibiotics can be recognized as sensitizing compounds. ► They affect the expression of metabolic enzymes, cytokines and transcription factors. ► Sulfamethoxazole has no measurable effect on THP-1 cells and moDCs.« less

Method for preparation and readout of polyatomic molecules in single quantum states

NASA Astrophysics Data System (ADS)

Patterson, David

2018-03-01

Polyatomic molecular ions contain many desirable attributes of a useful quantum system, including rich internal degrees of freedom and highly controllable coupling to the environment. To date, the vast majority of state-specific experimental work on molecular ions has concentrated on diatomic species. The ability to prepare and read out polyatomic molecules in single quantum states would enable diverse experimental avenues not available with diatomics, including new applications in precision measurement, sensitive chemical and chiral analysis at the single-molecule level, and precise studies of Hz-level molecular tunneling dynamics. While cooling the motional state of a polyatomic ion via sympathetic cooling with a laser-cooled atomic ion is straightforward, coupling this motional state to the internal state of the molecule has proven challenging. Here we propose a method for readout and projective measurement of the internal state of a trapped polyatomic ion. The method exploits the rich manifold of technically accessible rotational states in the molecule to realize robust state preparation and readout with far less stringent engineering than quantum logic methods recently demonstrated on diatomic molecules. The method can be applied to any reasonably small (≲10 atoms) polyatomic ion with an anisotropic polarizability.

Lowering the Spectral Detection Threshold for Molecular Impurities in Gas Mixtures by Interference Multiplexing

NASA Astrophysics Data System (ADS)

Ivanov, M. P.; Tolmachev, Yu. A.

2018-05-01

We consider the most feasible ways to significantly improve the sensitivity of spectroscopic methods for detection and measurement of trace concentrations of greenhouse gas molecules in the atmosphere. The proposed methods are based on combining light fluxes from a number of spectral components of the specified molecule on the same photodetector, taking into account the characteristic features of the transmission spectrum of devices utilizing multipath interference effects.

LAMPhimerus: A novel LAMP assay for detecting Amphimerus sp. DNA in human stool samples

PubMed Central

Calvopiña, Manuel; Fontecha-Cuenca, Cristina; Sugiyama, Hiromu; Sato, Megumi; López Abán, Julio; Vicente, Belén; Muro, Antonio

2017-01-01

Background Amphimeriasis is a fish-borne disease caused by the liver fluke Amphimerus spp. that has recently been reported as endemic in the tropical Pacific side of Ecuador with a high prevalence in humans and domestic animals. The diagnosis is based on the stool examination to identify parasite eggs, but it lacks sensitivity. Additionally, the morphology of the eggs may be confounded with other liver and intestinal flukes. No immunological or molecular methods have been developed to date. New diagnostic techniques for specific and sensitive detection of Amphimerus spp. DNA in clinical samples are needed. Methodology/Principal findings A LAMP targeting a sequence of the Amphimerus sp. internal transcribed spacer 2 region was designed. Amphimerus sp. DNA was obtained from adult worms recovered from animals and used to optimize the molecular assays. Conventional PCR was performed using outer primers F3-B3 to verify the proper amplification of the Amphimerus sp. DNA target sequence. LAMP was optimized using different reaction mixtures and temperatures, and it was finally set up as LAMPhimerus. The specificity and sensitivity of both PCR and LAMP were evaluated. The detection limit was 1 pg of genomic DNA. Field testing was done using 44 human stool samples collected from localities where fluke is endemic. Twenty-five samples were microscopy positive for Amphimerus sp. eggs detection. In molecular testing, PCR F3-B3 was ineffective when DNA from fecal samples was used. When testing all human stool samples included in our study, the diagnostic parameters for the sensitivity and specificity were calculated for our LAMPhimerus assay, which were 76.67% and 80.77%, respectively. Conclusions/Significance We have developed and evaluated, for the first time, a specific and sensitive LAMP assay for detecting Amphimerus sp. in human stool samples. The procedure has been named LAMPhimerus method and has the potential to be adapted for field diagnosis and disease surveillance in amphimeriasis-endemic areas. Future large-scale studies will assess the applicability of this novel LAMP assay. PMID:28628614

Clinical identification of bacteria in human chronic wound infections: culturing vs. 16S ribosomal DNA sequencing

PubMed Central

2012-01-01

Background Chronic wounds affect millions of people and cost billions of dollars in the United States each year. These wounds harbor polymicrobial biofilm communities, which can be difficult to elucidate using culturing methods. Clinical molecular microbiological methods are increasingly being employed to investigate the microbiota of chronic infections, including wounds, as part of standard patient care. However, molecular testing is more sensitive than culturing, which results in markedly different results being reported to clinicians. This study compares the results of aerobic culturing and molecular testing (culture-free 16S ribosomal DNA sequencing), and it examines the relative abundance score that is generated by the molecular test and the usefulness of the relative abundance score in predicting the likelihood that the same organism would be detected by culture. Methods Parallel samples from 51 chronic wounds were studied using aerobic culturing and 16S DNA sequencing for the identification of bacteria. Results One hundred forty-five (145) unique genera were identified using molecular methods, and 68 of these genera were aerotolerant. Fourteen (14) unique genera were identified using aerobic culture methods. One-third (31/92) of the cultures were determined to be < 1% of the relative abundance of the wound microbiota using molecular testing. At the genus level, molecular testing identified 85% (78/92) of the bacteria that were identified by culture. Conversely, culturing detected 15.7% (78/497) of the aerotolerant bacteria and detected 54.9% of the collective aerotolerant relative abundance of the samples. Aerotolerant bacterial genera (and individual species including Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis) with higher relative abundance scores were more likely to be detected by culture as demonstrated with regression modeling. Conclusion Discordance between molecular and culture testing is often observed. However, culture-free 16S ribosomal DNA sequencing and its relative abundance score can provide clinicians with insight into which bacteria are most abundant in a sample and which are most likely to be detected by culture. PMID:23176603

Tuning Piezo ion channels to detect molecular-scale movements relevant for fine touch

PubMed Central

Poole, Kate; Herget, Regina; Lapatsina, Liudmila; Ngo, Ha-Duong; Lewin, Gary R.

2014-01-01

In sensory neurons, mechanotransduction is sensitive, fast and requires mechanosensitive ion channels. Here we develop a new method to directly monitor mechanotransduction at defined regions of the cell-substrate interface. We show that molecular-scale (~13 nm) displacements are sufficient to gate mechanosensitive currents in mouse touch receptors. Using neurons from knockout mice, we show that displacement thresholds increase by one order of magnitude in the absence of stomatin-like protein 3 (STOML3). Piezo1 is the founding member of a class of mammalian stretch-activated ion channels, and we show that STOML3, but not other stomatin-domain proteins, brings the activation threshold for Piezo1 and Piezo2 currents down to ~10 nm. Structure–function experiments localize the Piezo modulatory activity of STOML3 to the stomatin domain, and higher-order scaffolds are a prerequisite for function. STOML3 is the first potent modulator of Piezo channels that tunes the sensitivity of mechanically gated channels to detect molecular-scale stimuli relevant for fine touch. PMID:24662763

Photothermal optical coherence tomography of epidermal growth factor receptor in live cells using immunotargeted gold nanospheres

NASA Astrophysics Data System (ADS)

Skala, Melissa C.; Crow, Matthew J.; Wax, Adam; Izatt, Joseph A.

2009-02-01

Molecular imaging is a powerful tool for investigating disease processes and potential therapies in both in vivo and in vitro systems. However, high resolution molecular imaging has been limited to relatively shallow penetration depths that can be accessed with microscopy. Optical coherence tomography (OCT) is an optical analogue to ultrasound with relatively good penetration depth (1-2 mm) and resolution (~1-10 μm). We have developed and characterized photothermal OCT as a molecular contrast mechanism that allows for high resolution molecular imaging at deeper penetration depths than microscopy. Our photothermal system consists of an amplitude-modulated heating beam that spatially overlaps with the focused spot of the sample arm of a spectral-domain OCT microscope. Validation experiments in tissue-like phantoms containing gold nanospheres that absorb at 532 nm revealed a sensitivity of 14 parts per million nanospheres (weight/weight) in a tissue-like environment. The nanospheres were then conjugated to anti-EGFR, and molecular targeting was confirmed in cells that over-express EGFR (MDA-MB-468) and cells that express low levels of EGFR (MDA-MB-435). Molecular imaging in three-dimensional tissue constructs was confirmed with a significantly lower photothermal signal (p<0.0001) from the constructs composed of cells that express low levels of EGFR compared to the over-expressing cell constructs (300% signal increase). This technique could potentially augment confocal and multiphoton microscopy as a method for deep-tissue, depth-resolved molecular imaging with relatively high resolution and target sensitivity, without photobleaching or cytotoxicity.

Theoretical insights into the structures and mechanical properties of HMX/NQ cocrystal explosives and their complexes, and the influence of molecular ratios on their bonding energies.

PubMed

Li, Yong-Xiang; Chen, Shu-Sen; Ren, Fu-de

2015-09-01

Molecular dynamics (MD) methods were employed to study the binding energies and mechanical properties of selected crystal planes of 1,3,5,7-tetranitro-1,3,5,7-tetrazacyclooctane (HMX)/nitroguanidine (NQ) cocrystals at different molecular molar ratios. The densities and detonation velocities of the cocrystals at different molar ratios were estimated. The intermolecular interaction and bond dissociation energy (BDE) of the N-NO2 bond in the HMX:NQ (1:1) complex were calculated using the B3LYP, MP2(full) and M06-2X methods with the 6-311++G(d,p) and 6-311++G(2df,2p) basis sets. The results indicated that the HMX/NQ cocrystal prefers cocrystalizing in a 1:1 molar ratio, and the cocrystallization is dominated by the (0 2 0) and (1 0 0) facets. The K, G, and E values of the ratio of 1:1 are smaller than those of the other ratios, and the 1:1 cocrystal has the best ductility. The N-NO2 bond becomes stronger upon the formation of the intermolecular H-bonding interaction and the sensitivity of HMX decreases in the cocrystal. This sensitivity change in the HMX/NQ cocrystal originates not only from the formation of the intermolecular interaction but also from the increment of the BDE of N-NO2 bond in comparison with isolated HMX. The HMX/NQ (1:1) cocrystal exhibits good detonation performance. Reduced density gradient (RDG) reveals the nature of cocrystallization. Analysis of the surface electrostatic potential further confirmed that the sensitivity decreases in complex (or cocrystal) in comparison with that in isolated HMX.

A molecular gram stain using broad range PCR and pyrosequencing technology: a potentially useful tool for diagnosing orthopaedic infections.

PubMed

Kobayashi, Naomi; Bauer, Thomas W; Togawa, Daisuke; Lieberman, Isador H; Sakai, Hiroshige; Fujishiro, Takaaki; Tuohy, Marion J; Procop, Gary W

2005-06-01

The bacteria associated with orthopaedic infections are usually common gram-positive and gram-negative bacteria. This fundamental grouping of bacteria is a necessary first step in the selection of appropriate antibiotics. Since polymerase chain reaction (PCR) is more rapid and may be more sensitive than culture, we developed a postamplification pyrosequencing method to subcategorize bacteria based on a few nucleotide polymorphisms in the 16S rRNA gene. We validated this method using well-characterized strains of bacteria and applied it to specimens from spinal surgery cases with suspected infections. Lysates of 114 bacteria including 75 species were created following standard cultivation to obtain DNA. The DNA was amplified by a broad-range real-time PCR. The amplicons were evaluated by pyrosequencing and were classified as gram-positive, gram-negative, or acid-fast bacilli based on the first three to five nucleotides sequenced. In addition, clinical cases of suspected infection were obtained from spinal surgery. The results of the "molecular Gram stain" were compared with the results of traditional Gram stain and culture. The lysates of 107 (93.9%) of the bacteria extracts tested were appropriately categorized as gram-positive and gram-negative or as acid-fast bacilli on the basis of this assay. The sensitivity and specificity of this assay were 100% and 97.4% for gram-positive and 88.3% and 100% for gram-negative isolates. All of the five clinical samples were appropriately categorized as containing gram-positive or gram-negative bacteria with this assay. This study demonstrates that high sensitivity and specificity of a molecular gram stain may be achieved using broad-range real-time PCR and pyrosequencing.

Fungal biodiversity in the periglacial soil of Dosdè Glacier (Valtellina, Northern Italy).

PubMed

Rodolfi, Marinella; Longa, Claudia Maria Oliveira; Pertot, Ilaria; Tosi, Solveig; Savino, Elena; Guglielminetti, Maria; Altobelli, Elisa; Del Frate, Giuseppe; Picco, Anna Maria

2016-03-01

Periglacial areas are one of the least studied habitats on Earth, especially in terms of their fungal communities. In this work, both molecular and culture-dependent methods have been used to analyse the microfungi in soils sampled on the front of the East Dosdè Glacier (Valtellina, Northern Italy). Although this survey revealed a community that was rich in fungal species, a distinct group of psychrophilic microfungi has not been detected. Most of the isolated microfungi were mesophiles, which are well adapted to the sensitive climatic changes that occur in this alpine environment. A discrepancy in the results that were obtained by means of the two diagnostic approaches suggests that the used molecular methods cannot entirely replace traditional culture-dependent methods, and vice versa. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of ALK gene rearrangement in central nervous system metastases of non-small-cell lung cancer using two-step RT-PCR technique.

PubMed

Nicoś, M; Krawczyk, P; Wojas-Krawczyk, K; Bożyk, A; Jarosz, B; Sawicki, M; Trojanowski, T; Milanowski, J

2017-12-01

RT-PCR technique has showed a promising value as pre-screening method for detection of mRNA containing abnormal ALK sequences, but its sensitivity and specificity is still discussable. Previously, we determined the incidence of ALK rearrangement in CNS metastases of NSCLC using IHC and FISH methods. We evaluated ALK gene rearrangement using two-step RT-PCR method with EML4-ALK Fusion Gene Detection Kit (Entrogen, USA). The studied group included 145 patients (45 females, 100 males) with CNS metastases of NSCLC and was heterogeneous in terms of histology and smoking status. 21% of CNS metastases of NSCLC (30/145) showed presence of mRNA containing abnormal ALK sequences. FISH and IHC tests confirmed the presence of ALK gene rearrangement and expression of ALK abnormal protein in seven patients with positive result of RT-PCR analysis (4.8% of all patients, 20% of RT-PCR positive patients). RT-PCR method compared to FISH analysis achieved 100% of sensitivity and only 82.7% of specificity. IHC method compared to FISH method indicated 100% of sensitivity and 97.8% of specificity. In comparison to IHC, RT-PCR showed identical sensitivity with high number of false positive results. Utility of RT-PCR technique in screening of ALK abnormalities and in qualification patients for molecularly targeted therapies needs further validation.

Comparison of Five Assays for Detection of Clostridium difficile Toxin

PubMed Central

Chapin, Kimberle C.; Dickenson, Roberta A.; Wu, Fongman; Andrea, Sarah B.

2011-01-01

Performance characteristics of five assays for detection of Clostridium difficile toxin were compared using fresh stool samples from patients with C. difficile infection (CDI). Assays were performed simultaneously and according to the manufacturers' instructions. Patients were included in the study if they exhibited clinical symptoms consistent with CDI. Nonmolecular assays included glutamate dehydrogenase antigen tests, with positive findings followed by the Premier Toxin A and B Enzyme Immunoassay (GDH/EIA), and the C. Diff Quik Chek Complete test. Molecular assays (PCR) included the BD GeneOhm Cdiff Assay, the Xpert C. difficile test, and the ProGastro Cd assay. Specimens were considered true positive if results were positive in two or more assays. For each method, the Youden index was calculated and cost-effectiveness was analyzed. Of 81 patients evaluated, 26 (32.1%) were positive for CDI. Sensitivity of the BD GeneOhm Cdiff assay, the Xpert C. difficile test, the ProGastro Cd assay, C. Diff Quik Chek Complete test, and two-step GDH/EIA was 96.2%, 96.2%, 88.5%, 61.5%, and 42.3%, respectively. Specificity of the Xpert C. difficile test was 96.4%, and for the other four assays was 100%. Compared with nonmolecular methods, molecular methods detected 34.7% more positive specimens. Assessment of performance characteristics and cost-effectiveness demonstrated that the BD GeneOhm Cdiff assay yielded the best results. While costly, the Xpert C. difficile test required limited processing and yielded rapid results. Because of discordant results, specimen processing, and extraction equipment requirements, the ProGastro Cd assay was the least favored molecular assay. The GDH/EIA method lacked sufficient sensitivity to be recommended. PMID:21704273

Theoretical Insight into the Influences of Molecular Ratios on Stabilities and Mechanical Properties, Solvent Effect of HMX/FOX-7 Cocrystal Explosive

NASA Astrophysics Data System (ADS)

Wei, Yan-Ju; Ren, Fu-De; Shi, Wen-Jing; Zhao, Qi

2016-10-01

A molecular dynamics method was employed to study the binding energies of the selected crystal planes of the 1,3,5,7-tetranitro-1,3,5,7-tetrazacyclooctane/1,1-diamino-2,2-dinitroethylene (HMX/FOX-7) cocrystal in different molecular molar ratios. Mechanical properties, densities, and detonation velocities of the cocrystals in different ratios were estimated. The intermolecular interactions and bond dissociation energies (BDEs) of the N-NO2 bond in the HMX:FOX-7 (1:1) complex were calculated using the B3LYP and MP2(full) methods at the 6-311++G (d,p) and 6-311++G(2df,2p) basis sets. Solvent effects on stability are discussed. The results indicate that HMX/FOX-7 cocrystals prefer cocrystalizing in a 1:1 molar ratio, which has good mechanical properties. The N-NO2 bond becomes strong upon the formation of a complex and the sensitivity of HMX might decrease in cocrystals. The sensitivity change of HMX/FOX-7 originates from not only the formation of intermolecular interaction but also the increment in the N-NO2 BDE. HMX/FOX-7 cocrystals exhibit good detonation performance and meet the requirements of high-density energetic materials. Solvents with low dielectric constants may be chosen to obtain stable HMX/FOX-7 cocrystals.

Exploring the Surface Sensitivity of ToF-SIMS by Measuring the Implantation and Sampling Depths of Bin and C60 Ions in Organic Films

PubMed Central

Muramoto, Shin; Brison, Jeremy; Castner, David G.

2011-01-01

The surface sensitivity of Binq+ (n = 1, 3, 5, q = 1, 2) and C60q+ (q = 1, 2) primary ions in static time-of-flight secondary ion mass spectrometry (ToF-SIMS) experiments were investigated for molecular trehalose and polymeric tetraglyme organic films. Parameters related to surface sensitivity (impact crater depth, implantation depth, and molecular escape depths) were measured. Under static ToF-SIMS conditions (primary ion doses of 1 × 1012 ions/cm2), the 25 keV Bi1+ primary ions were the most surface sensitive with a molecular escape depth of 1.8 nm for protein films with tetraglyme overlayers, but they had the deepest implantation depth (~18 and 26 nm in trehalose and tetraglyme films, respectively). The 20 keV C60++ primary ions were the second most surface sensitive with a slightly larger molecular escape depth of 2.3 nm. The most important factor that determined the surface sensitivity of the primary ion was its impact crater depth, or the amount of surface erosion. The most surface sensitive primary ions, Bi1+ and C60++, created impact craters with depths of 0.3 and 1.0 nm, respectively, in tetraglyme films. In contrast, Bi5++ primary ions created impact craters with a depth of 1.8 nm in tetraglyme films and were the least surface sensitive with a molecular escape depth of 4.7 nm. PMID:22084828

Detect the sensitivity and response of protein molecular structure of whole canola seed (yellow and brown) to different heat processing methods and relation to protein utilization and availability using ATR-FT/IR molecular spectroscopy with chemometrics.

PubMed

Samadi; Theodoridou, Katerina; Yu, Peiqiang

2013-03-15

The objectives of this experiment were to detect the sensitivity and response of protein molecular structure of whole canola seed to different heat processing [moisture (autoclaving) vs. dry (roasting) heating] and quantify heat-induced protein molecular structure changes in relation to protein utilization and availability. In this study, whole canola seeds were autoclaved (moisture heating) and dry (roasting) heated at 120 °C for 1h, respectively. The parameters assessed included changes in (1) chemical composition profile, (2) CNCPS protein subfractions (PA, PB1, PB2, PB3, PC), (3) intestinal absorbed true protein supply, (4) energy values, and (5) protein molecular structures (amide I, amide II, ratio of amide I to II, α-helix, β-sheet, ratio of α-helix to β-sheet). The results showed that autoclave heating significantly decreased (P<0.05) but dry heating increased (P<0.05) the ratio of protein α-helix to β-sheet (with the ratios of 1.07, 0.95, 1.10 for the control (raw), autoclave heating and dry heating, respectively). The multivariate molecular spectral analyses (PCA, CLA) showed that there were significantly molecular structural differences in the protein amide I and II fingerprint region (ca. 1714-1480 cm(-1)) among the control, autoclave and dry heating. These differences were indicated by the form of separate class (PCA) and group of separate ellipse (CLA) between the treatments. The correlation analysis with spearman method showed that there were significantly and highly positive correlation (P<0.05) between heat-induced protein molecular structure changes in terms of α-helix to β-sheet ratios and in situ protein degradation and significantly negative correlation between the protein α-helix to β-sheet ratios and intestinal digestibility of undegraded protein. The results indicated that heat-induced changes of protein molecular structure revealed by vibration molecular spectroscopy could be used as a potential predictor to protein degradation and intestinal protein digestion of whole canola seed. Future study is needed to study response and impact of heat processing to each inherent layer of canola seed from outside to inside tissues and between yellow canola and brown canola. Copyright © 2012 Elsevier B.V. All rights reserved.

Clustering the Orion B giant molecular cloud based on its molecular emission.

PubMed

Bron, Emeric; Daudon, Chloé; Pety, Jérôme; Levrier, François; Gerin, Maryvonne; Gratier, Pierre; Orkisz, Jan H; Guzman, Viviana; Bardeau, Sébastien; Goicoechea, Javier R; Liszt, Harvey; Öberg, Karin; Peretto, Nicolas; Sievers, Albrecht; Tremblin, Pascal

2018-02-01

Previous attempts at segmenting molecular line maps of molecular clouds have focused on using position-position-velocity data cubes of a single molecular line to separate the spatial components of the cloud. In contrast, wide field spectral imaging over a large spectral bandwidth in the (sub)mm domain now allows one to combine multiple molecular tracers to understand the different physical and chemical phases that constitute giant molecular clouds (GMCs). We aim at using multiple tracers (sensitive to different physical processes and conditions) to segment a molecular cloud into physically/chemically similar regions (rather than spatially connected components), thus disentangling the different physical/chemical phases present in the cloud. We use a machine learning clustering method, namely the Meanshift algorithm, to cluster pixels with similar molecular emission, ignoring spatial information. Clusters are defined around each maximum of the multidimensional Probability Density Function (PDF) of the line integrated intensities. Simple radiative transfer models were used to interpret the astrophysical information uncovered by the clustering analysis. A clustering analysis based only on the J = 1 - 0 lines of three isotopologues of CO proves suffcient to reveal distinct density/column density regimes ( n H ~ 100 cm -3 , ~ 500 cm -3 , and > 1000 cm -3 ), closely related to the usual definitions of diffuse, translucent and high-column-density regions. Adding two UV-sensitive tracers, the J = 1 - 0 line of HCO + and the N = 1 - 0 line of CN, allows us to distinguish two clearly distinct chemical regimes, characteristic of UV-illuminated and UV-shielded gas. The UV-illuminated regime shows overbright HCO + and CN emission, which we relate to a photochemical enrichment effect. We also find a tail of high CN/HCO + intensity ratio in UV-illuminated regions. Finer distinctions in density classes ( n H ~ 7 × 10 3 cm -3 ~ 4 × 10 4 cm -3 ) for the densest regions are also identified, likely related to the higher critical density of the CN and HCO + (1 - 0) lines. These distinctions are only possible because the high-density regions are spatially resolved. Molecules are versatile tracers of GMCs because their line intensities bear the signature of the physics and chemistry at play in the gas. The association of simultaneous multi-line, wide-field mapping and powerful machine learning methods such as the Meanshift clustering algorithm reveals how to decode the complex information available in these molecular tracers.

Supersonic molecular beam-hyperthermal surface ionisation coupled with time-of-flight mass spectrometry applied to trace level detection of polynuclear aromatic hydrocarbons in drinking water for reduced sample preparation and analysis time.

PubMed

Davis, S C; Makarov, A A; Hughes, J D

1999-01-01

Analysis of sub-ppb levels of polynuclear aromatic hydrocarbons (PAHs) in drinking water by high performance liquid chromatography (HPLC) fluorescence detection typically requires large water samples and lengthy extraction procedures. The detection itself, although selective, does not give compound identity confirmation. Benchtop gas chromatography/mass spectrometry (GC/MS) systems operating in the more sensitive selected ion monitoring (SIM) acquisition mode discard spectral information and, when operating in scanning mode, are less sensitive and scan too slowly. The selectivity of hyperthermal surface ionisation (HSI), the high column flow rate capacity of the supersonic molecular beam (SMB) GC/MS interface, and the high acquisition rate of time-of-flight (TOF) mass analysis, are combined here to facilitate a rapid, specific and sensitive technique for the analysis of trace levels of PAHs in water. This work reports the advantages gained by using the GC/HSI-TOF system over the HPLC fluorescence method, and discusses in some detail the nature of the instrumentation used.

Determination of amantadine and rimantadine using a sensitive fluorescent probe

NASA Astrophysics Data System (ADS)

Wang, Guang-Quan; Qin, Yan-Fang; Du, Li-Ming; Li, Jun-Fei; Jing, Xu; Chang, Yin-Xia; Wu, Hao

2012-12-01

Amantadine hydrochloride (AMA) and rimantadine hydrochloride (RIM) are non-fluorescent in aqueous solutions. This property makes their determination through direct fluorescent method difficult. The competing reactions and the supramolecular interaction mechanisms between the two drugs and coptisine (COP) as they fight for occupancy of the cucurbit[7]uril (CB[7]) cavity, were studied using spectrofluorimetry, 1H NMR, and molecular modeling calculations. Based on the significant quenching of the supramolecular complex fluorescence intensity, a fluorescent probe method of high sensitivity and selectivity was developed to determine AMA or RIM in their pharmaceutical dosage forms and in urine samples with good precision and accuracy. The linear range of the method was from 0.0040 to 1.0 μg mL-1 with a detection limit ranging from 0.0012 to 0.0013 μg mL-1. This shows that the proposed method has promising potential for therapeutic monitoring and pharmacokinetics and for clinical application.

The use of biochemical methods in extraterrestrial life detection

NASA Astrophysics Data System (ADS)

McDonald, Gene

2006-08-01

Instrument development for in situ extraterrestrial life detection focuses primarily on the ability to distinguish between biological and non-biological material, mostly through chemical analysis for potential biosignatures (e.g., biogenic minerals, enantiomeric excesses). In constrast, biochemical analysis techniques commonly applied to Earth life focus primarily on the exploration of cellular and molecular processes, not on the classification of a given system as biological or non-biological. This focus has developed because of the relatively large functional gap between life and non-life on Earth today. Life on Earth is very diverse from an environmental and physiological point of view, but is highly conserved from a molecular point of view. Biochemical analysis techniques take advantage of this similarity of all terrestrial life at the molecular level, particularly through the use of biologically-derived reagents (e.g., DNA polymerases, antibodies), to enable analytical methods with enormous sensitivity and selectivity. These capabilities encourage consideration of such reagents and methods for use in extraterrestrial life detection instruments. The utility of this approach depends in large part on the (unknown at this time) degree of molecular compositional differences between extraterrestrial and terrestrial life. The greater these differences, the less useful laboratory biochemical techniques will be without significant modification. Biochemistry and molecular biology methods may need to be "de-focused" in order to produce instruments capable of unambiguously detecting a sufficiently wide range of extraterrestrial biochemical systems. Modern biotechnology tools may make that possible in some cases.

Highly sensitive and selective hyphenated technique (molecularly imprinted polymer solid-phase microextraction-molecularly imprinted polymer sensor) for ultra trace analysis of aspartic acid enantiomers.

PubMed

Prasad, Bhim Bali; Srivastava, Amrita; Tiwari, Mahavir Prasad

2013-03-29

The present work is related to combination of molecularly imprinted solid-phase microextraction and complementary molecularly imprinted polymer-sensor. The molecularly imprinted polymer grafted on titanium dioxide modified silica fiber was used for microextraction, while the same polymer immobilized on multiwalled carbon nanotubes/titanium dioxide modified pencil graphite electrode served as a detection tool. In both cases, the surface initiated polymerization was found to be advantageous to obtain a nanometer thin imprinted film. The modified silica fiber exhibited high adsorption capacity and enantioselective diffusion of aspartic acid isomers into respective molecular cavities. This combination enabled double preconcentrations of d- and l-aspartic acid that helped sensing both isomers in real samples, without any cross-selectivity and matrix complications. Taking into account 6×10(4)-fold dilution of serum and 2×10(3)-fold dilution of cerebrospinal fluid required by the proposed method, the limit of detection for l-aspartic acid is 0.031ngmL(-1). Also, taking into account 50-fold dilution required by the proposed method, the limit of detection for d-aspartic acid is 0.031ngmL(-1) in cerebrospinal fluid. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular recognition by gold, silver and copper nanoparticles

PubMed Central

Tauran, Yannick; Brioude, Arnaud; Coleman, Anthony W; Rhimi, Moez; Kim, Beonjoom

2013-01-01

The intrinsic physical properties of the noble metal nanoparticles, which are highly sensitive to the nature of their local molecular environment, make such systems ideal for the detection of molecular recognition events. The current review describes the state of the art concerning molecular recognition of Noble metal nanoparticles. In the first part the preparation of such nanoparticles is discussed along with methods of capping and stabilization. A brief discussion of the three common methods of functionalization: Electrostatic adsorption; Chemisorption; Affinity-based coordination is given. In the second section a discussion of the optical and electrical properties of nanoparticles is given to aid the reader in understanding the use of such properties in molecular recognition. In the main section the various types of capping agents for molecular recognition; nucleic acid coatings, protein coatings and molecules from the family of supramolecular chemistry are described along with their numerous applications. Emphasis for the nucleic acids is on complementary oligonucleotide and aptamer recognition. For the proteins the recognition properties of antibodies form the core of the section. With respect to the supramolecular systems the cyclodextrins, calix[n]arenes, dendrimers, crown ethers and the cucurbitales are treated in depth. Finally a short section deals with the possible toxicity of the nanoparticles, a concern in public health. PMID:23977421

Microscopic lymph node tumor burden quantified by macroscopic dual-tracer molecular imaging

PubMed Central

Tichauer, Kenneth M.; Samkoe, Kimberley S.; Gunn, Jason R.; Kanick, Stephen C.; Hoopes, P. Jack; Barth, Richard J.; Kaufman, Peter A.; Hasan, Tayyaba; Pogue, Brian W.

2014-01-01

Lymph node biopsy (LNB) is employed in many cancer surgeries to identify metastatic disease and stage the cancer, yet morbidity and diagnostic delays associated with LNB could be avoided if non-invasive imaging of nodal involvement was reliable. Molecular imaging has potential in this regard; however, variable delivery and nonspecific uptake of imaging tracers has made conventional approaches ineffective clinically. A method of correcting for non-specific uptake with injection of a second untargeted tracer is presented, allowing tumor burden in lymph nodes to be quantified. The approach was confirmed in an athymic mouse model of metastatic human breast cancer targeting epidermal growth factor receptor, a cell surface receptor overexpressed by many cancers. A significant correlation was observed between in vivo (dual-tracer) and ex vivo measures of tumor burden (r = 0.97, p < 0.01), with an ultimate sensitivity of approximately 200 cells (potentially more sensitive than conventional LNB). PMID:25344739

Sensitive and specific identification by polymerase chain reaction of Eimeria tenella and Eimeria maxima, important protozoan pathogens in laboratory avian facilities.

PubMed

Lee, Hyun-A; Hong, Sunhwa; Chung, Yungho; Kim, Okjin

2011-09-01

Eimeria tenella and Eimeria maxima are important pathogens causing intracellular protozoa infections in laboratory avian animals and are known to affect experimental results obtained from contaminated animals. This study aimed to find a fast, sensitive, and efficient protocol for the molecular identification of E. tenella and E. maxima in experimental samples using chickens as laboratory avian animals. DNA was extracted from fecal samples collected from chickens and polymerase chain reaction (PCR) analysis was employed to detect E. tenella and E. maxima from the extracted DNA. The target nucleic acid fragments were specifically amplified by PCR. Feces secreting E. tenella and E. maxima were detected by a positive PCR reaction. In this study, we were able to successfully detect E. tenella and E. maxima using the molecular diagnostic method of PCR. As such, we recommended PCR for monitoring E. tenella and E. maxima in laboratory avian facilities.

High-throughput molecular identification of Staphylococcus spp. isolated from a clean room facility in an environmental monitoring program

PubMed Central

2010-01-01

Background The staphylococci are one of the most common environmental isolates found in clean room facility. Consequently, isolation followed by comprehensive and accurate identification is an essential step in any environmental monitoring program. Findings We have used the API Staph identification kit (bioMérieux, France) which depends on the expression of metabolic activities and or morphological features to identify the Staphylococcus isolates. The API staphylococci showed low sensitivity in the identification of some species, so we performed molecular methods based on PCR based fingerprinting of glyceraldehyde-3-phosphate dehydrogenase encoding gene as useful taxonomic tool for examining Staphylococcus isolates. Conclusions Our results showed that PCR protocol used in this study which depends on genotypic features was relatively accurate, rapid, sensitive and superior in the identification of at least 7 species of Staphylococcus than API Staph which depends on phenotypic features. PMID:21047438

High-throughput molecular identification of Staphylococcus spp. isolated from a clean room facility in an environmental monitoring program.

PubMed

Sheraba, Norhan S; Yassin, Aymen S; Amin, Magdy A

2010-11-04

The staphylococci are one of the most common environmental isolates found in clean room facility. Consequently, isolation followed by comprehensive and accurate identification is an essential step in any environmental monitoring program. We have used the API Staph identification kit (bioMérieux, France) which depends on the expression of metabolic activities and or morphological features to identify the Staphylococcus isolates. The API staphylococci showed low sensitivity in the identification of some species, so we performed molecular methods based on PCR based fingerprinting of glyceraldehyde-3-phosphate dehydrogenase encoding gene as useful taxonomic tool for examining Staphylococcus isolates. Our results showed that PCR protocol used in this study which depends on genotypic features was relatively accurate, rapid, sensitive and superior in the identification of at least 7 species of Staphylococcus than API Staph which depends on phenotypic features.

Molecular identification of Malassezia species isolated from dermatitis affections.

PubMed

Affes, M; Ben Salah, S; Makni, F; Sellami, H; Ayadi, A

2009-05-01

The lipophilic yeast of the genus Malassezia are opportunistic microorganisms of the skin microflora but they can be agents of various dermatomycoses. The aim of this study was to perform molecular identification of the commonly isolated Malassezia species from various dermatomycoses in our region. Thirty strains of Malassezia were isolated from different dermatologic affections: pityriasis versicolor (17), dandruff (5), seborrheic dermatitis (4), onyxis (2), folliculitis (1) and blepharitis (1). These species were identified by their morphological features and biochemical characterisation. The molecular identification was achieved by amplification of the internal transcribed spacer region by simple PCR. PCR technique was used for molecular characterisation of four Malassezia species: Malassezia globosa (270 bp), Malassezia furfur (230 bp), Malassezia sympodialis (190 bp) and Malassezia restricta (320 bp). We have detected the association between M. furfur and M. sympodialis in 16% and confirmed presumptive identification in 70% of the cases. The phenotypic identification based on microscopic and physiological method is difficult and time consuming. The application of a simple PCR method provides a sensitive and rapid identification system for Malassezia species, which may be applied in epidemiological surveys and routine practice.

Determination of phospholipids in soybean lecithin samples via the phosphorus monoxide molecule by high-resolution continuum source graphite furnace molecular absorption spectrometry.

PubMed

Pires, Laís N; Brandão, Geovani C; Teixeira, Leonardo S G

2017-06-15

This paper presents a method for determining phospholipids in soybean lecithin samples by phosphorus determination using high-resolution continuum source graphite furnace molecular absorption spectrometry (HR-CS GF MAS) via molecular absorption of phosphorus monoxide. Samples were diluted in methyl isobutyl ketone. The best conditions were found to be 213.561nm with a pyrolysis temperature of 1300°C, a volatilization temperature of 2300°C and Mg as a chemical modifier. To increase the analytical sensitivity, measurement of the absorbance signal was obtained by summing molecular transition lines for PO surrounding 213nm: 213.561, 213.526, 213.617 and 213.637nm. The limit of detection was 2.35mgg -1 and the precision, evaluated as relative standard deviation (RSD), was 2.47% (n=10) for a sample containing 2.2% (w/v) phosphorus. The developed method was applied for the analysis of commercial samples of soybean lecithin. The determined concentrations of phospholipids in the samples varied between 38.1 and 45% (w/v). Copyright © 2017 Elsevier Ltd. All rights reserved.

Scalable and fast heterogeneous molecular simulation with predictive parallelization schemes

NASA Astrophysics Data System (ADS)

Guzman, Horacio V.; Junghans, Christoph; Kremer, Kurt; Stuehn, Torsten

2017-11-01

Multiscale and inhomogeneous molecular systems are challenging topics in the field of molecular simulation. In particular, modeling biological systems in the context of multiscale simulations and exploring material properties are driving a permanent development of new simulation methods and optimization algorithms. In computational terms, those methods require parallelization schemes that make a productive use of computational resources for each simulation and from its genesis. Here, we introduce the heterogeneous domain decomposition approach, which is a combination of an heterogeneity-sensitive spatial domain decomposition with an a priori rearrangement of subdomain walls. Within this approach, the theoretical modeling and scaling laws for the force computation time are proposed and studied as a function of the number of particles and the spatial resolution ratio. We also show the new approach capabilities, by comparing it to both static domain decomposition algorithms and dynamic load-balancing schemes. Specifically, two representative molecular systems have been simulated and compared to the heterogeneous domain decomposition proposed in this work. These two systems comprise an adaptive resolution simulation of a biomolecule solvated in water and a phase-separated binary Lennard-Jones fluid.

Gastric Cancer Cells in Peritoneal Lavage Fluid: A Systematic Review Comparing Cytological with Molecular Detection for Diagnosis of Peritoneal Metastases and Prediction of Peritoneal Recurrences.

PubMed

Virgilio, Edoardo; Giarnieri, Enrico; Giovagnoli, Maria Rosaria; Montagnini, Monica; Proietti, Antonella; D'Urso, Rosaria; Mercantini, Paolo; Valabrega, Stefano; Balducci, Genoveffa; Cavallini, Marco

2018-03-01

Detecting free tumor cells in the peritoneal lavage fluid of gastric cancer patients permits to assess a more accurate prognosis, predict peritoneal recurrence and select cases for a more aggressive treatment. Currently, cytology and molecular biology comprise the two most popular methods of detection that are under constant study by researchers. We burrowed into the available literature comparing cytological with molecular detection of free intraperitoneal gastric cancer cells. PubMed, Science Direct, Scopus and Google Scholar were the search engines investigated. As of 2017, 51 dedicated studies have been published. Messenger RNA of carcinoembryonic antigen was the genetic target most frequently described. The genetic technique is usually superior to cytology in sensitivity (38-100% vs. 12.3-67% respectively), whereas cytological examination tends to show a slight pre-eminence in specificity (approximately 100%). So far, given the imperfection of each method, employment of both cytology and molecular examination seem to be mandatory. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Flexible Lab-Tailored Cut-Offs for Suitability of Formalin-Fixed Tumor Samples for Diagnostic Mutational Analyses

PubMed Central

Mariani, Sara; Tondat, Fabrizio; Pacchioni, Donatella; Molinaro, Luca; Barreca, Antonella; Macrì, Luigia; Chiusa, Luigi; di Celle, Paola Francia; Cassoni, Paola; Sapino, Anna

2015-01-01

The selection of proper tissues from formalin-fixed and paraffin-embedded tumors before diagnostic molecular testing is responsibility of the pathologist and represents a crucial step to produce reliable test results. The international guidelines suggest two cut-offs, one for the percentage and one for the number of tumor cells, in order to enrich the tumor content before DNA extraction. The aim of the present work was two-fold: to evaluate to what extent a low percentage or absolute number of tumor cells can be qualified for somatic mutation testing; and to determine how assay sensitivities can guide pathologists towards a better definition of morphology-based adequacy cut-offs. We tested 1797 tumor specimens from melanomas, colorectal and lung adenocarcinomas. Respectively, their BRAF, K-RAS and EGFR genes were analyzed at specific exons by mutation-enriched PCR, pyrosequencing, direct sequencing and real-time PCR methods. We demonstrate that poorly cellular specimens do not modify the frequency distribution of either mutated or wild-type DNA samples nor that of specific mutations. This observation suggests that currently recommended cut-offs for adequacy of specimens to be processed for molecular assays seem to be too much stringent in a laboratory context that performs highly sensitive routine analytical methods. In conclusion, new cut-offs are needed based on test sensitivities and documented tumor heterogeneity. PMID:25844806

Enzyme-free and label-free ultra-sensitive colorimetric detection of Pb(2+) using molecular beacon and DNAzyme based amplification strategy.

PubMed

Yun, Wen; Cai, Dingzhou; Jiang, JiaoLai; Zhao, Pengxiang; Huang, Yu; Sang, Ge

2016-06-15

An enzyme-free and label-free colorimetric Pb(2+) sensor based on DNAzyme and molecular beacon (MB) has been developed and demonstrated by recycle using enzyme strand for signal amplification. The substrate strand DNA (S-DNA) of DNAzyme could be converted into MB structure with base pairs of stem part at the both ends. The MB could hybridize with enzyme strand DNA (E-DNA) to form DNAzyme, and be activated and cleaved in the presence of Pb(2+). The cleaved MB is much less stable, releasing from the DNAzyme as two product pieces. The product pieces of MB are flexible and could bind to unmodified AuNPs to effectively stabilize them against salt-induced aggregation. Then, the E-DNA is liberated to catalyze the next reaction and amplify the response signal. By taking advantage of repeated using of E-DNA, our proposed method exhibited high sensitive for Pb(2+) detection in a linear range from 0.05 to 5 nM with detection limit of 20 pM by UV-vis spectrometer. Moreover, this method was also used for determination of Pb(2+) in river water samples with satisfying results. Importantly, this strategy could reach high sensitivity without any modification and complex enzymatic or hairpins based amplification procedures. Copyright © 2016 Elsevier B.V. All rights reserved.

Fluorescence background subtraction technique for hybrid fluorescence molecular tomography/x-ray computed tomography imaging of a mouse model of early stage lung cancer.

PubMed

Ale, Angelique; Ermolayev, Vladimir; Deliolanis, Nikolaos C; Ntziachristos, Vasilis

2013-05-01

The ability to visualize early stage lung cancer is important in the study of biomarkers and targeting agents that could lead to earlier diagnosis. The recent development of hybrid free-space 360-deg fluorescence molecular tomography (FMT) and x-ray computed tomography (XCT) imaging yields a superior optical imaging modality for three-dimensional small animal fluorescence imaging over stand-alone optical systems. Imaging accuracy was improved by using XCT information in the fluorescence reconstruction method. Despite this progress, the detection sensitivity of targeted fluorescence agents remains limited by nonspecific background accumulation of the fluorochrome employed, which complicates early detection of murine cancers. Therefore we examine whether x-ray CT information and bulk fluorescence detection can be combined to increase detection sensitivity. Correspondingly, we research the performance of a data-driven fluorescence background estimator employed for subtraction of background fluorescence from acquisition data. Using mice containing known fluorochromes ex vivo, we demonstrate the reduction of background signals from reconstructed images and sensitivity improvements. Finally, by applying the method to in vivo data from K-ras transgenic mice developing lung cancer, we find small tumors at an early stage compared with reconstructions performed using raw data. We conclude with the benefits of employing fluorescence subtraction in hybrid FMT-XCT for early detection studies.

A MURINE MODEL FOR LOW MOLECULAR WEIGHT CHEMICALS: DIFFERENTIATION OF RESPIRATORY SENSITIZERS (TMA) FROM CONTACT SENSITIZERS (DNFB)

EPA Science Inventory

Exposure to low molecular weight (LMW) chemicals contributes to both dermal and respiratory sensitization and is an important occupational health problem. Our goal was to establish an in vivo murine model for hazard identification of LMW chemicals that have the potential to indu...

Sensitivities of ionic explosives

NASA Astrophysics Data System (ADS)

Politzer, Peter; Lane, Pat; Murray, Jane S.

2017-03-01

We have investigated the relevance for ionic explosive sensitivity of three factors that have been demonstrated to be related to the sensitivities of molecular explosives. These are (1) the maximum available heat of detonation, (2) the amount of free space per molecule (or per formula unit) in the crystal lattice and (3) specific features of the electrostatic potential on the molecular or ionic surface. We find that for ionic explosives, just as for molecular ones, there is an overall tendency for impact sensitivity to increase as the maximum detonation heat release is greater. This means that the usual emphasis upon designing explosives with large heats of detonation needs to be tempered somewhat. We also show that a moderate detonation heat release does not preclude a high level of detonation performance for ionic explosives, as was already demonstrated for molecular ones. Relating the free space per formula unit to sensitivity may require a modified procedure for ionic explosives; this will continue to be investigated. Finally, an encouraging start has been made in linking impact sensitivities to the electrostatic potentials on ionic surfaces, although limited so far to ammonium salts.

Diagnostic accuracy of quantitative real-time PCR assay versus clinical and Gram stain identification of bacterial vaginosis.

PubMed

Menard, J-P; Mazouni, C; Fenollar, F; Raoult, D; Boubli, L; Bretelle, F

2010-12-01

The purpose of this investigation was to determine the diagnostic accuracy of quantitative real-time polymerase chain reaction (PCR) assay in diagnosing bacterial vaginosis versus the standard methods, the Amsel criteria and the Nugent score. The Amsel criteria, the Nugent score, and results from the molecular tool were obtained independently from vaginal samples of 163 pregnant women who reported abnormal vaginal symptoms before 20 weeks gestation. To determine the performance of the molecular tool, we calculated the kappa value, sensitivity, specificity, and positive and negative predictive values. Either or both of the Amsel criteria (≥3 criteria) and the Nugent score (score ≥7) indicated that 25 women (15%) had bacterial vaginosis, and the remaining 138 women did not. DNA levels of Gardnerella vaginalis or Atopobium vaginae exceeded 10(9) copies/mL or 10(8) copies/mL, respectively, in 34 (21%) of the 163 samples. Complete agreement between both reference methods and high concentrations of G. vaginalis and A. vaginae was found in 94.5% of women (154/163 samples, kappa value = 0.81, 95% confidence interval 0.70-0.81). The nine samples with discordant results were categorized as intermediate flora by the Nugent score. The molecular tool predicted bacterial vaginosis with a sensitivity of 100%, a specificity of 93%, a positive predictive value of 73%, and a negative predictive value of 100%. The quantitative real-time PCR assay shows excellent agreement with the results of both reference methods for the diagnosis of bacterial vaginosis.

Simultaneous extraction and determination of phthalate esters in aqueous solution by yolk-shell magnetic mesoporous carbon-molecularly imprinted composites based on solid-phase extraction coupled with gas chromatography-mass spectrometry.

PubMed

Yang, Rui; Liu, Yuxin; Yan, Xiangyang; Liu, Shaomin

2016-12-01

A rapid, sensitive and accurate method for the simultaneous extraction and determination of five types of trace phthalate esters (PAEs) in environmental water and beverage samples using magnetic molecularly imprinted solid-phase extraction (MMIP-SPE) coupled with gas chromatography-mass spectrometry (GC-MS) was developed. A novel type of molecularly imprinted polymers on the surface of yolk-shell magnetic mesoporous carbon (Fe 3 O 4 @void@C-MIPs) was used as an efficient adsorbent for selective adsorption of phthalate esters based on magnetic solid-phase extraction (MSPE). The real samples were first preconcentrated by Fe 3 O 4 @void@C-MIPs, subsequently extracted by eluent and finally determined by GC-MS after magnetic separation. Several variables affecting the extraction efficiency of the analytes, including the type and volume of the elution solvent, amount of adsorbent, extraction time, desorption time and pH of the sample solution, were investigated and optimized. Validation experiments indicated that the developed method presented good linearity (R 2 >0.9961), satisfactory precision (RSD<6.7%), and high recovery (86.1-103.1%). The limits of detection ranged from 1.6ng/L to 5.2ng/L and the enrichment factor was in the range of 822-1423. The results indicated that the novel method had the advantages of convenience, good sensitivity, and high efficiency, and it could also be successfully applied to the analysis of PAEs in real samples. Copyright © 2016. Published by Elsevier B.V.

Elucidation of differences in N-glycosylation between different molecular weight forms of recombinant CLEC-2 by LC MALDI tandem MS.

PubMed

Zhou, Lei; Qian, Yifan; Zhang, Xingwang; Ruan, Yuanyuan; Ren, Shifang; Gu, Jianxin

2015-01-30

C-type lectin-like receptor 2 (CLEC-2) is a newly identified receptor expressed on the platelet surface. It has been reported that CLEC-2 exists as a higher molecular weight (HMW) and a lower molecular weight (LMW) form, which share the same protein core but differ in glycans. The two forms appear to have different ligand-binding abilities, indicating that the differential glycosylation of CLEC-2 possibly produces functionally distinct glycoforms. This study aimed to explore an easy method to directly elucidate the N-glycosylation difference by employing a glycoproteomics approach. The off-line coupling of nano-LC with a MALDI-QIT-TOF mass spectrometer was demonstrated to be capable of sensitive and direct elucidation of the glycosylation difference between HMW and LMW CLEC-2, simultaneously providing information about their oligosaccharide structures and the glycosylation sites. The results reveal that a specific glycosylation site, Asn 134, is differently glycosylated in the two forms, with complex types of bi-antennary, tri-antennary and tetra-antennary, N-linked, fucosylated glycans identified at this site in the HMW form but not in the LMW form. The observed difference in glycosylation might provide new insights into the underlying mechanisms of biological functions of CLEC-2. Because of its simplicity and sensitivity, the method explored in this work suggests that it holds promise as a method of elucidating differences in direct N-glycosylation of target glycoprotein, even in small amount of samples. Copyright © 2014 Elsevier Ltd. All rights reserved.

Experimental and molecular docking investigation on metal-organic framework MIL-101(Cr) as a sorbent for vortex assisted dispersive micro-solid-phase extraction of trace 5-nitroimidazole residues in environmental water samples prior to UPLC-MS/MS analysis.

PubMed

Lu, Nan; Wang, Ting; Zhao, Pan; Zhang, Lianjun; Lun, Xiaowen; Zhang, Xueli; Hou, Xiaohong

2016-11-01

In the presented work, metal-organic framework (MOF) material MIL-101(Cr) (MIL, Matérial Institute Lavoisier) was used as a sorbent for vortex assisted dispersive micro-solid-phase extraction (VA-D-μ-SPE) of trace amount of metronidazole (MNZ), ronidazole (RNZ), secnidazole (SNZ), dimetridazole (DMZ), tinidazole (TNZ), and ornidazole (ONZ) in different environmental water samples. Ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) was used to quantify the target analytes. The extraction conditions, including type of sorbents, amount of MIL-101(Cr), solution pH, extraction method, extraction time, effect of salt, and elution conditions were investigated. Upon the optimal conditions, the developed method showed an excellent extraction performance with the average recovery ranging from 75.2 to 98.8 %. Good sensitivity levels were achieved with the detection limits of 0.03∼0.06 μg/L and the quantitation limits of 0.09∼0.20 μg/L. The linear ranges were varied from 0.1 to 20 for SNZ and ONZ and from 0.2 to 40 μg/L for MNZ, RNZ, DMZ, and TNZ (r 2  > 0.992), and repeatability of the method was satisfactory with the relative standard deviations (RSD) <8 %. Ultimately, the applicability of the method was successfully confirmed by the extraction and determination of 5-nitroimidazoles (5-NDZs) in 12 real water samples, showing the positive findings of MNZ and TNZ ranging from 0.3 to 1.0 μg/L. Furthermore, molecular docking was applied to explain the molecular interactions and free binding energies between MIL-101(Cr) and 5-NDZs, providing a deep insight into the adsorption mechanism. The proposed method exhibited the advantages of simplicity, rapidly, less solvent consumption, ease of operation, higher sensitivity, and lower matrix effect. Graphical abstract Schematic diagram of the extraction process and molecular docking investigation.

Strategies of molecular imprinting-based fluorescence sensors for chemical and biological analysis.

PubMed

Yang, Qian; Li, Jinhua; Wang, Xiaoyan; Peng, Hailong; Xiong, Hua; Chen, Lingxin

2018-07-30

One pressing concern today is to construct sensors that can withstand various disturbances for highly selective and sensitive detecting trace analytes in complicated samples. Molecularly imprinted polymers (MIPs) with tailor-made binding sites are preferred to be recognition elements in sensors for effective targets detection, and fluorescence measurement assists in highly sensitive detection and user-friendly control. Accordingly, molecular imprinting-based fluorescence sensors (MI-FL sensors) have attracted great research interest in many fields such as chemical and biological analysis. Herein, we comprehensively review the recent advances in MI-FL sensors construction and applications, giving insights on sensing principles and signal transduction mechanisms, focusing on general construction strategies for intrinsically fluorescent or nonfluorescent analytes and improvement strategies in sensing performance, particularly in sensitivity. Construction strategies are well overviewed, mainly including the traditional indirect methods of competitive binding against pre-bound fluorescent indicators, employment of fluorescent functional monomers and embedding of fluorescence substances, and novel rational designs of hierarchical architecture (core-shell/hollow and mesoporous structures), post-imprinting modification, and ratiometric fluorescence detection. Furthermore, MI-FL sensor based microdevices are discussed, involving micromotors, test strips and microfluidics, which are more portable for rapid point-of-care detection and in-field diagnosing. Finally, the current challenges and future perspectives of MI-FL sensors are proposed. Copyright © 2018 Elsevier B.V. All rights reserved.

Optical Brain Imaging: A Powerful Tool for Neuroscience.

PubMed

Zhu, Xinpei; Xia, Yanfang; Wang, Xuecen; Si, Ke; Gong, Wei

2017-02-01

As the control center of organisms, the brain remains little understood due to its complexity. Taking advantage of imaging methods, scientists have found an accessible approach to unraveling the mystery of neuroscience. Among these methods, optical imaging techniques are widely used due to their high molecular specificity and single-molecule sensitivity. Here, we overview several optical imaging techniques in neuroscience of recent years, including brain clearing, the micro-optical sectioning tomography system, and deep tissue imaging.

Multiplex PCR for rapid diagnosis and differentiation of pox and pox-like diseases in dromedary Camels.

PubMed

Khalafalla, Abdelmalik I; Al-Busada, Khalid A; El-Sabagh, Ibrahim M

2015-07-07

Pox and pox-like diseases of camels are a group of exanthematous skin conditions that have become increasingly important economically. Three distinct viruses may cause them: camelpox virus (CMLV), camel parapox virus (CPPV) and camelus dromedary papilloma virus (CdPV). These diseases are often difficult to differentiate based on clinical presentation in disease outbreaks. Molecular methods such as PCR targeting species-specific genes have been developed and used to identify these diseases, but not simultaneously in a single tube. Recently, multiplex PCR has gained reputation as a convenient diagnostic method with cost-and timesaving benefits. In the present communication, we describe the development, optimization and validation of a multiplex PCR assay able to detect simultaneously the genome of the three viruses in one single test allowing for rapid and efficient molecular diagnosis. The assay was developed based on the evaluation and combination of published and new primer sets and was validated with viral genomic DNA extracted from known virus strains (n = 14) and DNA extracted from homogenized clinical skin specimens (n = 86). The assay detects correctly the target pathogens by amplification of targeted genes, even in case of co-infection. The method showed high sensitivity, and the specificity was confirmed by PCR-product sequencing. This assay provide rapid, sensitive and specific method for identifying three important viruses in specimens collected from dromedary camels with varying clinical presentations.

Restricted active space calculations of L-edge X-ray absorption spectra: from molecular orbitals to multiplet states.

PubMed

Pinjari, Rahul V; Delcey, Mickaël G; Guo, Meiyuan; Odelius, Michael; Lundberg, Marcus

2014-09-28

The metal L-edge (2p → 3d) X-ray absorption spectra are affected by a number of different interactions: electron-electron repulsion, spin-orbit coupling, and charge transfer between metal and ligands, which makes the simulation of spectra challenging. The core restricted active space (RAS) method is an accurate and flexible approach that can be used to calculate X-ray spectra of a wide range of medium-sized systems without any symmetry constraints. Here, the applicability of the method is tested in detail by simulating three ferric (3d(5)) model systems with well-known electronic structure, viz., atomic Fe(3+), high-spin [FeCl6](3-) with ligand donor bonding, and low-spin [Fe(CN)6](3-) that also has metal backbonding. For these systems, the performance of the core RAS method, which does not require any system-dependent parameters, is comparable to that of the commonly used semi-empirical charge-transfer multiplet model. It handles orbitally degenerate ground states, accurately describes metal-ligand interactions, and includes both single and multiple excitations. The results are sensitive to the choice of orbitals in the active space and this sensitivity can be used to assign spectral features. A method has also been developed to analyze the calculated X-ray spectra using a chemically intuitive molecular orbital picture.

Assessment of real-time PCR method for detection of EGFR mutation using both supernatant and cell pellet of malignant pleural effusion samples from non-small-cell lung cancer patients.

PubMed

Shin, Saeam; Kim, Juwon; Kim, Yoonjung; Cho, Sun-Mi; Lee, Kyung-A

2017-10-26

EGFR mutation is an emerging biomarker for treatment selection in non-small-cell lung cancer (NSCLC) patients. However, optimal mutation detection is hindered by complications associated with the biopsy procedure, tumor heterogeneity and limited sensitivity of test methodology. In this study, we evaluated the diagnostic utility of real-time PCR using malignant pleural effusion samples. A total of 77 pleural fluid samples from 77 NSCLC patients were tested using the cobas EGFR mutation test (Roche Molecular Systems). Pleural fluid was centrifuged, and separated cell pellets and supernatants were tested in parallel. Results were compared with Sanger sequencing and/or peptide nucleic acid (PNA)-mediated PCR clamping of matched tumor tissue or pleural fluid samples. All samples showed valid real-time PCR results in one or more DNA samples extracted from cell pellets and supernatants. Compared with other molecular methods, the sensitivity of real-time PCR method was 100%. Concordance rate of real-time PCR and Sanger sequencing plus PNA-mediated PCR clamping was 98.7%. We have confirmed that real-time PCR using pleural fluid had a high concordance rate compared to conventional methods, with no failed samples. Our data demonstrated that the parallel real-time PCR testing using supernatant and cell pellet could offer reliable and robust surrogate strategy when tissue is not available.

Improving Sensitivity in Ultrasound Molecular Imaging by Tailoring Contrast Agent Size Distribution: In Vivo Studies

PubMed Central

Streeter, Jason E.; Gessner, Ryan; Miles, Iman; Dayton, Paul A.

2010-01-01

Molecular imaging with ultrasound relies on microbubble contrast agents (MCAs) selectively adhering to a ligand-specific target. Prior studies have shown that only small quantities of microbubbles are retained at their target sites, therefore, enhancing contrast sensitivity to low concentrations of microbubbles is essential to improve molecular imaging techniques. In order to assess the effect of MCA diameter on imaging sensitivity, perfusion and molecular imaging studies were performed with microbubbles of varying size distributions. To assess signal improvement and MCA circulation time as a function of size and concentration, blood perfusion was imaged in rat kidneys using nontargeted size-sorted MCAs with a Siemens Sequoia ultrasound system (Siemans, Mountain View, CA) in cadence pulse sequencing (CPS) mode. Molecular imaging sensitivity improvements were studied with size-sorted αvβ3-targeted bubbles in both fibrosarcoma and R3230 rat tumor models. In perfusion imaging studies, video intensity and contrast persistence was ≈8 times and ≈3 times greater respectively, for “sorted 3-micron” MCAs (diameter, 3.3 ± 1.95 μm) when compared to “unsorted” MCAs (diameter, 0.9 ± 0.45 μm) at low concentrations. In targeted experiments, application of sorted 3-micron MCAs resulted in a ≈20 times video intensity increase over unsorted populations. Tailoring size-distributions results in substantial imaging sensitivity improvement over unsorted populations, which is essential in maximizing sensitivity to small numbers of MCAs for molecular imaging. PMID:20236606

A sensitive chemiluminescence enzyme immunoassay based on molecularly imprinted polymers solid-phase extraction of parathion.

PubMed

Chen, Ge; Jin, Maojun; Du, Pengfei; Zhang, Chan; Cui, Xueyan; Zhang, Yudan; She, Yongxin; Shao, Hua; Jin, Fen; Wang, Shanshan; Zheng, Lufei; Wang, Jing

2017-08-01

The chemiluminescence enzyme immunoassay (CLEIA) method responds differently to various sample matrices because of the matrix effect. In this work, the CLEIA method was coupled with molecularly imprinted polymers (MIPs) synthesized by precipitation polymerization to study the matrix effect. The sample recoveries ranged from 72.62% to 121.89%, with a relative standard deviation (RSD) of 3.74-18.14%.The ratio of the sample matrix-matched standard curve slope rate to the solvent standard curve slope was 1.21, 1.12, 1.17, and 0.85 for apple, rice, orange and cabbage in samples pretreated with the mixture of PSA and C 18 . However, the ratio of sample (apple, rice, orange, and cabbage) matrix-matched standard-MIPs curve slope rate to the solvent standard curve was 1.05, 0.92, 1.09, and 1.05 in samples pretreated with MIPs, respectively. The results demonstrated that the matrices of the samples greatly interfered with the detection of parathion residues by CLEIA. The MIPs bound specifically to the parathion in the samples and eliminated the matrix interference effect. Therefore, the CLEIA method have successfully applied MIPs in sample pretreatment to eliminate matrix interference effects and provided a new sensitive assay for agro-products. Copyright © 2017 Elsevier Inc. All rights reserved.

The Global Neurological Burden of Tuberculosis.

PubMed

Thakur, Kiran; Das, Mitashee; Dooley, Kelly E; Gupta, Amita

2018-04-01

Central nervous system (CNS) involvement of tuberculosis (TB) is the most severe manifestation of TB and accounts for approximately 5 to 10% of all extrapulmonary TB (EPTB) cases and approximately 1% of all TB cases. TB meningitis (TBM) is the most common form of CNS TB, though other forms occur, often in conjunction with TBM, including intracranial tuberculomas, tuberculous brain abscesses, and spinal tubercular arachnoiditis. CNS TB often presents with nonspecific clinical features that mimic symptoms of other neurological conditions, often making diagnosis difficult. Defining neuroimaging characteristics of TBM include thick basal meningeal enhancement, hydrocephalus, and parenchymal infarctions most commonly involving the basal ganglia and internal capsule. Traditional cerebrospinal fluid sample analysis frequently requires lengthy times-to-result and have low sensitivity. Given the pitfalls of conventional CNS TB diagnostic methods, various molecular-based methods, including immunoassays and polymerase chain reaction (PCR)-based assays have emerged as alternative diagnostic tools due to their rapidity, sensitivity, and specificity. Expert panels on TBM have recently emphasized the need for standard research procedures with updated case definitions and standardized study methods, which will hopefully pave the way for more robust multicenter international studies. In this article, we review the epidemiology, diagnosis, molecular factors associated with disease presentation and outcome, and treatment of CNS TB. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

An intelligent clinical decision support system for patient-specific predictions to improve cervical intraepithelial neoplasia detection.

PubMed

Bountris, Panagiotis; Haritou, Maria; Pouliakis, Abraham; Margari, Niki; Kyrgiou, Maria; Spathis, Aris; Pappas, Asimakis; Panayiotides, Ioannis; Paraskevaidis, Evangelos A; Karakitsos, Petros; Koutsouris, Dimitrios-Dionyssios

2014-01-01

Nowadays, there are molecular biology techniques providing information related to cervical cancer and its cause: the human Papillomavirus (HPV), including DNA microarrays identifying HPV subtypes, mRNA techniques such as nucleic acid based amplification or flow cytometry identifying E6/E7 oncogenes, and immunocytochemistry techniques such as overexpression of p16. Each one of these techniques has its own performance, limitations and advantages, thus a combinatorial approach via computational intelligence methods could exploit the benefits of each method and produce more accurate results. In this article we propose a clinical decision support system (CDSS), composed by artificial neural networks, intelligently combining the results of classic and ancillary techniques for diagnostic accuracy improvement. We evaluated this method on 740 cases with complete series of cytological assessment, molecular tests, and colposcopy examination. The CDSS demonstrated high sensitivity (89.4%), high specificity (97.1%), high positive predictive value (89.4%), and high negative predictive value (97.1%), for detecting cervical intraepithelial neoplasia grade 2 or worse (CIN2+). In comparison to the tests involved in this study and their combinations, the CDSS produced the most balanced results in terms of sensitivity, specificity, PPV, and NPV. The proposed system may reduce the referral rate for colposcopy and guide personalised management and therapeutic interventions.

An Intelligent Clinical Decision Support System for Patient-Specific Predictions to Improve Cervical Intraepithelial Neoplasia Detection

PubMed Central

Bountris, Panagiotis; Haritou, Maria; Pouliakis, Abraham; Margari, Niki; Kyrgiou, Maria; Spathis, Aris; Pappas, Asimakis; Panayiotides, Ioannis; Paraskevaidis, Evangelos A.; Karakitsos, Petros; Koutsouris, Dimitrios-Dionyssios

2014-01-01

Nowadays, there are molecular biology techniques providing information related to cervical cancer and its cause: the human Papillomavirus (HPV), including DNA microarrays identifying HPV subtypes, mRNA techniques such as nucleic acid based amplification or flow cytometry identifying E6/E7 oncogenes, and immunocytochemistry techniques such as overexpression of p16. Each one of these techniques has its own performance, limitations and advantages, thus a combinatorial approach via computational intelligence methods could exploit the benefits of each method and produce more accurate results. In this article we propose a clinical decision support system (CDSS), composed by artificial neural networks, intelligently combining the results of classic and ancillary techniques for diagnostic accuracy improvement. We evaluated this method on 740 cases with complete series of cytological assessment, molecular tests, and colposcopy examination. The CDSS demonstrated high sensitivity (89.4%), high specificity (97.1%), high positive predictive value (89.4%), and high negative predictive value (97.1%), for detecting cervical intraepithelial neoplasia grade 2 or worse (CIN2+). In comparison to the tests involved in this study and their combinations, the CDSS produced the most balanced results in terms of sensitivity, specificity, PPV, and NPV. The proposed system may reduce the referral rate for colposcopy and guide personalised management and therapeutic interventions. PMID:24812614

Is biomedical nuclear magnetic resonance limited by a revisitable paradigm in physics?

PubMed

de Certaines, J D

2005-12-14

The history of nuclear magnetic resonance (NMR) can be divided generally into two phases: before the Second World War, molecular beam methods made it possible to detect the whole set of spins. However, these methods were destructive for the sample and had a very low precision. The publications of F. Bloch and E. Purcell in 1946 opened up a second phase for NMR with the study of condensed matter, but at the expense of an enormous loss in theoretical sensitivity. During more than half a century, the method of Bloch and Purcell, based on inductive detection of the NMR signal, has allowed many developments in biomedicine. But, curiously, this severely constraining limitation on sensitivity has not been called into question during this half-century, as if the pioneers of the pre-war period had been forgotten.

Reversible interactions with para-hydrogen enhance NMR sensitivity by polarization transfer.

PubMed

Adams, Ralph W; Aguilar, Juan A; Atkinson, Kevin D; Cowley, Michael J; Elliott, Paul I P; Duckett, Simon B; Green, Gary G R; Khazal, Iman G; López-Serrano, Joaquín; Williamson, David C

2009-03-27

The sensitivity of both nuclear magnetic resonance spectroscopy and magnetic resonance imaging is very low because the detected signal strength depends on the small population difference between spin states even in high magnetic fields. Hyperpolarization methods can be used to increase this difference and thereby enhance signal strength. This has been achieved previously by incorporating the molecular spin singlet para-hydrogen into hydrogenation reaction products. We show here that a metal complex can facilitate the reversible interaction of para-hydrogen with a suitable organic substrate such that up to an 800-fold increase in proton, carbon, and nitrogen signal strengths are seen for the substrate without its hydrogenation. These polarized signals can be selectively detected when combined with methods that suppress background signals.

Surface Acoustic Wave Nebulisation Mass Spectrometry for the Fast and Highly Sensitive Characterisation of Synthetic Dyes in Textile Samples

NASA Astrophysics Data System (ADS)

Astefanei, Alina; van Bommel, Maarten; Corthals, Garry L.

2017-10-01

Surface acoustic wave nebulisation (SAWN) mass spectrometry (MS) is a method to generate gaseous ions compatible with direct MS of minute samples at femtomole sensitivity. To perform SAWN, acoustic waves are propagated through a LiNbO3 sampling chip, and are conducted to the liquid sample, which ultimately leads to the generation of a fine mist containing droplets of nanometre to micrometre diameter. Through fission and evaporation, the droplets undergo a phase change from liquid to gaseous analyte ions in a non-destructive manner. We have developed SAWN technology for the characterisation of organic colourants in textiles. It generates electrospray-ionisation-like ions in a non-destructive manner during ionisation, as can be observed by the unmodified chemical structure. The sample size is decreased by tenfold to 1000-fold when compared with currently used liquid chromatography-MS methods, with equal or better sensitivity. This work underscores SAWN-MS as an ideal tool for molecular analysis of art objects as it is non-destructive, is rapid, involves minimally invasive sampling and is more sensitive than current MS-based methods. [Figure not available: see fulltext.

Molecularly imprinted solid-phase extraction for the determination of ten macrolide drugs residues in animal muscles by liquid chromatography-tandem mass spectrometry.

PubMed

Song, Xuqin; Zhou, Tong; Liu, Qingying; Zhang, Meiyu; Meng, Chenying; Li, Jiufeng; He, Limin

2016-10-01

A simple and sensitive method based on molecularly imprinted solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry was developed for the determination of the residues of ten macrolide drugs in swine, cattle and chicken muscles samples. The molecularly imprinted polymers (MIPs) were synthesized using tylosin as a template and methacrylic acid as a functional monomer. Samples were extracted with sodium borate buffer solution and ethyl acetate, and purified by the MIP cartridge. The results showed that the cartridge exhibited good recognition performance for macrolides, and better purification effect than the traditional solid-phase extraction cartridges. Recoveries of analytes at three spiking levels 1, 5 and 20μgkg(-1) ranged from 60.7% to 100.3% with the relative standard deviations less than 14%. The limits of detection of the method were between 0.1 and 0.4μgkg(-1). The method is useful for the routine monitoring of the residues of macrolide drugs in animal muscles. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular detection of pathogens in water--the pros and cons of molecular techniques.

PubMed

Girones, Rosina; Ferrús, Maria Antonia; Alonso, José Luis; Rodriguez-Manzano, Jesus; Calgua, Byron; Corrêa, Adriana de Abreu; Hundesa, Ayalkibet; Carratala, Anna; Bofill-Mas, Sílvia

2010-08-01

Pollution of water by sewage and run-off from farms produces a serious public health problem in many countries. Viruses, along with bacteria and protozoa in the intestine or in urine are shed and transported through the sewer system. Even in highly industrialized countries, pathogens, including viruses, are prevalent throughout the environment. Molecular methods are used to monitor viral, bacterial, and protozoan pathogens, and to track pathogen- and source-specific markers in the environment. Molecular techniques, specifically polymerase chain reaction-based methods, provide sensitive, rapid, and quantitative analytical tools with which to study such pathogens, including new or emerging strains. These techniques are used to evaluate the microbiological quality of food and water, and to assess the efficiency of virus removal in drinking and wastewater treatment plants. The range of methods available for the application of molecular techniques has increased, and the costs involved have fallen. These developments have allowed the potential standardization and automation of certain techniques. In some cases they facilitate the identification, genotyping, enumeration, viability assessment, and source-tracking of human and animal contamination. Additionally, recent improvements in detection technologies have allowed the simultaneous detection of multiple targets in a single assay. However, the molecular techniques available today and those under development require further refinement in order to be standardized and applicable to a diversity of matrices. Water disinfection treatments may have an effect on the viability of pathogens and the numbers obtained by molecular techniques may overestimate the quantification of infectious microorganisms. The pros and cons of molecular techniques for the detection and quantification of pathogens in water are discussed. (c) 2010 Elsevier Ltd. All rights reserved.

A novel method for extracting nucleic acids from dried blood spots for ultrasensitive detection of low-density Plasmodium falciparum and Plasmodium vivax infections.

PubMed

Zainabadi, Kayvan; Adams, Matthew; Han, Zay Yar; Lwin, Hnin Wai; Han, Kay Thwe; Ouattara, Amed; Thura, Si; Plowe, Christopher V; Nyunt, Myaing M

2017-09-18

Greater Mekong Subregion countries are committed to eliminating Plasmodium falciparum malaria by 2025. Current elimination interventions target infections at parasite densities that can be detected by standard microscopy or rapid diagnostic tests (RDTs). More sensitive detection methods have been developed to detect lower density "asymptomatic" infections that may represent an important transmission reservoir. These ultrasensitive polymerase chain reaction (usPCR) tests have been used to identify target populations for mass drug administration (MDA). To date, malaria usPCR tests have used either venous or capillary blood sampling, which entails complex sample collection, processing and shipping requirements. An ultrasensitive method performed on standard dried blood spots (DBS) would greatly facilitate the molecular surveillance studies needed for targeting elimination interventions. A highly sensitive method for detecting Plasmodium falciparum and P. vivax 18S ribosomal RNA from DBS was developed by empirically optimizing nucleic acid extraction conditions. The limit of detection (LoD) was determined using spiked DBS samples that were dried and stored under simulated field conditions. Further, to assess its utility for routine molecular surveillance, two cross-sectional surveys were performed in Myanmar during the wet and dry seasons. The lower LoD of the DBS-based ultrasensitive assay was 20 parasites/mL for DBS collected on Whatman 3MM filter paper and 23 parasites/mL for Whatman 903 Protein Saver cards-equivalent to 1 parasite per 50 µL DBS. This is about 5000-fold more sensitive than standard RDTs and similar to the LoD of ≤16-22 parasites/mL reported for other ultrasensitive methods based on whole blood. In two cross-sectional surveys in Myanmar, nearly identical prevalence estimates were obtained from contemporaneous DBS samples and capillary blood samples collected during the wet and dry season. The DBS-based ultrasensitive method described in this study shows equal sensitivity as previously described methods based on whole blood, both in its limit of detection and prevalence estimates in two field surveys. The reduced cost and complexity of this method will allow for the scale-up of surveillance studies to target MDA and other malaria elimination interventions, and help lead to a better understanding of the epidemiology of low-density malaria infections.

A surface enhanced Raman scattering quantitative analytical platform for detection of trace Cu coupled the catalytic reaction and gold nanoparticle aggregation with label-free Victoria blue B molecular probe.

PubMed

Li, Chongning; Ouyang, Huixiang; Tang, Xueping; Wen, Guiqing; Liang, Aihui; Jiang, Zhiliang

2017-01-15

With development of economy and society, there is an urgent need to develop convenient and sensitive methods for detection of Cu 2+ pollution in water. In this article, a simple and sensitive SERS sensor was proposed to quantitative analysis of trace Cu 2+ in water. The SERS sensor platform was prepared a common gold nanoparticle (AuNP)-SiO 2 sol substrate platform by adsorbing HSA, coupling with the catalytic reaction of Cu 2+ -ascorbic acid (H 2 A)-dissolved oxygen, and using label-free Victoria blue B (VBB) as SERS molecular probes. The SERS sensor platform response to the AuNP aggregations by hydroxyl radicals (•OH) oxidizing from the Cu 2+ catalytic reaction, which caused the SERS signal enhancement. Therefore, by monitoring the increase of SERS signal, Cu 2+ in water can be determined accurately. The results show that the SERS sensor platforms owns a linear response with a range from 0.025 to 25μmol/L Cu 2+ , and with a detection limit of 0.008μmol/L. In addition, the SERS method demonstrated good specificity for Cu 2+ , which can determined accurately trace Cu 2+ in water samples, and good recovery and accuracy are obtained for the water samples. With its high selectivity and good accuracy, the sensitive SERS quantitative analysis method is expected to be a promising candidate for determining copper ions in environmental monitoring and food safety. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular detection of Borrelia burgdorferi sensu lato – An analytical comparison of real-time PCR protocols from five different Scandinavian laboratories

PubMed Central

Faller, Maximilian; Wilhelmsson, Peter; Kjelland, Vivian; Andreassen, Åshild; Dargis, Rimtas; Quarsten, Hanne; Dessau, Ram; Fingerle, Volker; Margos, Gabriele; Noraas, Sølvi; Ornstein, Katharina; Petersson, Ann-Cathrine; Matussek, Andreas; Lindgren, Per-Eric; Henningsson, Anna J.

2017-01-01

Introduction Lyme borreliosis (LB) is the most common tick transmitted disease in Europe. The diagnosis of LB today is based on the patient´s medical history, clinical presentation and laboratory findings. The laboratory diagnostics are mainly based on antibody detection, but in certain conditions molecular detection by polymerase chain reaction (PCR) may serve as a complement. Aim The purpose of this study was to evaluate the analytical sensitivity, analytical specificity and concordance of eight different real-time PCR methods at five laboratories in Sweden, Norway and Denmark. Method Each participating laboratory was asked to analyse three different sets of samples (reference panels; all blinded) i) cDNA extracted and transcribed from water spiked with cultured Borrelia strains, ii) cerebrospinal fluid spiked with cultured Borrelia strains, and iii) DNA dilution series extracted from cultured Borrelia and relapsing fever strains. The results and the method descriptions of each laboratory were systematically evaluated. Results and conclusions The analytical sensitivities and the concordance between the eight protocols were in general high. The concordance was especially high between the protocols using 16S rRNA as the target gene, however, this concordance was mainly related to cDNA as the type of template. When comparing cDNA and DNA as the type of template the analytical sensitivity was in general higher for the protocols using DNA as template regardless of the use of target gene. The analytical specificity for all eight protocols was high. However, some protocols were not able to detect Borrelia spielmanii, Borrelia lusitaniae or Borrelia japonica. PMID:28937997

Novel aptamer-linked nanoconjugate approach for detection of waterborne bacterial pathogens: an update

NASA Astrophysics Data System (ADS)

Singh, Gulshan; Manohar, Murli; Adegoke, Anthony Ayodeji; Stenström, Thor Axel; Shanker, Rishi

2017-01-01

The lack of microbiologically safe water in underdeveloped nations is the prime cause of infectious disease outbreaks. The need for the specific identification and detection of microorganisms encourages the development of advanced, rapid, sensitive and highly specific methods for the monitoring of pathogens and management of potential risk to human health. The rapid molecular assays based on detection of specific molecular signatures offer advantages over conventional methods in terms of specificity and sensitivity but require complex instrumentation and skilled personnel. Nanotechnology is an emerging area and provides a robust approach for the identification of pathogenic microorganism utilizing the peculiar properties of nanomaterials, i.e. small size (1-100 nm) and large surface area. This emerging technology promises to fulfill the urgent need of a novel strategy to enhance the bacterial identification and quantitation in the environment. In this context, the peculiar properties of gold nanoparticles, their plasmonic shifts, and changes in magnetic properties have been utilized for the simple and cost-effective detection of bacterial nucleic acids, antigens and toxins with quite improved sensitivity. One of the promising leads to develop an advance detection method might be the coupling of nucleic acid aptamers (capable of interacting specifically with bacteria, protozoa, and viruses) with nanomaterials. Such aptamer-nano conjugate can be used for the specific recognition of infectious agents in different environmental matrices. This review summarizes the application of nanotechnology in the area of pathogen detection and discusses the prospects of coupling nucleic acid aptamers with nanoparticles for the specific detection of targeted pathogens.

Molecular typing of uropathogenic E. coli strains by the ERIC-PCR method.

PubMed

Ardakani, Maryam Afkhami; Ranjbar, Reza

2016-04-01

Escherichia coli (E. coli) is the most common cause of urinary infections in hospitals. The aim of this study was to evaluate the ERIC-PCR method for molecular typing of uropathogenic E. coli strains isolated from hospitalized patients. In a cross sectional study, 98 E. coli samples were collected from urine samples taken from patients admitted to Baqiyatallah Hospital from June 2014 to January 2015. The disk agar diffusion method was used to determine antibiotic sensitivity. DNA proliferation based on repetitive intergenic consensus was used to classify the E. coli strains. The products of proliferation were electrophoresed on 1.5% agarose gel, and their dendrograms were drawn. The data were analyzed by online Insillico software. The method used in this research proliferated numerous bands (4-17 bands), ranging from 100 to 3000 base pairs. The detected strains were classified into six clusters (E1-E6) with 70% similarity between them. In this study, uropathogenic E. coli strains belonged to different genotypic clusters. It was found that ERIC-PCR had good differentiation power for molecular typing of uropathogenic E. coli strains isolated from the patients in the study.

Molecular Tools To Study Preharvest Food Safety Challenges.

PubMed

Kumar, Deepak; Thakur, Siddhartha

2018-02-01

Preharvest food safety research and activities have advanced over time with the recognition of the importance and complicated nature of the preharvest phase of food production. In developed nations, implementation of preharvest food safety procedures along with strict monitoring and containment at various postharvest stages such as slaughter, processing, storage, and distribution have remarkably reduced the burden of foodborne pathogens in humans. Early detection and adequate surveillance of pathogens at the preharvest stage is of the utmost importance to ensure a safe meat supply. There is an urgent need to develop rapid, cost-effective, and point-of-care diagnostics which could be used at the preharvest stage and would complement postmortem and other quality checks performed at the postharvest stage. With newer methods and technologies, more efforts need to be directed toward developing rapid, sensitive, and specific methods for detection or screening of foodborne pathogens at the preharvest stage. In this review, we will discuss the molecular methods available for detection and molecular typing of bacterial foodborne pathogens at the farm. Such methods include conventional techniques such as endpoint PCR, real-time PCR, DNA microarray, and more advanced techniques such as matrix-assisted layer desorption ionization-time of flight mass spectrometry and whole-genome sequencing.

Refined method for predicting electrochemical windows of ionic liquids and experimental validation studies.

PubMed

Zhang, Yong; Shi, Chaojun; Brennecke, Joan F; Maginn, Edward J

2014-06-12

A combined classical molecular dynamics (MD) and ab initio MD (AIMD) method was developed for the calculation of electrochemical windows (ECWs) of ionic liquids. In the method, the liquid phase of ionic liquid is explicitly sampled using classical MD. The electrochemical window, estimated by the energy difference between the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), is calculated at the density functional theory (DFT) level based on snapshots obtained from classical MD trajectories. The snapshots were relaxed using AIMD and quenched to their local energy minima, which assures that the HOMO/LUMO calculations are based on stable configurations on the same potential energy surface. The new procedure was applied to a group of ionic liquids for which the ECWs were also experimentally measured in a self-consistent manner. It was found that the predicted ECWs not only agree with the experimental trend very well but also the values are quantitatively accurate. The proposed method provides an efficient way to compare ECWs of ionic liquids in the same context, which has been difficult in experiments or simulation due to the fact that ECW values sensitively depend on experimental setup and conditions.

Simple and Sensitive Molecularly Imprinted Polymer - Mn-Doped ZnS Quantum Dots Based Fluorescence Probe for Cocaine and Metabolites Determination in Urine.

PubMed

Chantada-Vázquez, María Pilar; Sánchez-González, Juan; Peña-Vázquez, Elena; Tabernero, María Jesús; Bermejo, Ana María; Bermejo-Barrera, Pilar; Moreda-Piñeiro, Antonio

2016-03-01

A new molecularly imprinted polymer (MIP)-based fluorescent artificial receptor has been prepared by anchoring a selective MIP for cocaine (COC) on the surface of polyethylene glycol (PEG) modified Mn-doped ZnS quantum dots (QDs). The prepared material combines the high selectivity attributed to MIPs and the sensitive fluorescent property of the Mn-doped ZnS QDs. Simple and low cost methods have therefore been optimized for assessing cocaine abuse in urine by monitoring the fluorescence quenching when the template (COC) and also metabolites from COC [benzoylecgonine (BZE) and ecgonine methyl ester (EME)] are present. Fluorescence quenching was not observed when performing experiments with other drugs of abuse (and their metabolites) or when using nonimprinted polymer (NIP)-coated QDs. Under optimized operating conditions (1.5 mL of 200 mg L(-1) MIP-coated QDs solution, pH 5.5, and 15 min before fluorescence scanning) two analytical methods were developed/validated. One of the procedures (direct method) consisted of urine sample 1:20 dilution before fluorescence measurements. The method has been found to be fast, precise, and accurate, but the standard addition technique for performing the analysis was required because of the existence of matrix effect. The second procedure performed a solid phase extraction (SPE) first, avoiding matrix effect and allowing external calibration. The limits of detection of the methods were 0.076 mg L(-1) (direct method) and 0.0042 mg L(-1) (SPE based method), which are lower than the cutoff values for confirmative conclusions regarding cocaine abuse.

Clustering the Orion B giant molecular cloud based on its molecular emission

NASA Astrophysics Data System (ADS)

Bron, Emeric; Daudon, Chloé; Pety, Jérôme; Levrier, François; Gerin, Maryvonne; Gratier, Pierre; Orkisz, Jan H.; Guzman, Viviana; Bardeau, Sébastien; Goicoechea, Javier R.; Liszt, Harvey; Öberg, Karin; Peretto, Nicolas; Sievers, Albrecht; Tremblin, Pascal

2018-02-01

Context. Previous attempts at segmenting molecular line maps of molecular clouds have focused on using position-position-velocity data cubes of a single molecular line to separate the spatial components of the cloud. In contrast, wide field spectral imaging over a large spectral bandwidth in the (sub)mm domain now allows one to combine multiple molecular tracers to understand the different physical and chemical phases that constitute giant molecular clouds (GMCs). Aims: We aim at using multiple tracers (sensitive to different physical processes and conditions) to segment a molecular cloud into physically/chemically similar regions (rather than spatially connected components), thus disentangling the different physical/chemical phases present in the cloud. Methods: We use a machine learning clustering method, namely the Meanshift algorithm, to cluster pixels with similar molecular emission, ignoring spatial information. Clusters are defined around each maximum of the multidimensional probability density function (PDF) of the line integrated intensities. Simple radiative transfer models were used to interpret the astrophysical information uncovered by the clustering analysis. Results: A clustering analysis based only on the J = 1-0 lines of three isotopologues of CO proves sufficient to reveal distinct density/column density regimes (nH 100 cm-3, 500 cm-3, and >1000 cm-3), closely related to the usual definitions of diffuse, translucent and high-column-density regions. Adding two UV-sensitive tracers, the J = 1-0 line of HCO+ and the N = 1-0 line of CN, allows us to distinguish two clearly distinct chemical regimes, characteristic of UV-illuminated and UV-shielded gas. The UV-illuminated regime shows overbright HCO+ and CN emission, which we relate to a photochemical enrichment effect. We also find a tail of high CN/HCO+ intensity ratio in UV-illuminated regions. Finer distinctions in density classes (nH 7 × 103 cm-3, 4 × 104 cm-3) for the densest regions are also identified, likely related to the higher critical density of the CN and HCO+ (1-0) lines. These distinctions are only possible because the high-density regions are spatially resolved. Conclusions: Molecules are versatile tracers of GMCs because their line intensities bear the signature of the physics and chemistry at play in the gas. The association of simultaneous multi-line, wide-field mapping and powerful machine learning methods such as the Meanshift clustering algorithm reveals how to decode the complex information available in these molecular tracers. Data products associated with this paper are available at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (http://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/610/A12 and at http://www.iram.fr/ pety/ORION-B

A Highly Sensitive Diagnostic System for Detecting Dengue Viruses Using the Interaction between a Sulfated Sugar Chain and a Virion.

PubMed

Saksono, Budi; Dewi, Beti Ernawati; Nainggolan, Leonardo; Suda, Yasuo

2015-01-01

We propose a novel method of detecting trace amounts of dengue virus (DENVs) from serum. Our method is based on the interaction between a sulfated sugar chain and a DENV surface glycoprotein. After capturing DENV with the sulfated sugar chain-immobilized gold nanoparticles (SGNPs), the resulting complex is precipitated and viral RNA content is measured using the reverse-transcription quantitative polymerase chain reaction SYBR Green I (RT-qPCR-Syb) method. Sugar chains that bind to DENVs were identified using the array-type sugar chain immobilized chip (Sugar Chip) and surface plasmon resonance (SPR) imaging. Heparin and low-molecular-weight dextran sulfate were identified as binding partners, and immobilized on gold nanoparticles to prepare 3 types of SGNPs. The capacity of these SGNPs to capture and concentrate trace amounts of DENVs was evaluated in vitro. The SGNP with greatest sensitivity was tested using clinical samples in Indonesia in 2013-2014. As a result, the novel method was able to detect low concentrations of DENVs using only 6 μL of serum, with similar sensitivity to that of a Qiagen RNA extraction kit using 140 μL of serum. In addition, this method allows for multiplex-like identification of serotypes of DENVs. This feature is important for good healthcare management of DENV infection in order to safely diagnose the dangerous, highly contagious disease quickly, with high sensitivity.

A novel automatic molecular test for detection of multidrug resistance tuberculosis in sputum specimen: A case control study.

PubMed

Li, Qiang; Ou, Xi C; Pang, Yu; Xia, Hui; Huang, Hai R; Zhao, Bing; Wang, Sheng F; Zhao, Yan L

2017-07-01

MiniLab tuberculosis (ML TB) assay is a new automatic diagnostic tool for diagnosis of multidrug resistance tuberculosis (MDR-TB). This study was conducted with aims to know the performance of this assay. Sputum sample from 224 TB suspects was collected from tuberculosis suspects seeking medical care at Beijing Chest hospital. The sputum samples were directly used for smear and ML TB test. The left sputum sample was used to conduct Xpert MTB/RIF, Bactec MGIT culture and drug susceptibility test (DST). All discrepancies between the results from DST, molecular and phenotypic methods were confirmed by DNA Sequencing. The sensitivity and specificity of ML TB test for detecting MTBC from TB suspects were 95.1% and 88.9%, respectively. The sensitivity for smear negative TB suspects was 64.3%. For detection of RIF resistance, the sensitivity and specificity of ML TB test were 89.2% and 95.7%, respectively. For detection of INH resistance, the sensitivity and specificity of ML TB test were 78.3% and 98.1%, respectively. ML TB test showed similar performance to Xpert MTB/RIF for detection of MTBC and RIF resistance. In addition, ML TB also had good performance for INH resistance detection. Copyright © 2017. Published by Elsevier Ltd.

Molecular Tools for the Detection of Nitrogen Cycling Archaea

PubMed Central

Rusch, Antje

2013-01-01

Archaea are widespread in extreme and temperate environments, and cultured representatives cover a broad spectrum of metabolic capacities, which sets them up for potentially major roles in the biogeochemistry of their ecosystems. The detection, characterization, and quantification of archaeal functions in mixed communities require Archaea-specific primers or probes for the corresponding metabolic genes. Five pairs of degenerate primers were designed to target archaeal genes encoding key enzymes of nitrogen cycling: nitrite reductases NirA and NirB, nitrous oxide reductase (NosZ), nitrogenase reductase (NifH), and nitrate reductases NapA/NarG. Sensitivity towards their archaeal target gene, phylogenetic specificity, and gene specificity were evaluated in silico and in vitro. Owing to their moderate sensitivity/coverage, the novel nirB-targeted primers are suitable for pure culture studies only. The nirA-targeted primers showed sufficient sensitivity and phylogenetic specificity, but poor gene specificity. The primers designed for amplification of archaeal nosZ performed well in all 3 criteria; their discrimination against bacterial homologs appears to be weakened when Archaea are strongly outnumbered by bacteria in a mixed community. The novel nifH-targeted primers showed high sensitivity and gene specificity, but failed to discriminate against bacterial homologs. Despite limitations, 4 of the new primer pairs are suitable tools in several molecular methods applied in archaeal ecology. PMID:23365509

Molecular and Microscopical Investigation of the Microflora Inhabiting a Deteriorated Italian Manuscript Dated from the Thirteenth Century

PubMed Central

Michaelsen, Astrid; Piñar, Guadalupe

2010-01-01

This case study shows the application of nontraditional diagnostic methods to investigate the microbial consortia inhabiting an ancient manuscript. The manuscript was suspected to be biologically deteriorated and SEM observations showed the presence of fungal spores attached to fibers, but classic culturing methods did not succeed in isolating microbial contaminants. Therefore, molecular methods, including PCR, denaturing gradient gel electrophoresis (DGGE), and clone libraries, were used as a sensitive alternative to conventional cultivation techniques. DGGE fingerprints revealed a high biodiversity of both bacteria and fungi inhabiting the manuscript. DNA sequence analysis confirmed the existence of fungi and bacteria in manuscript samples. A number of fungal clones identified on the manuscript showed similarity to fungal species inhabiting dry or saline environments, suggesting that the manuscript environment selects for osmophilic or xerophilic fungal species. Most of the bacterial sequences retrieved from the manuscript belong to phylotypes with cellulolytic activities. PMID:20449583

Shaping the Atomic-Scale Geometries of Electrodes to Control Optical and Electrical Performance of Molecular Devices.

PubMed

Zhao, Zhikai; Liu, Ran; Mayer, Dirk; Coppola, Maristella; Sun, Lu; Kim, Youngsang; Wang, Chuankui; Ni, Lifa; Chen, Xing; Wang, Maoning; Li, Zongliang; Lee, Takhee; Xiang, Dong

2018-04-01

A straightforward method to generate both atomic-scale sharp and atomic-scale planar electrodes is reported. The atomic-scale sharp electrodes are generated by precisely stretching a suspended nanowire, while the atomic-scale planar electrodes are obtained via mechanically controllable interelectrodes compression followed by a thermal-driven atom migration process. Notably, the gap size between the electrodes can be precisely controlled at subangstrom accuracy with this method. These two types of electrodes are subsequently employed to investigate the properties of single molecular junctions. It is found, for the first time, that the conductance of the amine-linked molecular junctions can be enhanced ≈50% as the atomic-scale sharp electrodes are used. However, the atomic-scale planar electrodes show great advantages to enhance the sensitivity of Raman scattering upon the variation of nanogap size. The underlying mechanisms for these two interesting observations are clarified with the help of density functional theory calculation and finite-element method simulation. These findings not only provide a strategy to control the electron transport through the molecule junction, but also pave a way to modulate the optical response as well as to improve the stability of single molecular devices via the rational design of electrodes geometries. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lessons learned with molecular methods targeting the BCSP-31 membrane protein for diagnosis of human brucellosis.

PubMed

Sanjuan-Jimenez, Rocio; Colmenero, Juan D; Morata, Pilar

2017-06-01

Brucellosis remains an emerging and re-emerging zoonosis worldwide causing high human morbidity. It usually affects persons who are permanently exposed to fastidious microorganisms of the Brucella genus and has a nonspecific clinical picture. Thus, diagnosis of brucellosis can sometimes be difficult. Molecular techniques have recently been found very useful in the diagnosis of brucellosis together with its common and very diverse focal complications. We herein review all the lessons learned by our group concerning the molecular diagnosis of human brucellosis over the last twenty years. The results, initially using one-step conventional PCR, later PCR-ELISA and more recently real-time PCR, using both fluorescent intercalating reagents (SYBR-Green I) and specific probes (Taqman), have shown that these techniques are all much more sensitive than bacteriological methods and more specific than the usual serological techniques for the diagnosis of primary infection, the post-treatment control of the disease, early detection of relapse and the diagnosis of focal complications. Optimization of the technique and improvements introduced over the years show that molecular methods, currently accessible for most clinical laboratories, enable easy rapid diagnosis of brucellosis at the same time as they avoid any risk to laboratory personnel while handling live Brucella spp. Copyright © 2017 Elsevier B.V. All rights reserved.

Measuring protein dynamics in live cells: protocols and practical considerations for fluorescence fluctuation microscopy

PubMed Central

Youker, Robert T.; Teng, Haibing

2014-01-01

Abstract. Quantitative analysis of protein complex stoichiometries and mobilities are critical for elucidating the mechanisms that regulate cellular pathways. Fluorescence fluctuation spectroscopy (FFS) techniques can measure protein dynamics, such as diffusion coefficients and formation of complexes, with extraordinary precision and sensitivity. Complete calibration and characterization of the microscope instrument is necessary in order to avoid artifacts during data acquisition and to capitalize on the full capabilities of FFS techniques. We provide an overview of the theory behind FFS techniques, discuss calibration procedures, provide protocols, and give practical considerations for performing FFS experiments. One important parameter recovered from FFS measurements is the relative molecular brightness that can correlate with oligomerization. Three methods for measuring molecular brightness (fluorescence correlation spectroscopy, photon-counting histogram, and number and brightness analysis) recover similar values when measuring samples under ideal conditions in vitro. However, examples are given illustrating that these different methods used for calculating molecular brightness of fluorescent molecules in cells are not always equivalent. Methods relying on spot measurements are more prone to bleaching and movement artifacts that can lead to underestimation of brightness values. We advocate for the use of multiple FFS techniques to study molecular brightnesses to overcome and compliment limitations of individual techniques. PMID:25260867

DNA detection and single nucleotide mutation identification using SERS for molecular diagnostics and global health

NASA Astrophysics Data System (ADS)

Ngo, Hoan T.; Gandra, Naveen; Fales, Andrew M.; Taylor, Steve M.; Vo-Dinh, Tuan

2017-02-01

Nucleic acid-based molecular diagnostics at the point-of-care (POC) and in resource-limited settings is still a challenge. We present a sensitive yet simple DNA detection method with single nucleotide polymorphism (SNP) identification capability. The detection scheme involves sandwich hybridization of magnetic beads conjugated with capture probes, target sequences, and ultrabright surface-enhanced Raman Scattering (SERS) nanorattles conjugated with reporter probes. Upon hybridization, the sandwich probes are concentrated at the detection focus controlled by a magnetic system for SERS measurements. The ultrabright SERS nanorattles, consisting of a core and a shell with resonance Raman reporters loaded in the gap space between the core and the shell, serve as SERS tags for ultrasensitive signal detection. Specific DNA sequences of the malaria parasite Plasmodium falciparum and dengue virus 1 (DENV1) were used as the model marker system. Detection limit of approximately 100 attomoles was achieved. Single nucleotide polymorphism (SNP) discrimination of wild type malaria DNA and mutant malaria DNA, which confers resistance to artemisinin drugs, was also demonstrated. The results demonstrate the molecular diagnostic potential of the nanorattle-based method to both detect and genotype infectious pathogens. The method's simplicity makes it a suitable candidate for molecular diagnosis at the POC and in resource-limited settings.

Molecular Mechanisms Underlying Occult Hepatitis B Virus Infection

PubMed Central

Samal, Jasmine; Kandpal, Manish

2012-01-01

Summary: Chronic hepatitis B virus (HBV) infection is a complex clinical entity frequently associated with cirrhosis and hepatocellular carcinoma (HCC). The persistence of HBV genomes in the absence of detectable surface antigenemia is termed occult HBV infection. Mutations in the surface gene rendering HBsAg undetectable by commercial assays and inhibition of HBV by suppression of viral replication and viral proteins represent two fundamentally different mechanisms that lead to occult HBV infections. The molecular mechanisms underlying occult HBV infections, including recently identified mechanisms associated with the suppression of HBV replication and inhibition of HBV proteins, are reviewed in detail. The availability of highly sensitive molecular methods has led to increased detection of occult HBV infections in various clinical settings. The clinical relevance of occult HBV infection and the utility of appropriate diagnostic methods to detect occult HBV infection are discussed. The need for specific guidelines on the diagnosis and management of occult HBV infection is being increasingly recognized; the aspects of mechanistic studies that warrant further investigation are discussed in the final section. PMID:22232374

[Yeast species in vulvovaginitis candidosa].

PubMed

Nemes-Nikodém, Éva; Tamási, Béla; Mihalik, Noémi; Ostorházi, Eszter

2015-01-04

Vulvovaginal candidiasis is the most common mycosis, however, the available information about antifungal susceptibilities of these yeasts is limited. To compare the gold standard fungal culture with a new molecular identification method and report the incidence of yeast species in vulvovaginitis candidosa. The authors studied 370 yeasts isolated from vulvovaginal candidiasis and identified them by phenotypic and molecular methods. The most common species was Candida albicans (85%), followed by Candida glabrata, and other Candida species. At present there are no recommendations for the evaluation of antifungal susceptibility of pathogenic fungal species occurring in vulvovaginal candidiasis and the natural antifungal resistance of the different species is known only. Matrix Assisted Laser Desorption Ionization Time of Flight identification can be used to differentiate the fluconazole resistant Candida dubliniensis and the sensitive Candida albicans strains.

New molecular methods for the detection of hepatitis A and Norwalk viruses in shellfish.

PubMed

Romalde, J L

1996-12-01

Outbreaks of viral enteric diseases after consumption of shellfish are a major health risk. Methodological problems (such as toxicity for cell cultures and low viral concentrations) and the unculturability of some strains (i.e. hepatitis A virus, Norwalk virus) have made it difficult to study those viruses in the environmental samples. Currently, the analysis of the hygienic quality of marketable shellfish is determined by the use of fecal indicator bacteria, but their reliability in determining viral pollution of shellfish is very low. Recent biotechnology developments are providing available rapid, sensitive, and specific tools for detecting food-borne viruses in shellfish and in shellfish-growing waters. In this paper, a review of these new molecular methods is carried out, discussing their advantages and possible applications.

Molecular engineering of fluorescein dyes as complementary absorbers in dye co-sensitized solar cells

DOE PAGES

Pepe, Giulio; Cole, Jacqueline M.; Waddell, Paul G.; ...

2016-09-22

Fluorescein dye derivatives exhibit extended optical absorption up to 500 nm, rendering these compounds suitable as co-absorbers in dye-sensitized solar cells (DSCs). A molecular engineering approach is presented, which embraces this intrinsic optical attribute of fluoresceins, while modifying the dye chemistry to enhance their light harvesting efficiency, in order to effectively tailor them for DSC applications. This approach first realizes relationships between the molecular structure and the optoelectronic properties for a series of five a priori known (parent) fluorescein dyes: 5-carboxyfluorescein (1), a mixture of m-carboxyfluorescein where m = 5 or 6 (2), 5-carboxyfluorescein diacetate (3), 6-carboxyfluorescein diacetate (4), amore » mixture of n-carboxy-2',7'-dichlorofluorescein diacetate where n = 5 or 6 (5). The first step in this approach combines, where available, experimental and computational methods so that electronic structure calculations can also be validated for representative fluorescein dyes. Such calculations can then be used reliably to predict the structure and properties of fluorescein dyes for cases where experimental data are lacking. Structure-function relationships established from this initial step inform the selection of parent dye 1 that is taken forward to the second step in molecular engineering: in silico chemical derivation to re-functionalize 1 for DSC applications. For this purpose, computational calculations are used to extend the charge conjugation in 1 between its donor and acceptor moieties. These structural modifications result in a bathochromic shift of the lowest excitation by ~1.3-1.9 eV (100-170 nm), making the dye optically absorb in the visible region. Further calculations on dye molecules adsorbed onto the surface of a TiO 2 cluster are used to investigate the dye sensitization behavior via dye adsorption energies and anchoring modes. The results of this theoretical investigation lead to two molecularly engineered fluoresceins being proposed to act as co-sensitizers together with a rhodamine dye. This combination of three dyes ensures chemical compatibility, panchromatic absorption, and restores optical absorption dipping otherwise observed in a DSC device at ~350-400 nm owing to the I-/I- 3 electrolyte. Altogether, the results of this study demonstrate that molecular engineering can be used to identify suitable chemical modifications for organic dyes with improved light harvesting properties for photovoltaic applications.« less

Molecular engineering of fluorescein dyes as complementary absorbers in dye co-sensitized solar cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pepe, Giulio; Cole, Jacqueline M.; Waddell, Paul G.

Fluorescein dye derivatives exhibit extended optical absorption up to 500 nm, rendering these compounds suitable as co-absorbers in dye-sensitized solar cells (DSCs). A molecular engineering approach is presented, which embraces this intrinsic optical attribute of fluoresceins, while modifying the dye chemistry to enhance their light harvesting efficiency, in order to effectively tailor them for DSC applications. This approach first realizes relationships between the molecular structure and the optoelectronic properties for a series of five a priori known (parent) fluorescein dyes: 5-carboxyfluorescein (1), a mixture of m-carboxyfluorescein where m = 5 or 6 (2), 5-carboxyfluorescein diacetate (3), 6-carboxyfluorescein diacetate (4), amore » mixture of n-carboxy-2',7'-dichlorofluorescein diacetate where n = 5 or 6 (5). The first step in this approach combines, where available, experimental and computational methods so that electronic structure calculations can also be validated for representative fluorescein dyes. Such calculations can then be used reliably to predict the structure and properties of fluorescein dyes for cases where experimental data are lacking. Structure-function relationships established from this initial step inform the selection of parent dye 1 that is taken forward to the second step in molecular engineering: in silico chemical derivation to re-functionalize 1 for DSC applications. For this purpose, computational calculations are used to extend the charge conjugation in 1 between its donor and acceptor moieties. These structural modifications result in a bathochromic shift of the lowest excitation by ~1.3-1.9 eV (100-170 nm), making the dye optically absorb in the visible region. Further calculations on dye molecules adsorbed onto the surface of a TiO 2 cluster are used to investigate the dye sensitization behavior via dye adsorption energies and anchoring modes. The results of this theoretical investigation lead to two molecularly engineered fluoresceins being proposed to act as co-sensitizers together with a rhodamine dye. This combination of three dyes ensures chemical compatibility, panchromatic absorption, and restores optical absorption dipping otherwise observed in a DSC device at ~350-400 nm owing to the I-/I- 3 electrolyte. Altogether, the results of this study demonstrate that molecular engineering can be used to identify suitable chemical modifications for organic dyes with improved light harvesting properties for photovoltaic applications.« less

Highly sensitive and rapid bacteria detection using molecular beacon-Au nanoparticles hybrid nanoprobes.

PubMed

Cao, Jing; Feng, Chao; Liu, Yan; Wang, Shouyu; Liu, Fei

2014-07-15

Since many diseases are caused by pathogenic bacterial infections, accurate and rapid detection of pathogenic bacteria is in urgent need to timely apply appropriate treatments and to reduce economic costs. To end this, we designed molecular beacon-Au nanoparticle hybrid nanoprobes to improve the bacterial detection efficiency and sensitivity. Here, we show that the designed molecular beacon modified Au nanoparticles could specifically recognize synthetic DNAs targets and can readily detect targets in clinical samples. Moreover, the hybrid nanoprobes can recognize Escherichia coli within an hour at a concentration of 10(2) cfu/ml, which is 1000-folds sensitive than using molecular beacon directly. Our results show that the molecular beacon-Au nanoparticle hybrid nanoprobes have great potential in medical and biological applications. Copyright © 2014 Elsevier B.V. All rights reserved.

The development of methods for the detection of Salmonella in chickens by a combination of immunomagnetic separation and PCRs.

PubMed

Dai, Fengying; Zhang, Miao; Xu, Dixin; Yang, Yin; Wang, Jiaxiao; Li, Mingzhen; Du, Meihong

2017-11-01

Micro- and nanoimmunomagnetic beads (MIMBs and NIMBs) used for immunomagnetic separation (IMS) with PCR were studied for the rapid detection of Salmonella. The capture efficiency of the two different IMBs was evaluated by a conventional plate counting method, and the binding pattern was studied using scanning electron microscopy. The specificity of the IMBs was tested with Salmonella, Shigella flexneri, enterohemorrhagic Escherichia coli O157:H7, and Listeria monocytogenes. By comparing the pre-enrichment IMS and the IMS enrichment steps with a 5.5-H enrichment time, this study developed a rapid and sensitive method for the detection of Salmonella in chicken. The method was implemented by IMS enrichment and PCR with MIMBs and NIMBs, with a total analysis time of 8 H. We showed that the method was sensitive based on NIMBs with a detection limit of 10° CFU for Salmonella in 25 g of chicken. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Selective and sensitive fluorimetric determination of carbendazim in apple and orange after preconcentration with magnetite-molecularly imprinted polymer

NASA Astrophysics Data System (ADS)

İlktaç, Raif; Aksuner, Nur; Henden, Emur

2017-03-01

In this study, magnetite-molecularly imprinted polymer has been used for the first time as selective adsorbent before the fluorimetric determination of carbendazim. Adsorption capacity of the magnetite-molecularly imprinted polymer was found to be 2.31 ± 0.63 mg g- 1 (n = 3). Limit of detection (LOD) and limit of quantification (LOQ) of the method were found to be 2.3 and 7.8 μg L- 1, respectively. Calibration graph was linear in the range of 10-1000 μg L- 1. Rapidity is an important advantage of the method where re-binding and recovery processes of carbendazim can be completed within an hour. The same imprinted polymer can be used for the determination of carbendazim without any capacity loss repeatedly for at least ten times. Proposed method has been successfully applied to determine carbendazim residues in apple and orange, where the recoveries of the spiked samples were found to be in the range of 95.7-103%. Characterization of the adsorbent and the effects of some potential interferences were also evaluated. With the reasonably high capacity and reusability of the adsorbent, dynamic calibration range, rapidity, simplicity, cost-effectiveness and with suitable LOD and LOQ, the proposed method is an ideal method for the determination of carbendazim.

Local bias-induced phase transitions

DOE PAGES

Seal, Katyayani; Baddorf, Arthur P.; Jesse, Stephen; ...

2008-11-27

Electrical bias-induced phase transitions underpin a wide range of applications from data storage to energy generation and conversion. The mechanisms behind these transitions are often quite complex and in many cases are extremely sensitive to local defects that act as centers for local transformations or pinning. Furthermore, using ferroelectrics as an example, we review methods for probing bias-induced phase transitions and discuss the current limitations and challenges for extending the methods to field-induced phase transitions and electrochemical reactions in energy storage, biological and molecular systems.

Molecular Analysis of Endocrine Disruption in Hornyhead Turbot at Wastewater Outfalls in Southern California Using a Second Generation Multi-Species Microarray

PubMed Central

Baker, Michael E.; Vidal-Dorsch, Doris E.; Ribecco, Cataldo; Sprague, L. James; Angert, Mila; Lekmine, Narimene; Ludka, Colleen; Martella, Andrea; Ricciardelli, Eugenia; Bay, Steven M.; Gully, Joseph R.; Kelley, Kevin M.; Schlenk, Daniel; Carnevali, Oliana; Šášik, Roman; Hardiman, Gary

2013-01-01

Sentinel fish hornyhead turbot ( Pleuronichthys verticalis ) captured near wastewater outfalls are used for monitoring exposure to industrial and agricultural chemicals of ~ 20 million people living in coastal Southern California. Although analyses of hormones in blood and organ morphology and histology are useful for assessing contaminant exposure, there is a need for quantitative and sensitive molecular measurements, since contaminants of emerging concern are known to produce subtle effects. We developed a second generation multi-species microarray with expanded content and sensitivity to investigate endocrine disruption in turbot captured near wastewater outfalls in San Diego, Orange County and Los Angeles California. Analysis of expression of genes involved in hormone [e.g., estrogen, androgen, thyroid] responses and xenobiotic metabolism in turbot livers was correlated with a series of phenotypic end points. Molecular analyses of turbot livers uncovered altered expression of vitellogenin and zona pellucida protein, indicating exposure to one or more estrogenic chemicals, as well as, alterations in cytochrome P450 (CYP) 1A, CYP3A and glutathione S-transferase-α indicating induction of the detoxification response. Molecular responses indicative of exposure to endocrine disruptors were observed in field-caught hornyhead turbot captured in Southern California demonstrating the utility of molecular methods for monitoring environmental chemicals in wastewater outfalls. Moreover, this approach can be adapted to monitor other sites for contaminants of emerging concern in other fish species for which there are few available gene sequences. PMID:24086568

Development of a cyclic voltammetry method for the detection of Clostridium novyi in black disease.

PubMed

Liu, L L; Jiang, D N; Xiang, G M; Liu, C; Yu, J C; Pu, X Y

2014-03-17

Black disease is an acute disease of sheep and cattle. The pathogen is the obligate anaerobe, Clostridium novyi. Due to difficulties of anaerobic culturing in the country or disaster sites, a simple, rapid, and sensitive method is required. In this study, an electrochemical method, the cyclic voltammetry method, basing on loop-mediated isothermal amplification (LAMP), electrochemical ion bonding (positive dye, methylene blue), was introduced. DNA extracted from C. novyi specimens was amplified through the LAMP reaction. Then the products combined were with methylene blue, which lead to a reduction in the oxidation peak current (ipA) and the reduction peak current (ipC) of the cyclic voltammetry. The changes of ipA/ipC were real-time measured by special designed electrode, so the DNA was quantitatively detected. The results displayed that this electrochemical detection of C. novyi could be completed in 1-2 h with the lowest bacterial concentration of 10(2) colony forming units/mL, and high accuracy (96.5%), sensitivity (96%), and specificity (97%) compared to polymerase chain reation. The cyclic voltammetry method was a simple and fast method, with high sensitivity and high specificity, and has great potential to be a usable molecular tool for fast diagnosis of Black disease.

INDUCTION OF GENE EXPRESSION IN SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS) TREATED WITH 17B-ESTRADIOL, DIETHYLSTILBESTROL, OR ETHINYLESTRADIOL: THE USE OF MRNA FINGERPRINTS AS AN INDICATOR OF GENE REGULATION

EPA Science Inventory

The recent interest in hormonally active environmental contaminants has sparked a drive to find sensitive methods to measure their effects on wildlife. A molecular-based assay has been developed to measure the induction of gene expression in sheepshead minnows (Cyprinodon variega...

Molecular Methods and Platforms for Infectious Diseases Testing

PubMed Central

Emmadi, Rajyasree; Boonyaratanakornkit, Jerry B.; Selvarangan, Rangaraj; Shyamala, Venkatakrishna; Zimmer, Barbara L.; Williams, Laurina; Bryant, Bonita; Schutzbank, Ted; Schoonmaker, Michele M.; Amos Wilson, Jean A.; Hall, Leslie; Pancholi, Preeti; Bernard, Kathryn

2011-01-01

The superior sensitivity and specificity associated with the use of molecular assays has greatly improved the field of infectious disease diagnostics by providing clinicians with results that are both accurate and rapidly obtained. Herein, we review molecularly based infectious disease diagnostic tests that are Food and Drug Administration approved or cleared and commercially available in the United States as of December 31, 2010. We describe specific assays and their performance, as stated in the Food and Drug Administration's Summary of Safety and Effectiveness Data or the Office of In Vitro Diagnostic Device Evaluation and Safety's decision summaries, product inserts, or peer-reviewed literature. We summarize indications for testing, limitations, and challenges related to implementation in a clinical laboratory setting for a wide variety of common pathogens. The information presented in this review will be particularly useful for laboratories that plan to implement or expand their molecular offerings in the near term. PMID:21871973

Error correction in multi-fidelity molecular dynamics simulations using functional uncertainty quantification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reeve, Samuel Temple; Strachan, Alejandro, E-mail: strachan@purdue.edu

We use functional, Fréchet, derivatives to quantify how thermodynamic outputs of a molecular dynamics (MD) simulation depend on the potential used to compute atomic interactions. Our approach quantifies the sensitivity of the quantities of interest with respect to the input functions as opposed to its parameters as is done in typical uncertainty quantification methods. We show that the functional sensitivity of the average potential energy and pressure in isothermal, isochoric MD simulations using Lennard–Jones two-body interactions can be used to accurately predict those properties for other interatomic potentials (with different functional forms) without re-running the simulations. This is demonstrated undermore » three different thermodynamic conditions, namely a crystal at room temperature, a liquid at ambient pressure, and a high pressure liquid. The method provides accurate predictions as long as the change in potential can be reasonably described to first order and does not significantly affect the region in phase space explored by the simulation. The functional uncertainty quantification approach can be used to estimate the uncertainties associated with constitutive models used in the simulation and to correct predictions if a more accurate representation becomes available.« less

Size-exclusion chromatography of perfluorosulfonated ionomers.

PubMed

Mourey, T H; Slater, L A; Galipo, R C; Koestner, R J

2011-08-26

A size-exclusion chromatography (SEC) method in N,N-dimethylformamide containing 0.1 M LiNO(3) is shown to be suitable for the determination of molar mass distributions of three classes of perfluorosulfonated ionomers, including Nafion(®). Autoclaving sample preparation is optimized to prepare molecular solutions free of aggregates, and a solvent exchange method concentrates the autoclaved samples to enable the use of molar-mass-sensitive detection. Calibration curves obtained from light scattering and viscometry detection suggest minor variation in the specific refractive index increment across the molecular size distributions, which introduces inaccuracies in the calculation of local absolute molar masses and intrinsic viscosities. Conformation plots that combine apparent molar masses from light scattering detection with apparent intrinsic viscosities from viscometry detection partially compensate for the variations in refractive index increment. The conformation plots are consistent with compact polymer conformations, and they provide Mark-Houwink-Sakurada constants that can be used to calculate molar mass distributions without molar-mass-sensitive detection. Unperturbed dimensions and characteristic ratios calculated from viscosity-molar mass relationships indicate unusually free rotation of the perfluoroalkane backbones and may suggest limitations to applying two-parameter excluded volume theories for these ionomers. Copyright © 2011 Elsevier B.V. All rights reserved.

Recent advances in rice genome and chromosome structure research by fluorescence in situ hybridization (FISH).

PubMed

Ohmido, Nobuko; Fukui, Kiichi; Kinoshita, Toshiro

2010-01-01

Fluorescence in situ hybridization (FISH) is an effective method for the physical mapping of genes and repetitive DNA sequences on chromosomes. Physical mapping of unique nucleotide sequences on specific rice chromosome regions was performed using a combination of chromosome identification and highly sensitive FISH. Increases in the detection sensitivity of smaller DNA sequences and improvements in spatial resolution have ushered in a new phase in FISH technology. Thus, it is now possible to perform in situ hybridization on somatic chromosomes, pachytene chromosomes, and even on extended DNA fibers (EDFs). Pachytene-FISH allows the integration of genetic linkage maps and quantitative chromosome maps. Visualization methods using FISH can reveal the spatial organization of the centromere, heterochromatin/euchromatin, and the terminal structures of rice chromosomes. Furthermore, EDF-FISH and the DNA combing technique can resolve a spatial distance of 1 kb between adjacent DNA sequences, and the detection of even a 300-bp target is now feasible. The copy numbers of various repetitive sequences and the sizes of various DNA molecules were quantitatively measured using the molecular combing technique. This review describes the significance of these advances in molecular cytology in rice and discusses future applications in plant studies using visualization techniques.

Comparison of the Performances of Five Primer Sets for the Detection and Quantification of Plasmodium in Anopheline Vectors by Real-Time PCR.

PubMed

Chaumeau, V; Andolina, C; Fustec, B; Tuikue Ndam, N; Brengues, C; Herder, S; Cerqueira, D; Chareonviriyaphap, T; Nosten, F; Corbel, V

2016-01-01

Quantitative real-time polymerase chain reaction (qrtPCR) has made a significant improvement for the detection of Plasmodium in anopheline vectors. A wide variety of primers has been used in different assays, mostly adapted from molecular diagnosis of malaria in human. However, such an adaptation can impact the sensitivity of the PCR. Therefore we compared the sensitivity of five primer sets with different molecular targets on blood stages, sporozoites and oocysts standards of Plasmodium falciparum (Pf) and P. vivax (Pv). Dilution series of standard DNA were used to discriminate between methods at low concentrations of parasite and to generate standard curves suitable for the absolute quantification of Plasmodium sporozoites. Our results showed that the best primers to detect blood stages were not necessarily the best ones to detect sporozoites. Absolute detection threshold of our qrtPCR assay varied between 3.6 and 360 Pv sporozoites and between 6 and 600 Pf sporozoites per mosquito according to the primer set used in the reaction mix. In this paper, we discuss the general performance of each primer set and highlight the need to use efficient detection methods for transmission studies.

Comparison of the Performances of Five Primer Sets for the Detection and Quantification of Plasmodium in Anopheline Vectors by Real-Time PCR

PubMed Central

Chaumeau, V.; Andolina, C.; Fustec, B.; Tuikue Ndam, N.; Brengues, C.; Herder, S.; Cerqueira, D.; Chareonviriyaphap, T.; Nosten, F.; Corbel, V.

2016-01-01

Quantitative real-time polymerase chain reaction (qrtPCR) has made a significant improvement for the detection of Plasmodium in anopheline vectors. A wide variety of primers has been used in different assays, mostly adapted from molecular diagnosis of malaria in human. However, such an adaptation can impact the sensitivity of the PCR. Therefore we compared the sensitivity of five primer sets with different molecular targets on blood stages, sporozoites and oocysts standards of Plasmodium falciparum (Pf) and P. vivax (Pv). Dilution series of standard DNA were used to discriminate between methods at low concentrations of parasite and to generate standard curves suitable for the absolute quantification of Plasmodium sporozoites. Our results showed that the best primers to detect blood stages were not necessarily the best ones to detect sporozoites. Absolute detection threshold of our qrtPCR assay varied between 3.6 and 360 Pv sporozoites and between 6 and 600 Pf sporozoites per mosquito according to the primer set used in the reaction mix. In this paper, we discuss the general performance of each primer set and highlight the need to use efficient detection methods for transmission studies. PMID:27441839

Gating Charge Calculations by Computational Electrophysiology Simulations.

PubMed

Machtens, Jan-Philipp; Briones, Rodolfo; Alleva, Claudia; de Groot, Bert L; Fahlke, Christoph

2017-04-11

Electrical cell signaling requires adjustment of ion channel, receptor, or transporter function in response to changes in membrane potential. For the majority of such membrane proteins, the molecular details of voltage sensing remain insufficiently understood. Here, we present a molecular dynamics simulation-based method to determine the underlying charge movement across the membrane-the gating charge-by measuring electrical capacitor properties of membrane-embedded proteins. We illustrate the approach by calculating the charge transfer upon membrane insertion of the HIV gp41 fusion peptide, and validate the method on two prototypical voltage-dependent proteins, the Kv1.2 K + channel and the voltage sensor of the Ciona intestinalis voltage-sensitive phosphatase, against experimental data. We then use the gating charge analysis to study how the T1 domain modifies voltage sensing in Kv1.2 channels and to investigate the voltage dependence of the initial binding of two Na + ions in Na + -coupled glutamate transporters. Our simulation approach quantifies various mechanisms of voltage sensing, enables direct comparison with experiments, and supports mechanistic interpretation of voltage sensitivity by fractional amino acid contributions. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A Paramagnetic Molecular Voltmeter

PubMed Central

Surek, Jack T.; Thomas, David D.

2008-01-01

We have developed a general electron paramagnetic resonance (EPR) method to measure electrostatic potential at spin labels on proteins to millivolt accuracy. Electrostatic potential is fundamental to energy-transducing proteins like myosin, because molecular energy storage and retrieval is primarily electrostatic. Quantitative analysis of protein electrostatics demands a site-specific spectroscopic method sensitive to millivolt changes. Previous electrostatic potential studies on macromolecules fell short in sensitivity, accuracy and/or specificity. Our approach uses fast-relaxing charged and neutral paramagnetic relaxation agents (PRAs) to increase nitroxide spin label relaxation rate solely through collisional spin exchange. These PRAs were calibrated in experiments on small nitroxides of known structure and charge to account for differences in their relaxation efficiency. Nitroxide longitudinal (R1) and transverse (R2) relaxation rates were separated by applying lineshape analysis to progressive saturation spectra. The ratio of measured R1 increases for each pair of charged and neutral PRAs measures the shift in local PRA concentration due to electrostatic potential. Voltage at the spin label is then calculated using the Boltzmann equation. Measured voltages for two small charged nitroxides agree with Debye-Hückel calculations. Voltage for spin-labeled myosin fragment S1 also agrees with calculation based on the pK shift of the reacted cysteine. PMID:17964835

Diagnosis of Strongyloides stercoralis by morphological characteristics combine with molecular biological methods.

PubMed

Wang, Li-Fu; Xu, Lian; Luo, Shi-Qi; Xie, Hui; Chen, Wei; Wu, Zhong-Dao; Sun, Xi

2017-04-01

Strongyloidiasis is one of the neglected tropical diseases caused by infection with the nematode Strongyloides genus and distributed worldwide. Strongyloidiasis can be fatal in immunosuppressed patients induced hyperinfection or disseminated strongyloidiasis. Unfortunately, until now, due to the unspecific clinical symptom in infected individuals and the low sensitivity diagnosis of strongyloidiasis, many patients were misdiagnosed every year. Furthermore, the larvae of the Strongyloides stercoralis (S. stercoralis) is similar to other nematodes such as hookworm, Trichostrongylus increased the difficulty of diagnosis. In this case, the patient is a 63-year-old male person, who had a nearly 30 years medical history of asthma and emphysema, and 4-5-year medical history of diabetes. The sputum examination found some parasite larvae, then we identify the larvae using clinical observation and morphological characteristics combine with examined cytochrome oxidase subunit 1 (COX1) and 18S rRNA genes by PCR, sequence analysis and finally classified by phylogenetic analysis, the larvae were diagnosed as S. stercoralis. Our results showed that diagnosis with strongyloidiasis by morphological characteristics combine with molecular biological methods can improve the sensitive of diagnosis and provide a final diagnosis for the disease in the clinics.

SISGR: Room Temperature Single-Molecule Detection and Imaging by Stimulated Emission Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Xiaoliang Sunney

Single-molecule spectroscopy has made considerable impact on many disciplines including chemistry, physics, and biology. To date, most single-molecule spectroscopy work is accomplished by detecting fluorescence. On the other hand, many naturally occurring chromophores, such as retinal, hemoglobin and cytochromes, do not have detectable fluorescence. There is an emerging need for single-molecule spectroscopy techniques that do not require fluorescence. In the last proposal period, we have successfully demonstrated stimulated emission microscopy, single molecule absorption, and stimulated Raman microscopy based on a high-frequency modulation transfer technique. These first-of-a- kind new spectroscopy/microscopy methods tremendously improved our ability to observe molecules that fluorescence weakly,more » even to the limit of single molecule detection for absorption measurement. All of these methods employ two laser beams: one (pump beam) excites a single molecule to a real or virtual excited state, and the other (probe beam) monitors the absorption/emission property of the single. We extract the intensity change of the probe beam with high sensitivity by implementing a high-frequency phase-sensitive detection scheme, which offers orders of magnitude improvement in detection sensitivity over direct absorption/emission measurement. However, single molecule detection based on fluorescence or absorption is fundamentally limited due to their broad spectral response. It is important to explore other avenues in single molecule detection and imaging which provides higher molecular specificity for studying a wide variety of heterogeneous chemical and biological systems. This proposal aimed to achieve single-molecule detection sensitivity with near resonance stimulated Raman scattering (SRS) microscopy. SRS microscopy was developed in our lab as a powerful technique for imaging heterogeneous samples based on their intrinsic vibrational contrasts, which provides much higher molecular specificity than absorption and fluorescence. Current sensitivity limit of SRS microscopy has not yet reached single molecule detection. We proposed to capitalize on our state-of-the-art SRS microscopy and develop near-resonance enhanced SRS for single molecule detection of carotenoids and heme proteins. The specific aims we pursued are: (1) building the next SRS generation microscope that utilizes near resonance enhancement to allow detection and imaging of single molecules with undetectable fluorescence, such as -carotene. (2) using near-resonance SRS as a contrast mechanism to study dye-sensitize semiconductor interface, elucidating the heterogeneous electron ejection kinetics with high spatial and temporal resolution. (3) studying the binding and unbinding of oxygen in single hemoglobin molecules in order to gain molecular level understanding of the long-standing issue of cooperativity. The new methods developed in the fund period of this grant have advanced the detection sensitivity in many aspects. Near-resonance SRS improved the signal by using shorter wavelengths for SRS microscopy. Frequency modulation and multi-color SRS target the reduction of background to improve the chemical specificity of SRS while maintaining the high imaging speed. Time-domain coherent Raman scattering microscopy targets to reduce the noise floor of coherent Raman microscopy. These methods have already demonstrated first-of-a-kind new applications in biology and medical research. However, we are still one order of magnitude away from single molecule limit. It is important to continue to improve the laser specification and develop new imaging methods to finally achieve label-free single molecule microscopy.« less

Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules

PubMed Central

Kühnemund, Malte; Hernández-Neuta, Iván; Sharif, Mohd Istiaq; Cornaglia, Matteo; Gijs, Martin A.M.

2017-01-01

Abstract Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope. PMID:28077562

Synthesis and Characterization of a New Co-Crystal Explosive with High Energy and Good Sensitivity

NASA Astrophysics Data System (ADS)

Gao, Han; Jiang, Wei; Liu, Jie; Hao, Gazi; Xiao, Lei; Ke, Xiang; Chen, Teng

2017-10-01

A new energetic co-crystal consisting of one of the most powerful explosive molecules 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20) and the military explosive cyclotrimethylenetrinitramine (RDX) was prepared with a simple solvent evaporation method. Scanning electron microscopy (SEM) revealed the morphology of the bar-shaped product, which differed greatly from the morphology of the individual components. Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray diffraction spectrum (XRD), and differential scanning calorimetry (DSC) proved the formation of the co-crystal at the molecular level. The result of mechanical sensitivity test indicated the sensitivity was effectively reduced compared to raw CL-20. Finally, a possible crystallization mechanism was discussed.

Nanotechnology: a promising method for oral cancer detection and diagnosis.

PubMed

Chen, Xiao-Jie; Zhang, Xue-Qiong; Liu, Qi; Zhang, Jing; Zhou, Gang

2018-06-11

Oral cancer is a common and aggressive cancer with high morbidity, mortality, and recurrence rate globally. Early detection is of utmost importance for cancer prevention and disease management. Currently, tissue biopsy remains the gold standard for oral cancer diagnosis, but it is invasive, which may cause patient discomfort. The application of traditional noninvasive methods-such as vital staining, exfoliative cytology, and molecular imaging-is limited by insufficient sensitivity and specificity. Thus, there is an urgent need for exploring noninvasive, highly sensitive, and specific diagnostic techniques. Nano detection systems are known as new emerging noninvasive strategies that bring the detection sensitivity of biomarkers to nano-scale. Moreover, compared to current imaging contrast agents, nanoparticles are more biocompatible, easier to synthesize, and able to target specific surface molecules. Nanoparticles generate localized surface plasmon resonances at near-infrared wavelengths, providing higher image contrast and resolution. Therefore, using nano-based techniques can help clinicians to detect and better monitor diseases during different phases of oral malignancy. Here, we review the progress of nanotechnology-based methods in oral cancer detection and diagnosis.

Giardiasis: an update review on sensitivity and specificity of methods for laboratorial diagnosis.

PubMed

Soares, Renata; Tasca, Tiana

2016-10-01

Giardiasis is a major cause of diarrhoea transmitted by ingestion of contaminated water and food with cysts, and it has been spread among people with poor oral hygiene. The traditional diagnosis is performed by identifying trophozoites and cysts of Giardia duodenalis through microscopy of faecal samples. In addition to microscopy, different methods have been validated for giardiasis diagnosis which are based on immunologic and molecular analyses. The aim of this study was to conduct a review of the main methods applied in clinical laboratory for diagnosis of giardiasis, in the last 10years, regarding the specificity and sensitivity criteria. It was observed high variability in the performance of the same methodology across studies; however, several techniques have been considered better than microscopy. The later, although gold standard, presents low sensitivity in cases of low number of cysts in the sample, and the experience of the microscopist must also be considered. We conclude that microscopy should still be held and complementary technique is recommended, in order to provide a reliable diagnosis and a proper treatment of the patient. Copyright © 2016 Elsevier B.V. All rights reserved.

Integrated digital error suppression for improved detection of circulating tumor DNA

PubMed Central

Kurtz, David M.; Chabon, Jacob J.; Scherer, Florian; Stehr, Henning; Liu, Chih Long; Bratman, Scott V.; Say, Carmen; Zhou, Li; Carter, Justin N.; West, Robert B.; Sledge, George W.; Shrager, Joseph B.; Loo, Billy W.; Neal, Joel W.; Wakelee, Heather A.; Diehn, Maximilian; Alizadeh, Ash A.

2016-01-01

High-throughput sequencing of circulating tumor DNA (ctDNA) promises to facilitate personalized cancer therapy. However, low quantities of cell-free DNA (cfDNA) in the blood and sequencing artifacts currently limit analytical sensitivity. To overcome these limitations, we introduce an approach for integrated digital error suppression (iDES). Our method combines in silico elimination of highly stereotypical background artifacts with a molecular barcoding strategy for the efficient recovery of cfDNA molecules. Individually, these two methods each improve the sensitivity of cancer personalized profiling by deep sequencing (CAPP-Seq) by ~3 fold, and synergize when combined to yield ~15-fold improvements. As a result, iDES-enhanced CAPP-Seq facilitates noninvasive variant detection across hundreds of kilobases. Applied to clinical non-small cell lung cancer (NSCLC) samples, our method enabled biopsy-free profiling of EGFR kinase domain mutations with 92% sensitivity and 96% specificity and detection of ctDNA down to 4 in 105 cfDNA molecules. We anticipate that iDES will aid the noninvasive genotyping and detection of ctDNA in research and clinical settings. PMID:27018799

Detection and Characterization of Viral Species/Subspecies Using Isothermal Recombinase Polymerase Amplification (RPA) Assays.

PubMed

Glais, Laurent; Jacquot, Emmanuel

2015-01-01

Numerous molecular-based detection protocols include an amplification step of the targeted nucleic acids. This step is important to reach the expected sensitive detection of pathogens in diagnostic procedures. Amplifications of nucleic acid sequences are generally performed, in the presence of appropriate primers, using thermocyclers. However, the time requested to amplify molecular targets and the cost of the thermocycler machines could impair the use of these methods in routine diagnostics. Recombinase polymerase amplification (RPA) technique allows rapid (short-term incubation of sample and primers in an enzymatic mixture) and simple (isothermal) amplification of molecular targets. RPA protocol requires only basic molecular steps such as extraction procedures and agarose gel electrophoresis. Thus, RPA can be considered as an interesting alternative to standard molecular-based diagnostic tools. In this paper, the complete procedures to set up an RPA assay, applied to detection of RNA (Potato virus Y, Potyvirus) and DNA (Wheat dwarf virus, Mastrevirus) viruses, are described. The proposed procedure allows developing species- or subspecies-specific detection assay.

Method Designed to Respect Molecular Heterogeneity Can Profoundly Correct Present Data Interpretations for Genome-Wide Expression Analysis

PubMed Central

Chen, Chih-Hao; Hsu, Chueh-Lin; Huang, Shih-Hao; Chen, Shih-Yuan; Hung, Yi-Lin; Chen, Hsiao-Rong; Wu, Yu-Chung

2015-01-01

Although genome-wide expression analysis has become a routine tool for gaining insight into molecular mechanisms, extraction of information remains a major challenge. It has been unclear why standard statistical methods, such as the t-test and ANOVA, often lead to low levels of reproducibility, how likely applying fold-change cutoffs to enhance reproducibility is to miss key signals, and how adversely using such methods has affected data interpretations. We broadly examined expression data to investigate the reproducibility problem and discovered that molecular heterogeneity, a biological property of genetically different samples, has been improperly handled by the statistical methods. Here we give a mathematical description of the discovery and report the development of a statistical method, named HTA, for better handling molecular heterogeneity. We broadly demonstrate the improved sensitivity and specificity of HTA over the conventional methods and show that using fold-change cutoffs has lost much information. We illustrate the especial usefulness of HTA for heterogeneous diseases, by applying it to existing data sets of schizophrenia, bipolar disorder and Parkinson’s disease, and show it can abundantly and reproducibly uncover disease signatures not previously detectable. Based on 156 biological data sets, we estimate that the methodological issue has affected over 96% of expression studies and that HTA can profoundly correct 86% of the affected data interpretations. The methodological advancement can better facilitate systems understandings of biological processes, render biological inferences that are more reliable than they have hitherto been and engender translational medical applications, such as identifying diagnostic biomarkers and drug prediction, which are more robust. PMID:25793610

Non destructive examination of interface of molecular assembly

NASA Astrophysics Data System (ADS)

Perez, Guy; Richard, Isaline; Lecomte, Jean-Claude

2017-11-01

Molecular assembly interfaces can be characterised by mechanical testing and/or the interaction between waves and the interface. The disadvantage of the mechanical approach is that new defects may be produced at the interface, or existing defects may be destroyed. Using the interaction between waves and the interface is a non-destructive approach. But what kind of waves should be used? Electromagnetic waves in the visible range depend on wave attenuation in the material, infrared waves also depend on the thickness and X-ray waves have a too short a wave length to detect interface defects. In this article, the use of acoustic waves is proposed for non-destructive examination of molecular assembly interfaces. Acoustic wave propagation is very sensitive to variations in interface characteristics depending on whether the waves are reflected or transmitted. To improve the sensitivity and resolution of this technique, small wave lengths have been used with a scanning acoustic microscope (S.A.M.) with a band width from 1MHz to 400 MHz. After a short description of the principle of the method, results are given for different types of components. Different applications of acoustic microscopy are proposed for non-destructive examination of interfaces and defect detection in materials.

Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia.

PubMed

Giustacchini, Alice; Thongjuea, Supat; Barkas, Nikolaos; Woll, Petter S; Povinelli, Benjamin J; Booth, Christopher A G; Sopp, Paul; Norfo, Ruggiero; Rodriguez-Meira, Alba; Ashley, Neil; Jamieson, Lauren; Vyas, Paresh; Anderson, Kristina; Segerstolpe, Åsa; Qian, Hong; Olsson-Strömberg, Ulla; Mustjoki, Satu; Sandberg, Rickard; Jacobsen, Sten Eirik W; Mead, Adam J

2017-06-01

Recent advances in single-cell transcriptomics are ideally placed to unravel intratumoral heterogeneity and selective resistance of cancer stem cell (SC) subpopulations to molecularly targeted cancer therapies. However, current single-cell RNA-sequencing approaches lack the sensitivity required to reliably detect somatic mutations. We developed a method that combines high-sensitivity mutation detection with whole-transcriptome analysis of the same single cell. We applied this technique to analyze more than 2,000 SCs from patients with chronic myeloid leukemia (CML) throughout the disease course, revealing heterogeneity of CML-SCs, including the identification of a subgroup of CML-SCs with a distinct molecular signature that selectively persisted during prolonged therapy. Analysis of nonleukemic SCs from patients with CML also provided new insights into cell-extrinsic disruption of hematopoiesis in CML associated with clinical outcome. Furthermore, we used this single-cell approach to identify a blast-crisis-specific SC population, which was also present in a subclone of CML-SCs during the chronic phase in a patient who subsequently developed blast crisis. This approach, which might be broadly applied to any malignancy, illustrates how single-cell analysis can identify subpopulations of therapy-resistant SCs that are not apparent through cell-population analysis.

Development of a polymerase chain reaction applicable to rapid and sensitive detection of Clonorchis sinensis eggs in human stool samples

PubMed Central

Cho, Pyo Yun; Na, Byoung-Kuk; Mi Choi, Kyung; Kim, Jin Su; Cho, Shin-Hyeong; Lee, Won-Ja; Lim, Sung-Bin; Cha, Seok Ho; Park, Yun-Kyu; Pak, Jhang Ho; Lee, Hyeong-Woo; Hong, Sung-Jong; Kim, Tong-Soo

2013-01-01

Microscopic examination of eggs of parasitic helminths in stool samples has been the most widely used classical diagnostic method for infections, but tiny and low numbers of eggs in stool samples often hamper diagnosis of helminthic infections with classical microscopic examination. Moreover, it is also difficult to differentiate parasite eggs by the classical method, if they have similar morphological characteristics. In this study, we developed a rapid and sensitive polymerase chain reaction (PCR)-based molecular diagnostic method for detection of Clonorchis sinensis eggs in stool samples. Nine primers were designed based on the long-terminal repeat (LTR) of C. sinensis retrotransposon1 (CsRn1) gene, and seven PCR primer sets were paired. Polymerase chain reaction with each primer pair produced specific amplicons for C. sinensis, but not for other trematodes including Metagonimus yokogawai and Paragonimus westermani. Particularly, three primer sets were able to detect 10 C. sinensis eggs and were applicable to amplify specific amplicons from DNA samples purified from stool of C. sinensis-infected patients. This PCR method could be useful for diagnosis of C. sinensis infections in human stool samples with a high level of specificity and sensitivity. PMID:23916334

Performance Comparison of Bench-Top Next Generation Sequencers Using Microdroplet PCR-Based Enrichment for Targeted Sequencing in Patients with Autism Spectrum Disorder

PubMed Central

Okamoto, Nobuhiko; Nakashima, Mitsuko; Tsurusaki, Yoshinori; Miyake, Noriko; Saitsu, Hirotomo; Matsumoto, Naomichi

2013-01-01

Next-generation sequencing (NGS) combined with enrichment of target genes enables highly efficient and low-cost sequencing of multiple genes for genetic diseases. The aim of this study was to validate the accuracy and sensitivity of our method for comprehensive mutation detection in autism spectrum disorder (ASD). We assessed the performance of the bench-top Ion Torrent PGM and Illumina MiSeq platforms as optimized solutions for mutation detection, using microdroplet PCR-based enrichment of 62 ASD associated genes. Ten patients with known mutations were sequenced using NGS to validate the sensitivity of our method. The overall read quality was better with MiSeq, largely because of the increased indel-related error associated with PGM. The sensitivity of SNV detection was similar between the two platforms, suggesting they are both suitable for SNV detection in the human genome. Next, we used these methods to analyze 28 patients with ASD, and identified 22 novel variants in genes associated with ASD, with one mutation detected by MiSeq only. Thus, our results support the combination of target gene enrichment and NGS as a valuable molecular method for investigating rare variants in ASD. PMID:24066114

Molecular detection of cytomegalovirus, herpes simplex virus 2, human papillomavirus 16-18 in Turkish pregnants.

PubMed

Dinc, Bedia; Bozdayi, Gulendam; Biri, Aydan; Kalkanci, Ayse; Dogan, Bora; Bozkurt, Nuray; Rota, Seyyal

2010-01-01

Human cytomegalovirus (CMV) is the most common cause of viral intrauterine infections in the world. Herpes simplex virus type 2 (HSV-2) and human papillomavirus (HPV) are the main agents of viral sexually transmitted diseases, which cause genital ulcers and genital warts, respectively. HPV infection has been linked to the majority of the anogenital malignancies. The aim of this study was to detect the existence of CMV, HSV-2 and HPV type 16-18 in Turkish pregnants by using sensitive molecular assays. One hundred thirty-four women (18-41 years old; mean age ± SD: 27 ± 8) applied to outpatient clinic of Obstetrics and Gynecology, in between 18th - 22nd weeks of their pregnancy and a control group of 99 healthy women (15-39 years old; mean age ± SD: 24 ± 8) were included in the study. Cervical smear samples were used for DNA extraction. CMV, HSV-2 and HPV 16-18 detections were carried out by real time PCR and in house PCR method, respectively. Three patients (3/134; 2.2%) were found to be positive for each HPV and HSV-2. Dual infection with HPV and HSV was found in just one patient. HPV 18 was detected in all positive samples. CMV was found to be positive in two patients (2/134; 1.4 %). HPV, HSV and CMV must be screened due to high prevalence of these viruses in pregnants by using sensitive molecular methods.

Voltammetric determination of attomolar levels of a sequence derived from the genom of hepatitis B virus by using molecular beacon mediated circular strand displacement and rolling circle amplification.

PubMed

Huang, Shan; Feng, Mengmeng; Li, Jiawen; Liu, Yi; Xiao, Qi

2018-03-03

The authors describe an electrochemical method for the determination of the single-stranded DNA (ssDNA) oligonucleotide with a sequence derived from the genom of hepatitis B virus (HBV). It is making use of circular strand displacement (CSD) and rolling circle amplification (RCA) strategies mediated by a molecular beacon (MB). This ssDNA hybridizes with the loop portion of the MB immobilized on the surface of a gold electrode, while primer DNA also hybridizes with the rest of partial DNA sequences of MB. This triggers the MB-mediated CSD. The RCA is then initiated to produce a long DNA strand with multiple tandem-repeat sequences, and this results in a significant increase of the differential pulse voltammetric response of the electrochemical probe Methylene Blue at a rather low working potential of -0.24 V (vs. Ag/AgCl). Under optimal experimental conditions, the assay displays an ultrahigh sensitivity (with a 2.6 aM detection limit) and excellent selectivity. Response is linear in the 10 to 700 aM DNA concentration range. Graphical abstract Schematic of a voltammetric method for the determination of attomolar levels of target DNA. It is based on molecular beacon mediated circular strand displacement and rolling circle amplification strategies. Under optimal experimental conditions, the assay displays an ultrahigh sensitivity with a 2.6 aM detection limit and excellent selectivity.

Plasmon Resonance Methods in GPCR Signaling and Other Membrane Events

PubMed Central

Alves, I.D.; Park, C.K.; Hruby, V.J.

2005-01-01

The existence of surface guided electromagnetic waves has been theoretically predicted from Maxwell’s equations and investigated during the first decades of the 20th century. However, it is only since the late 1960’s that they have attracted the interest of surface physicists and earned the moniker of “surface plasmon”. With the advent of commercially available instruments and well established theories, the technique has been used to study a wide variety of biochemical and biotechnological phenomena. Spectral response of the resonance condition serves as a sensitive indicator of the optical properties of thin films immobilized within a wavelength of the surface. This enhanced surface sensitivity has provided a boon to the surface sciences, and fosters collaboration between surface chemistry, physics and the ongoing biological and biotechnological revolution. Since then, techniques based on surface plasmons such as Surface Plasmon Resonance (SPR), SPR Imaging, Plasmon Waveguide Resonance (PWR) and others, have been increasingly used to determine the affinity and kinetics of a wide variety of real time molecular interactions such as protein-protein, lipid-protein and ligand-protein, without the need for a molecular tag or label. The physical-chemical methodologies used to immobilize membranes at the surface of these optical devices are reviewed, pointing out advantages and limitations of each method. The paper serves to summarize both historical and more recent developments of these technologies for investigating structure-function aspects of these molecular interactions, and regulation of specific events in signal transduction by G-protein coupled receptors (GPCRs). PMID:16101432

Recent advances in immunosensor for narcotic drug detection

PubMed Central

Gandhi, Sonu; Suman, Pankaj; Kumar, Ashok; Sharma, Prince; Capalash, Neena; Suri, C. Raman

2015-01-01

Introduction: Immunosensor for illicit drugs have gained immense interest and have found several applications for drug abuse monitoring. This technology has offered a low cost detection of narcotics; thereby, providing a confirmatory platform to compliment the existing analytical methods. Methods: In this minireview, we define the basic concept of transducer for immunosensor development that utilizes antibodies and low molecular mass hapten (opiate) molecules. Results: This article emphasizes on recent advances in immunoanalytical techniques for monitoring of opiate drugs. Our results demonstrate that high quality antibodies can be used for immunosensor development against target analyte with greater sensitivity, specificity and precision than other available analytical methods. Conclusion: In this review we highlight the fundamentals of different transducer technologies and its applications for immunosensor development currently being developed in our laboratory using rapid screening via immunochromatographic kit, label free optical detection via enzyme, fluorescence, gold nanoparticles and carbon nanotubes based immunosensing for sensitive and specific monitoring of opiates. PMID:26929925

Multiscale Mathematics for Biomass Conversion to Renewable Hydrogen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Katsoulakis, Markos

2014-08-09

Our two key accomplishments in the first three years were towards the development of, (1) a mathematically rigorous and at the same time computationally flexible framework for parallelization of Kinetic Monte Carlo methods, and its implementation on GPUs, and (2) spatial multilevel coarse-graining methods for Monte Carlo sampling and molecular simulation. A common underlying theme in both these lines of our work is the development of numerical methods which are at the same time both computationally efficient and reliable, the latter in the sense that they provide controlled-error approximations for coarse observables of the simulated molecular systems. Finally, our keymore » accomplishment in the last year of the grant is that we started developing (3) pathwise information theory-based and goal-oriented sensitivity analysis and parameter identification methods for complex high-dimensional dynamics and in particular of nonequilibrium extended (high-dimensional) systems. We discuss these three research directions in some detail below, along with the related publications.« less

Application of molecularly imprinted polymer based matrix solid phase dispersion for determination of fluoroquinolones, tetracyclines and sulfonamides in meat.

PubMed

Wang, Geng Nan; Zhang, Lei; Song, Yi Ping; Liu, Ju Xiang; Wang, Jian Ping

2017-10-15

In this study, a type of novel mixed-template molecularly imprinted polymer was synthesized that was able to recognize 8 fluoroquinolones, 8 sulfonamides and 4 tetracyclines simultaneously with recoveries higher than 92%. Then the polymer was used to develop a matrix solid phase dispersion method for simultaneous extraction of the 20 drugs in pork followed by determination with ultra performance liquid chromatography. During the experiments, the MMIP amount, washing solvent and elution solvent were optimized respectively. The limits of detection of this method for the 20 drugs in pork were in the range of 0.5-3.0ngg -1 , and the intra-day and inter-day recoveries from the fortified blank samples were in the range of 74.5%-102.7%. Therefore, this method could be used as a rapid, simple, specific and sensitive method for multi-determination of the residues of the three classes of drugs in meat. Copyright © 2017 Elsevier B.V. All rights reserved.

Scrambled eggs: A highly sensitive molecular diagnostic workflow for Fasciola species specific detection from faecal samples.

PubMed

Calvani, Nichola Eliza Davies; Windsor, Peter Andrew; Bush, Russell David; Šlapeta, Jan

2017-09-01

Fasciolosis, due to Fasciola hepatica and Fasciola gigantica, is a re-emerging zoonotic parasitic disease of worldwide importance. Human and animal infections are commonly diagnosed by the traditional sedimentation and faecal egg-counting technique. However, this technique is time-consuming and prone to sensitivity errors when a large number of samples must be processed or if the operator lacks sufficient experience. Additionally, diagnosis can only be made once the 12-week pre-patent period has passed. Recently, a commercially available coprological antigen ELISA has enabled detection of F. hepatica prior to the completion of the pre-patent period, providing earlier diagnosis and increased throughput, although species differentiation is not possible in areas of parasite sympatry. Real-time PCR offers the combined benefits of highly sensitive species differentiation for medium to large sample sizes. However, no molecular diagnostic workflow currently exists for the identification of Fasciola spp. in faecal samples. A new molecular diagnostic workflow for the highly-sensitive detection and quantification of Fasciola spp. in faecal samples was developed. The technique involves sedimenting and pelleting the samples prior to DNA isolation in order to concentrate the eggs, followed by disruption by bead-beating in a benchtop homogeniser to ensure access to DNA. Although both the new molecular workflow and the traditional sedimentation technique were sensitive and specific, the new molecular workflow enabled faster sample throughput in medium to large epidemiological studies, and provided the additional benefit of speciation. Further, good correlation (R2 = 0.74-0.76) was observed between the real-time PCR values and the faecal egg count (FEC) using the new molecular workflow for all herds and sampling periods. Finally, no effect of storage in 70% ethanol was detected on sedimentation and DNA isolation outcomes; enabling transport of samples from endemic to non-endemic countries without the requirement of a complete cold chain. The commercially-available ELISA displayed poorer sensitivity, even after adjustment of the positive threshold (65-88%), compared to the sensitivity (91-100%) of the new molecular diagnostic workflow. Species-specific assays for sensitive detection of Fasciola spp. enable ante-mortem diagnosis in both human and animal settings. This includes Southeast Asia where there are potentially many undocumented human cases and where post-mortem examination of production animals can be difficult. The new molecular workflow provides a sensitive and quantitative diagnostic approach for the rapid testing of medium to large sample sizes, potentially superseding the traditional sedimentation and FEC technique and enabling surveillance programs in locations where animal and human health funding is limited.

High sensitivity optical molecular imaging system

NASA Astrophysics Data System (ADS)

An, Yu; Yuan, Gao; Huang, Chao; Jiang, Shixin; Zhang, Peng; Wang, Kun; Tian, Jie

2018-02-01

Optical Molecular Imaging (OMI) has the advantages of high sensitivity, low cost and ease of use. By labeling the regions of interest with fluorescent or bioluminescence probes, OMI can noninvasively obtain the distribution of the probes in vivo, which play the key role in cancer research, pharmacokinetics and other biological studies. In preclinical and clinical application, the image depth, resolution and sensitivity are the key factors for researchers to use OMI. In this paper, we report a high sensitivity optical molecular imaging system developed by our group, which can improve the imaging depth in phantom to nearly 5cm, high resolution at 2cm depth, and high image sensitivity. To validate the performance of the system, special designed phantom experiments and weak light detection experiment were implemented. The results shows that cooperated with high performance electron-multiplying charge coupled device (EMCCD) camera, precision design of light path system and high efficient image techniques, our OMI system can simultaneously collect the light-emitted signals generated by fluorescence molecular imaging, bioluminescence imaging, Cherenkov luminance and other optical imaging modality, and observe the internal distribution of light-emitting agents fast and accurately.

Redox-Triggered Bonding-Induced Emission of Thiol-Functionalized Gold Nanoclusters for Luminescence Turn-On Detection of Molecular Oxygen.

PubMed

Ao, Hang; Feng, Hui; Zhao, Mengting; Zhao, Meizhi; Chen, Jianrong; Qian, Zhaosheng

2017-11-22

Most optical sensors for molecular oxygen were developed based on the quenching effect of the luminescence of oxygen-sensitive probes; however, the signal turn-off mode of these probes is undesirable to quantify and visualize molecular oxygen. Herein, we report a novel luminescence turn-on detection strategy for molecular oxygen via the specific oxygen-triggered bonding-induced emission of thiol-functionalized gold nanoclusters. Thiol-functionalized gold nanoclusters were prepared by a facile one-step synthesis, and as-prepared gold nanoclusters possess significant aggregation-induced emission (AIE) property. It is the first time to discover the oxygen-triggered bonding-induced emission (BIE) behavior of gold nanoclusters, which results in disulfide-linked covalent bonding assemblies with intensely red luminescence. This specific redox-triggered BIE is capable of quantitatively detecting dissolved oxygen in aqueous solution in a light-up manner, and trace amount of dissolved oxygen at ppb level is achieved based on this detection method. A facile and convenient test strip for oxygen detection was also developed to monitor molecular oxygen in a gas matrix. Covalent bonding-induced emission is proven to be a more efficient way to attain high brightness of AIEgens than a physical aggregation-induced emission process, and provides a more convenient and desirable detection method for molecular oxygen than the previous sensors.

Molecular signatures of transgenerational response to ocean acidification in a species of reef fish

NASA Astrophysics Data System (ADS)

Schunter, Celia; Welch, Megan J.; Ryu, Taewoo; Zhang, Huoming; Berumen, Michael L.; Nilsson, Göran E.; Munday, Philip L.; Ravasi, Timothy

2016-11-01

The impact of ocean acidification on marine ecosystems will depend on species capacity to adapt. Recent studies show that the behaviour of reef fishes is impaired at projected CO 2 levels; however, individual variation exists that might promote adaptation. Here, we show a clear signature of parental sensitivity to high CO 2 in the brain molecular phenotype of juvenile spiny damselfish, Acanthochromis polyacanthus, primarily driven by circadian rhythm genes. Offspring of CO 2-tolerant and CO 2-sensitive parents were reared at near-future CO 2 (754 μatm) or present-day control levels (414 μatm). By integrating 33 brain transcriptomes and proteomes with a de novo assembled genome we investigate the molecular responses of the fish brain to increased CO 2 and the expression of parental tolerance to high CO 2 in the offspring molecular phenotype. Exposure to high CO 2 resulted in differential regulation of 173 and 62 genes and 109 and 68 proteins in the tolerant and sensitive groups, respectively. Importantly, the majority of differences between offspring of tolerant and sensitive parents occurred in high CO 2 conditions. This transgenerational molecular signature suggests that individual variation in CO 2 sensitivity could facilitate adaptation of fish populations to ocean acidification.

Methicillin-Resistant Staphylococcus aureus (MRSA) Detection: Comparison of Two Molecular Methods (IDI-MRSA PCR Assay and GenoType MRSA Direct PCR Assay) with Three Selective MRSA Agars (MRSA ID, MRSASelect, and CHROMagar MRSA) for Use with Infection-Control Swabs▿

PubMed Central

van Hal, S. J.; Stark, D.; Lockwood, B.; Marriott, D.; Harkness, J.

2007-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) is an increasing problem. Rapid detection of MRSA-colonized patients has the potential to limit spread of the organism. We evaluated the sensitivities and specificities of MRSA detection by two molecular methods (IDI-MRSA PCR assay and GenoType MRSA Direct PCR assay) and three selective MRSA agars (MRSA ID, MRSASelect, and CHROMagar MRSA), using 205 (101 nasal, 52 groin, and 52 axillary samples) samples from consecutive known MRSA-infected and/or -colonized patients. All detection methods had higher MRSA detection rates for nasal swabs than for axillary and groin swabs. Detection of MRSA by IDI-MRSA was the most sensitive method, independent of the site (94% for nasal samples, 80% for nonnasal samples, and 90% overall). The sensitivities of the GenoType MRSA Direct assay and the MRSA ID, MRSASelect, and CHROMagar MRSA agars with nasal swabs were 70%, 72%, 68%, and 75%, respectively. All detection methods had high specificities (95 to 99%), independent of the swab site. Extended incubation for a further 24 h with selective MRSA agars increased the detection of MRSA, with a corresponding decline in specificity secondary to a significant increase in false-positive results. There was a noticeable difference in test performance of the GenoType MRSA Direct assay in detection of MRSA (28/38 samples [74%]) compared with detection of nonmultiresistant MRSA (17/31 samples [55%]) (susceptible to two or more non-β-lactam antibiotics). This was not observed with selective MRSA agar plates or IDI-MRSA. Although it is more expensive, in addition to rapid turnaround times of 2 to 4 h, IDI-MRSA offers greater detection of MRSA colonization, independent of the swab site, than do conventional selective agars and GenoType MRSA Direct. PMID:17537949

Retrieving transient conformational molecular structure information from inner-shell photoionization of laser-aligned molecules

PubMed Central

Wang, Xu; Le, Anh-Thu; Yu, Chao; Lucchese, R. R.; Lin, C. D.

2016-01-01

We discuss a scheme to retrieve transient conformational molecular structure information using photoelectron angular distributions (PADs) that have averaged over partial alignments of isolated molecules. The photoelectron is pulled out from a localized inner-shell molecular orbital by an X-ray photon. We show that a transient change in the atomic positions from their equilibrium will lead to a sensitive change in the alignment-averaged PADs, which can be measured and used to retrieve the former. Exploiting the experimental convenience of changing the photon polarization direction, we show that it is advantageous to use PADs obtained from multiple photon polarization directions. A simple single-scattering model is proposed and benchmarked to describe the photoionization process and to do the retrieval using a multiple-parameter fitting method. PMID:27025410

Retrieving transient conformational molecular structure information from inner-shell photoionization of laser-aligned molecules

NASA Astrophysics Data System (ADS)

Wang, Xu; Le, Anh-Thu; Yu, Chao; Lucchese, R. R.; Lin, C. D.

2016-03-01

We discuss a scheme to retrieve transient conformational molecular structure information using photoelectron angular distributions (PADs) that have averaged over partial alignments of isolated molecules. The photoelectron is pulled out from a localized inner-shell molecular orbital by an X-ray photon. We show that a transient change in the atomic positions from their equilibrium will lead to a sensitive change in the alignment-averaged PADs, which can be measured and used to retrieve the former. Exploiting the experimental convenience of changing the photon polarization direction, we show that it is advantageous to use PADs obtained from multiple photon polarization directions. A simple single-scattering model is proposed and benchmarked to describe the photoionization process and to do the retrieval using a multiple-parameter fitting method.

HSQC-1,n-ADEQUATE: a new approach to long-range 13C-13C correlation by covariance processing.

PubMed

Martin, Gary E; Hilton, Bruce D; Willcott, M Robert; Blinov, Kirill A

2011-10-01

Long-range, two-dimensional heteronuclear shift correlation NMR methods play a pivotal role in the assembly of novel molecular structures. The well-established GHMBC method is a high-sensitivity mainstay technique, affording connectivity information via (n)J(CH) coupling pathways. Unfortunately, there is no simple way of determining the value of n and hence no way of differentiating two-bond from three- and occasionally four-bond correlations. Three-bond correlations, however, generally predominate. Recent work has shown that the unsymmetrical indirect covariance or generalized indirect covariance processing of multiplicity edited GHSQC and 1,1-ADEQUATE spectra provides high-sensitivity access to a (13)C-(13) C connectivity map in the form of an HSQC-1,1-ADEQUATE spectrum. Covariance processing of these data allows the 1,1-ADEQUATE connectivity information to be exploited with the inherent sensitivity of the GHSQC spectrum rather than the intrinsically lower sensitivity of the 1,1-ADEQUATE spectrum itself. Data acquisition times and/or sample size can be substantially reduced when covariance processing is to be employed. In an extension of that work, 1,n-ADEQUATE spectra can likewise be subjected to covariance processing to afford high-sensitivity access to the equivalent of (4)J(CH) GHMBC connectivity information. The method is illustrated using strychnine as a model compound. Copyright © 2011 John Wiley & Sons, Ltd.

Structure-matched Phthalocyanine Ion Pair as a Red-emitting Fluorescent Optical Probe for the Analysis of Sodium Dodecylbenzenesulfonate with High Specificity and Sensitivity.

PubMed

Yu, Fei; Guo, Menglin; Deng, Yabin; Lu, Yin; Chen, Lin; Huang, Ping; Li, Donghui

2016-01-01

We have found that a positively charged cationic copper phthalocyanine, Alcian blue (Alcian blue 8GX), can efficiently quench the fluorescence of an oppositely charged red fluorescent phthalocyanine compound with a matched molecular structure, tetrasulfonated aluminum phthalocyanine (AlS4Pc), because of the formation of an ion pair complex (AlS4Pc-Alcian blue 8GX) that exhibits almost no fluorescence. An investigation was carried out on the fluorescence recovery of AlS4Pc-Alcian blue 8GX caused by a series of anionic surfactants containing a sulfonic group (sodium dodecylbenzenesulfonate (SDBS), sodium lauryl sulfate (SLS), and sodium dodecyl sulfate (SDS)). The results showed that SDBS exhibited a significant response, and the highest sensitivity among the surfactants. Due to its high efficiency of fluorescence quenching and the high level of fluorescence recovery, direct observes can even be performed by the naked eye. The results revealed that the Alcian blue 8GX-AlS4Pc ion-pair complex fluorescent probe only responded to SDBS in the low-concentration range. Based on the new founding, this study proposed a novel principle and method of fluorescence enhancement to specifically measure the concentration of SDBS, thereby achieving a highly sensitive and highly specific determination of SDBS. Under the optimal conditions, the fluorescence intensity (I(f)) of the system and the concentration of SDBS in the range of 1 × 10(-7) - 1 × 10(-5) mol/dm(3) exhibited a good linear relationship. This method is highly sensitive, and the operation is simple and rapid. It had been applied for the quantitative analysis of SDBS in environmental water, while achieving satisfactory results compared with those of the standard method. This study developed a new application of the fluorescent phthalocyanine compounds used as molecular probes in analytical sciences.

Visualizing tissue molecular structure of a black type of canola (Brassica) seed with a thick seed coat after heat-related processing in a chemical way.

PubMed

Yu, Peiqiang

2013-02-20

Heat-related processing of cereal grains, legume seeds, and oil seeds could be used to improve nutrient availability in ruminants. However, different types of processing may have a different impact on intrinsic structure of tissues. To date, there is little research on structure changes after processing within intact tissues. The synchrotron-based molecular imaging technique enables us to detect inherent structure change on a molecular level. The objective of this study was to visualize tissue of black-type canola (Brassica) seed with a thick seed coat after heat-related processing in a chemical way using the synchrotron imaging technique. The results showed that the chemical images of protein amides were obtained through the imaging technique for the raw, wet, and dry heated black type of canola seed tissues. It seems that different types of processing have a different impact on the protein spectral profile in the black type of canola tissues. Wet heating had a greater impact on the protein α-helix to β-sheet ratio than dry heating. Both dry and wet heating resulted in different patterns in amide I, the second derivative, and FSD spectra. However, the exact differences in the tissue images are relatively difficult to be obtained through visual comparison. Future studies should focus on (1) comparing the response and sensitivity of canola seeds to various processing methods between the yellow-type and black-type of canola seeds; (2) developing a sensitive method to compare the image difference between tissues and between treatments; (3) developing a method to link images to nutrient digestion, and (4) revealing how structure changes affect nutrient absorption in humans and animals.

Greek PDO saffron authentication studies using species specific molecular markers.

PubMed

Bosmali, I; Ordoudi, S A; Tsimidou, M Z; Madesis, P

2017-10-01

Saffron, the spice produced from the red stigmas of the flower of Crocus sativus L. is a frequent target of fraud and mislabeling practices that cannot be fully traced using the ISO 3632 trade standard specifications and test methods. A molecular approach is proposed herein as a promising branding strategy for the authentication of highly esteemed saffron brands such as the Greek Protected Designation of Origin (PDO) "Krokos Kozanis". Specific ISSR (inter-simple sequence repeat) markers were used to assess for the first time, the within species variability of several populations of C. sativus L. from the cultivation area of "Krokos Kozanis" as well as the potential differences with the band pattern produced by other Crocus species. Then, species-specific markers were developed taking advantage of an advanced molecular technique such as the HRM analysis coupled with universal DNA barcoding regions (trnL) (Bar-HRM) and applied to saffron admixtures with some of the most common plant adulterants (Calendula officinalis, Carthamus tinctorius, Gardenia jasminoides, Zea mays and Curcuma longa). The sensitivity of the procedure was tested for turmeric as a case study whereas HPLC-fluorescence determination of secondary metabolites was also employed for comparison. The overall results indicated that the Bar-HRM approach is quite effective in terms of specificity and sensitivity. Its effectiveness regarding the detection of turmeric was comparable to that of a conventional HPLC method (0.5% vs 1.0%, w/w). Yet, the proposed DNA-based method is much faster, cost-effective and can be used even by non-geneticists, in any laboratory having access to an HRM-capable real-time PCR instrumentation. It can be, thus, regarded as a strong analytical tool in saffron authentication studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Diagnostic Accuracy of Molecular Amplification Tests for Human African Trypanosomiasis—Systematic Review

PubMed Central

Boer, Kimberly R.; Dyserinck, Heleen C.; Büscher, Philippe; Schallig, Henk D. H. F.; Leeflang, Mariska M. G.

2012-01-01

Background A range of molecular amplification techniques have been developed for the diagnosis of Human African Trypanosomiasis (HAT); however, careful evaluation of these tests must precede implementation to ensure their high clinical accuracy. Here, we investigated the diagnostic accuracy of molecular amplification tests for HAT, the quality of articles and reasons for variation in accuracy. Methodology Data from studies assessing diagnostic molecular amplification tests were extracted and pooled to calculate accuracy. Articles were included if they reported sensitivity and specificity or data whereby values could be calculated. Study quality was assessed using QUADAS and selected studies were analysed using the bivariate random effects model. Results 16 articles evaluating molecular amplification tests fulfilled the inclusion criteria: PCR (n = 12), NASBA (n = 2), LAMP (n = 1) and a study comparing PCR and NASBA (n = 1). Fourteen articles, including 19 different studies were included in the meta-analysis. Summary sensitivity for PCR on blood was 99.0% (95% CI 92.8 to 99.9) and the specificity was 97.7% (95% CI 93.0 to 99.3). Differences in study design and readout method did not significantly change estimates although use of satellite DNA as a target significantly lowers specificity. Sensitivity and specificity of PCR on CSF for staging varied from 87.6% to 100%, and 55.6% to 82.9% respectively. Conclusion Here, PCR seems to have sufficient accuracy to replace microscopy where facilities allow, although this conclusion is based on multiple reference standards and a patient population that was not always representative. Future studies should, therefore, include patients for which PCR may become the test of choice and consider well designed diagnostic accuracy studies to provide extra evidence on the value of PCR in practice. Another use of PCR for control of disease could be to screen samples collected from rural areas and test in reference laboratories, to spot epidemics quickly and direct resources appropriately. PMID:22253934

Diagnostic accuracy of molecular amplification tests for human African trypanosomiasis--systematic review.

PubMed

Mugasa, Claire M; Adams, Emily R; Boer, Kimberly R; Dyserinck, Heleen C; Büscher, Philippe; Schallig, Henk D H F; Leeflang, Mariska M G

2012-01-01

A range of molecular amplification techniques have been developed for the diagnosis of Human African Trypanosomiasis (HAT); however, careful evaluation of these tests must precede implementation to ensure their high clinical accuracy. Here, we investigated the diagnostic accuracy of molecular amplification tests for HAT, the quality of articles and reasons for variation in accuracy. Data from studies assessing diagnostic molecular amplification tests were extracted and pooled to calculate accuracy. Articles were included if they reported sensitivity and specificity or data whereby values could be calculated. Study quality was assessed using QUADAS and selected studies were analysed using the bivariate random effects model. 16 articles evaluating molecular amplification tests fulfilled the inclusion criteria: PCR (n = 12), NASBA (n = 2), LAMP (n = 1) and a study comparing PCR and NASBA (n = 1). Fourteen articles, including 19 different studies were included in the meta-analysis. Summary sensitivity for PCR on blood was 99.0% (95% CI 92.8 to 99.9) and the specificity was 97.7% (95% CI 93.0 to 99.3). Differences in study design and readout method did not significantly change estimates although use of satellite DNA as a target significantly lowers specificity. Sensitivity and specificity of PCR on CSF for staging varied from 87.6% to 100%, and 55.6% to 82.9% respectively. Here, PCR seems to have sufficient accuracy to replace microscopy where facilities allow, although this conclusion is based on multiple reference standards and a patient population that was not always representative. Future studies should, therefore, include patients for which PCR may become the test of choice and consider well designed diagnostic accuracy studies to provide extra evidence on the value of PCR in practice. Another use of PCR for control of disease could be to screen samples collected from rural areas and test in reference laboratories, to spot epidemics quickly and direct resources appropriately.

The Unique Molecular Signatures of Contact Dermatitis and Implications for Treatment.

PubMed

Leonard, Alexandra; Guttman-Yassky, Emma

2018-05-12

Irritant contact dermatitis (ICD) and allergic contact dermatitis (ACD) are common skin disorders that are characterized by inflammation, oozing, crusting, and pruritus. Atopic dermatitis (AD) is an inflammatory skin disease characterized by immune and barrier abnormalities and is additionally a risk factor for acquiring ICD and ACD. New work on allergic sensitization to common allergens (e.g., nickel and fragrance) in human skin has shown that different allergens have distinct molecular fingerprinting. For example, nickel promotes strong Th1/Th17 polarization, whereas fragrance allergy causes Th2/Th22 skewing, which is similar to the phenotype of AD. While ACD has previously been considered to be constant across all allergens, largely based on mouse models involving strong sensitizers, these new data suggest that ACD differs mechanistically according to allergen. Further, ACD in the setting of concurrent AD shows a different and attenuated phenotype as compared to healthy individuals with ACD, which influences the way AD patients respond to vaccination and other treatment modalities. As in contact sensitization, skin challenged by food patch testing shows that common food allergens (e.g., peanut and barley) also cause distinct immune polarizations in the skin. Additionally, house dust mite reactions in human skin have been profiled to show unique Th2, Th9, and Th17/22 activation as compared to controls, which are similar to the phenotype of psoriasis and contact responses to nickel. Given this information, ACD patients should be treated based on their unique allergen polarity. Refined understanding of the molecular behavior of contact dermatitis and related diseases translates to improved methods of inducing tolerance in sensitized allergic patients, such as with targeted drug therapy and epicutaneous immunotherapy.

Molecular packing and magnetic properties of lithium naphthalocyanine crystals: hollow channels enabling permeability and paramagnetic sensitivity to molecular oxygen

PubMed Central

Pandian, Ramasamy P.; Dolgos, Michelle; Marginean, Camelia; Woodward, Patrick M.; Hammel, P. Chris; Manoharan, Periakaruppan T.; Kuppusamy, Periannan

2009-01-01

The synthesis, structural framework, magnetic and oxygen-sensing properties of a lithium naphthalocyanine (LiNc) radical probe are presented. LiNc was synthesized in the form of a microcrystalline powder using a chemical method and characterized by electron paramagnetic resonance (EPR) spectroscopy, magnetic susceptibility, powder X-ray diffraction analysis, and mass spectrometry. X-Ray powder diffraction studies revealed a structural framework that possesses long, hollow channels running parallel to the packing direction. The channels measured approximately 5.0 × 5.4 Å2 in the two-dimensional plane perpendicular to the length of the channel, enabling diffusion of oxygen molecules (2.9 × 3.9 Å2) through the channel. The powdered LiNc exhibited a single, sharp EPR line under anoxic conditions, with a peak-to-peak linewidth of 630 mG at room temperature. The linewidth was sensitive to surrounding molecular oxygen, showing a linear increase in pO2 with an oxygen sensitivity of 31.2 mG per mmHg. The LiNc microcrystals can be further prepared as nano-sized crystals without the loss of its high oxygen-sensing properties. The thermal variation of the magnetic properties of LiNc, such as the EPR linewidth, EPR intensity and magnetic susceptibility revealed the existence of two different temperature regimes of magnetic coupling and hence differing columnar packing, both being one-dimensional antiferromagnetic chains but with differing magnitudes of exchange coupling constants. At a temperature of ∼50 K, LiNc crystals undergo a reversible phase transition. The high degree of oxygen-sensitivity of micro- and nano-sized crystals of LiNc, combined with excellent stability, should enable precise and accurate measurements of oxygen concentration in biological systems using EPR spectroscopy. PMID:19809598

Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood

PubMed Central

Bacconi, Andrea; Richmond, Gregory S.; Baroldi, Michelle A.; Laffler, Thomas G.; Blyn, Lawrence B.; Carolan, Heather E.; Frinder, Mark R.; Toleno, Donna M.; Metzgar, David; Gutierrez, Jose R.; Massire, Christian; Rounds, Megan; Kennel, Natalie J.; Rothman, Richard E.; Peterson, Stephen; Carroll, Karen C.; Wakefield, Teresa; Ecker, David J.

2014-01-01

The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml whole-blood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections. PMID:24951806

Use of long term dermal sensitization followed by intratracheal challenge method to identify low-dose chemical-induced respiratory allergic responses in mice.

PubMed

Fukuyama, Tomoki; Ueda, Hideo; Hayashi, Koichi; Tajima, Yukari; Shuto, Yasufumi; Saito, Toru R; Harada, Takanori; Kosaka, Tadashi

2008-10-01

The inhalation of many types of chemicals, including pesticides, perfumes, and other low-molecular weight chemicals, is a leading cause of allergic respiratory diseases. We attempted to develop a new test protocol to detect environmental chemical-related respiratory hypersensitivity at low and weakly immunogenic doses. We used long-term dermal sensitization followed by a low-dose intratracheal challenge to evaluate sensitization by the well-known respiratory sensitizers trimellitic anhydride (TMA) and toluene diisocyanate (TDI) and the contact sensitizer 2,4-dinitrochlorobenzene (DNCB). After topically sensitizing BALB/c mice (9 times in 3 weeks) and challenging them intratracheally with TMA, TDI, or DNCB, we assayed differential cell counts and chemokine levels in bronchoalveolar lavage fluid (BALF); lymphocyte counts, surface antigen expression of B cells, and local cytokine production in lung-associated lymph nodes (LNs); and antigen-specific IgE levels in serum and BALF. TMA induced marked increases in antigen-specific IgE levels in both serum and BALF, proliferation of eosinophils and chemokines (MCP-1, eotaxin, and MIP-1beta) in BALF, and proliferation of Th2 cytokines (interleukin (IL)-4, IL-10, and IL-13) in restimulated LN cells. TDI induced marked increases in levels of cytokines (IL-4, IL-10, IL-13, and IFN-gamma) produced by restimulated LN cells. In contrast, DNCB treatment yielded, at most, small, nonsignificant increases in all parameters. Our protocol thus detected respiratory allergic responses to low-molecular weight chemicals and may be useful for detecting environmental chemical-related respiratory allergy.

Evaluation of phenotypic detection methods for metallo-β-lactamases (MBLs) in clinical isolates of Pseudomonas aeruginosa.

PubMed

Peter, S; Lacher, A; Marschal, M; Hölzl, F; Buhl, M; Autenrieth, I; Kaase, M; Willmann, M

2014-07-01

Metallo-beta-lactamase (MBL) production in Pseudomonas aeruginosa is a growing issue across the globe. Fast and reliable diagnostic tools are needed for appropriate implementation of infection control measures. In this study we evaluated the performance of three commercial combined disk tests, two EDTA based in-house combined disk tests and the Carba NP test in comparison to molecular detection of MBL genes on 133 meropenem non-susceptible non-duplicate P. aeruginosa clinical isolates. The meropenem/DPA based commercial KPC + MBL-confirm ID kit (Rosco Diagnostica, Denmark) and the MASTDISCS™ ID carbapenemase (Enterobacteriaceae) detection disc set (MAST Diagnostics, UK) showed sensitivities of 31.1 % and 28.8 % and specificities of 69.3 % and 79.6 %, respectively. The total MBL confirm kit (Rosco Diagnostica, Denmark) contains imipenem/DPA and imipenem/EDTA combination disks. Evaluation of the single disk combinations revealed 84.4 % sensitivity and 81.8 % specificity for the imipenem/DPA assay and 86.7 % sensitivity and 51.1 % specificity for the imipenem/EDTA test. Applying both tests simultaneously resulted in a slightly higher sensitivity of 88.9 % but a lower specificity of 48.9 % when compared to the single tests alone. The Carba NP test showed 93.3 % sensitivity and 96.6 % specificity. All phenotypic combined disk tests lacked either sensitivity or specificity for the detection of MBL in P. aeruginosa. The Carba NP test showed excellent test properties, but suffers from drawbacks in handling and high costs. The optimal diagnostic approach needs to be chosen depending on the epidemiological situation, laboratory resources and availability of molecular confirmation tests.

Potential transducers based man-tailored biomimetic sensors for selective recognition of dextromethorphan as an antitussive drug.

PubMed

El-Naby, Eman H; Kamel, Ayman H

2015-09-01

A biomimetic potentiometric sensor for specific recognition of dextromethorphan (DXM), a drug classified according to the Drug Enforcement Administration (DEA) as a "drug of concern", is designed and characterized. A molecularly imprinted polymer (MIP), with special molecular recognition properties of DXM, was prepared by thermal polymerization in which DXM acted as template molecule, methacrylic acid (MAA) and acrylonitrile (AN) acted as functional monomers in the presence of ethylene glycol dimethacrylate (EGDMA) as crosslinker. The sensors showed a high selectivity and a sensitive response to the template in aqueous system. Electrochemical evaluation of these sensors revealed near-Nernstian response with slopes of 49.6±0.5 and 53.4±0.5 mV decade(-1) with a detection limit of 1.9×10(-6), and 1.0×10(-6) mol L(-1) DXM with MIP/MAA and MIP/AN membrane based sensors, respectively. Significantly improved accuracy, precision, response time, stability, selectivity and sensitivity were offered by these simple and cost-effective potentiometric sensors compared with other standard techniques. The method has the requisite accuracy, sensitivity and precision to assay DXM in pharmaceutical products. Copyright © 2015 Elsevier B.V. All rights reserved.

Smart dual-mode fluorescent gold nanoparticle agents.

PubMed

Kang, Kyung A; Wang, Jianting

2014-01-01

Fluorophore-mediated, molecular sensing is one of the most popular and important technique in biomedical studies. As in any sensing technique, the two most important factors in this sensing are the sensitivity and specificity. Since the fluorescence of a fluorophore is emitted in the process of fluorophore electrons returning from their excited to ground state, a tool that can locally manipulate the electron state can be useful to maximize the sensitivity and specificity. A good tool candidate for this purpose is nanosized metal particles that can form an electromagnetic (EM) field at a sufficiently strong level, upon receiving a particular wavelength that fits the excitation wavelength of the fluorophore to be used. There are several metal nanoparticle types that can generate a sufficiently strong EM field for this purpose. Nevertheless, for the biomedical studies, which require minimal toxicity, gold nanoparticles (GNPs) are known to be the most suitable. In this article, various methods for fluorescence alteration using GNPs, which can be beneficially utilized for biomarker-specific, highly sensitive molecular sensing and imaging, are discussed. For further resources related to this article, please visit the WIREs website. The authors have declared no conflicts of interest for this article. © 2014 Wiley Periodicals, Inc.

Spectroscopic (FT-IR, FT-Raman and UV-Visible) investigations, NMR chemical shielding anisotropy (CSA) parameters of 2,6-Diamino-4-chloropyrimidine for dye sensitized solar cells using density functional theory.

PubMed

Gladis Anitha, E; Joseph Vedhagiri, S; Parimala, K

2015-02-05

The molecular structure, geometry optimization, vibrational frequencies of organic dye sensitizer 2,6-Diamino-4-chloropyrimidine (DACP) were studied based on Hartree-Fock (HF) and density functional theory (DFT) using B3LYP methods with 6-311++G(d,p) basis set. Ultraviolet-Visible (UV-Vis) spectrum was investigated by time dependent DFT (TD-DFT). Features of the electronic absorption spectrum in the UV-Visible regions were assigned based on TD-DFT calculation. The absorption bands are assigned to transitions. The interfacial electron transfer between semiconductor TiO2 electrode and dye sensitizer DACP is due to an electron injection process from excited dye to the semiconductor's conduction band. The observed and the calculated frequencies are found to be in good agreement. The energies of the frontier molecular orbitals (FMOS) have also been determined. The chemical shielding anisotropic (CSA) parameters are calculated from the NMR analysis, Stability of the molecule arising from hyperconjugative interactions and charge delocalization has been analyzed using natural bond orbital (NBO) analysis. Copyright © 2014 Elsevier B.V. All rights reserved.

Technical basis for inner container leak detection sensitivity goals in 3013 DE surveillance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Berg, John M.

Helium leak checking of 3013 inner container lids is under consideration for addition to DE Surveillance tasks as an improved means to detect any through-wall flaws that may have formed during storage. This white paper evaluates whether leak checking at DE could replace and improve upon the current method of comparing gas compositions and pressures within the inner and outer containers. We have used viscous and molecular flow equations in ANSI N14.5 to calculate what the measured standard helium leak rate would be for hypothetical leaks of three different sizes. For comparison, we have also calculated the effects on gasmore » composition and pressure differences as a function of pre-DE storage time for the same three leak sizes, using molecular and viscous flow equations as well as diffusion equations to predict the relevant gas transport. For a hypothetical leak that would be measured at 1x10 -7 std cc/sec, likely an achievable sensitivity using helium leak checking at DE, the calculations predict no measurable effect on pressure difference or gas composition as measured by DE gas analysis. We also calculate that it would take over 200 years for water vapor to diffuse through a 10 -7 std cc/sec leak enough to raise the RH outer container to half the RH value in the inner container. A leak 100 times larger, which would be measured at 1x10 -5 std cc/sec, the same water vapor diffusion would take at least 14 years. Our conclusion is that helium leak checking will be useful even at a sensitivity of 1x10 -5 std cc/sec, and a significant improvement over current DE methods at a sensitivity of 1x10 -7 std cc/sec.« less

State of the art in non-animal approaches for skin sensitization testing: from individual test methods towards testing strategies.

PubMed

Ezendam, Janine; Braakhuis, Hedwig M; Vandebriel, Rob J

2016-12-01

The hazard assessment of skin sensitizers relies mainly on animal testing, but much progress is made in the development, validation and regulatory acceptance and implementation of non-animal predictive approaches. In this review, we provide an update on the available computational tools and animal-free test methods for the prediction of skin sensitization hazard. These individual test methods address mostly one mechanistic step of the process of skin sensitization induction. The adverse outcome pathway (AOP) for skin sensitization describes the key events (KEs) that lead to skin sensitization. In our review, we have clustered the available test methods according to the KE they inform: the molecular initiating event (MIE/KE1)-protein binding, KE2-keratinocyte activation, KE3-dendritic cell activation and KE4-T cell activation and proliferation. In recent years, most progress has been made in the development and validation of in vitro assays that address KE2 and KE3. No standardized in vitro assays for T cell activation are available; thus, KE4 cannot be measured in vitro. Three non-animal test methods, addressing either the MIE, KE2 or KE3, are accepted as OECD test guidelines, and this has accelerated the development of integrated or defined approaches for testing and assessment (e.g. testing strategies). The majority of these approaches are mechanism-based, since they combine results from multiple test methods and/or computational tools that address different KEs of the AOP to estimate skin sensitization potential and sometimes potency. Other approaches are based on statistical tools. Until now, eleven different testing strategies have been published, the majority using the same individual information sources. Our review shows that some of the defined approaches to testing and assessment are able to accurately predict skin sensitization hazard, sometimes even more accurate than the currently used animal test. A few defined approaches are developed to provide an estimate of the potency sub-category of a skin sensitizer as well, but these approaches need further independent evaluation with a new dataset of chemicals. To conclude, this update shows that the field of non-animal approaches for skin sensitization has evolved greatly in recent years and that it is possible to predict skin sensitization hazard without animal testing.

Molecularly Imprinted Core-Shell CdSe@SiO2/CDs as a Ratiometric Fluorescent Probe for 4-Nitrophenol Sensing

NASA Astrophysics Data System (ADS)

Liu, Mingyue; Gao, Zhao; Yu, Yanjun; Su, Rongxin; Huang, Renliang; Qi, Wei; He, Zhimin

2018-01-01

4-Nitrophenol (4-NP) is a priority pollutant in water and is both carcinogenic and genotoxic to humans and wildlife even at very low concentrations. Thus, we herein fabricated a novel molecularly imprinted core-shell nanohybrid as a ratiometric fluorescent sensor for the highly sensitive and selective detection of 4-NP. This sensor was functioned by the transfer of fluorescence resonance energy between photoluminescent carbon dots (CDs) and 4-NP. This sensor was synthesized by linking organosilane-functionalized CDs to silica-coated CdSe quantum dots (CdSe@SiO2) via Si-O bonds. The nanohybrids were further modified by anchoring a molecularly imprinted polymer (MIP) layer on the ratiometric fluorescent sensor through a facile sol-gel polymerization method. The morphology, chemical structure, and optical properties of the resulting molecularly imprinted dual-emission fluorescent probe were characterized by transmission electron microscopy and spectroscopic analysis. The probe was then applied in the detection of 4-NP and exhibited good linearity between 0.051 and 13.7 μg/mL, in addition to a low detection limit of 0.026 μg/mL. Furthermore, the simplicity, reliability, high selectivity, and high sensitivity of the developed sensor demonstrate that the combination of MIPs and ratiometric fluorescence allows the preparation of excellent fluorescent sensors for the detection of trace or ultra-trace analytes.

A nanocomposite optosensor containing carboxylic functionalized multiwall carbon nanotubes and quantum dots incorporated into a molecularly imprinted polymer for highly selective and sensitive detection of ciprofloxacin.

PubMed

Yuphintharakun, Naphat; Nurerk, Piyaluk; Chullasat, Kochaporn; Kanatharana, Proespichaya; Davis, Frank; Sooksawat, Dhassida; Bunkoed, Opas

2018-08-05

A nanocomposite optosensor consisting of carboxylic acid functionalized multiwall carbon nanotubes and CdTe quantum dots embedded inside a molecularly imprinted polymer (COOH@MWCNT-MIP-QDs) was developed for trace ciprofloxacin detection. The COOH@MWCNT-MIP-QDs were synthesized through a facile sol-gel process using ciprofloxacin as a template molecule, 3-aminopropylethoxysilane as a functional monomer and tetraethoxysilane as a cross-linker at a molar ratio of 1:8:20. The synthesized nanocomposite optosensor had high sensitivity, excellent specificity and high binding affinity to ciprofloxacin. Under optimal conditions, the fluorescence intensity of the optosensor decreased in a linear fashion with the concentration of ciprofloxacin and two linear dynamic ranges were obtained, 0.10-1.0 μg L -1 and 1.0-100.0 μg L -1 with a very low limit of detection of 0.066 μg L -1 . The imprinting factors of the two linear range were 17.67 and 4.28, respectively. The developed nanocomposite fluorescence probe was applied towards the determination of ciprofloxacin levels in chicken muscle and milk samples with satisfactory recoveries being obtained in the range of 82.6 to 98.4%. The results were also in good agreement with a HPLC method which indicates that the optosensor can be used as a sensitive, selective and rapid method to detect ciprofloxacin in chicken and milk samples. Copyright © 2018 Elsevier B.V. All rights reserved.

Molecular analysis of dolphin morbillivirus: A new sensitive detection method based on nested RT-PCR.

PubMed

Centelleghe, Cinzia; Beffagna, Giorgia; Zanetti, Rossella; Zappulli, Valentina; Di Guardo, Giovanni; Mazzariol, Sandro

2016-09-01

Cetacean Morbillivirus (CeMV) has been identified as the most pathogenic virus for cetaceans. Over the past three decades, this RNA virus has caused several outbreaks of lethal disease in odontocetes and mysticetes worldwide. Isolation and identification of CeMV RNA is very challenging in whales because of the poor preservation status frequently shown by tissues from stranded animals. Nested reverse transcription polymerase chain reaction (nested RT-PCR) is used instead of conventional RT-PCR when it is necessary to increase the sensitivity and the specificity of the reaction. This study describes a new nested RT-PCR technique useful to amplify small amounts of the cDNA copy of Cetacean morbillivirus (CeMV) when it is present in scant quantity in whales' biological specimens. This technique was used to analyze different tissues (lung, brain, spleen and other lymphoid tissues) from one under human care seal and seven cetaceans stranded along the Italian coastline between October 2011 and September 2015. A well-characterized, 200 base pair (bp) fragment of the dolphin Morbillivirus (DMV) haemagglutinin (H) gene, obtained by nested RT-PCR, was sequenced and used to confirm DMV positivity in all the eight marine mammals under study. In conclusion, this nested RT-PCR protocol can represent a sensitive detection method to identify CeMV-positive, poorly preserved tissue samples. Furthermore, this is also a rather inexpensive molecular technique, relatively easy to apply. Copyright © 2016 Elsevier B.V. All rights reserved.

Experimental investigation of a HOPG crystal fan for x-ray fluorescence molecular imaging

NASA Astrophysics Data System (ADS)

Rosentreter, Tanja; Müller, Bernhard; Schlattl, Helmut; Hoeschen, Christoph

2017-03-01

Imaging x-ray fluorescence generally generates a conflict between the best image quality or highest sensitivity and lowest possible radiation dose. Consequently many experimental studies investigating the feasibility of this molecular imaging method, deal with either monochromatic x-ray sources that are not practical in clinical environment or accept high x-ray doses in order to maintain the advantage of high sensitivity and producing high quality images. In this work we present a x-ray fluorescence imaging setup using a HOPG crystal fan construction consisting of a Bragg reflecting analyzer array together with a scatter reducing radial collimator. This method allows for the use of polychromatic x-ray tubes that are in general easily accessible in contrast to monochromatic x-ray sources such as synchrotron facilities. Moreover this energy-selecting device minimizes the amount of Compton scattered photons while simultaneously increasing the fluorescence signal yield, thus significantly reducing the signal to noise ratio. The aim is to show the feasibility of this approach by measuring the Bragg reflected Kα fluorescence signal of an object containing an iodine solution using a large area detector with moderate energy resolution. Contemplating the anisotropic energy distribution of background scattered x-rays we compare the detection sensitivity, applying two different detector angular configurations. Our results show that even for large area detectors with limited energy resolution, iodine concentrations of 0.12 % can be detected. However, the potentially large scan times and therefore high radiation dose need to be decreased in further investigations.

Molecularly imprinted coated graphene oxide solid-phase extraction monolithic capillary column for selective extraction and sensitive determination of phloxine B in coffee bean.

PubMed

Zhai, Haiyun; Su, Zihao; Chen, Zuanguang; Liu, Zhenping; Yuan, Kaisong; Huang, Lu

2015-03-20

A method was developed to sensitively determine phloxine B in coffee bean by molecularly imprinted polymers (MIPs) coated graphene oxide (GO) solid-phase extraction (GO-MISPE) coupled with high-performance liquid chromatography and laser-induced fluorescence detection (HPLC-LIF). The GO-MISPE capillary monolithic column was prepared by water-bath in situ polymerization, using GO as supporting material, phloxine B, methacrylic acid (MAA), and ethylene dimethacrylate (EDMA) as template, functional monomer, and cross-linker, respectively. The properties of the homemade GO-MISPE capillary monolithic column, including capacity and specificity, were investigated under optimized conditions. The GO-MIPs were characterized by scanning electron microscopy (SEM) and Fourier transform-infrared spectroscopy (FT-IR). The mean recoveries of phloxine B in coffee bean ranged from 89.5% to 91.4% and the intra-day and inter-day relative standard deviation (RSD) values all ranged from 3.6% to 4.7%. Good linearity was obtained over 0.001-2.0 μg mL(-1) (r=0.9995) with the detection limit (S/N=3) of 0.075 ng mL(-1). Under the selected conditions, enrichment factors of over 90-fold were obtained and extraction on the monolithic column effectively cleaned up the coffee bean matrix. The results demonstrated that the proposed GO-MISPE HPLC-LIF method can be applied to sensitively determine phloxine B in coffee bean. Copyright © 2015 Elsevier B.V. All rights reserved.

Competitive amplification of differentially melting amplicons (CADMA) enables sensitive and direct detection of all mutation types by high-resolution melting analysis.

PubMed

Kristensen, Lasse S; Andersen, Gitte B; Hager, Henrik; Hansen, Lise Lotte

2012-01-01

Sensitive and specific mutation detection is of particular importance in cancer diagnostics, prognostics, and individualized patient treatment. However, the majority of molecular methodologies that have been developed with the aim of increasing the sensitivity of mutation testing have drawbacks in terms of specificity, convenience, or costs. Here, we have established a new method, Competitive Amplification of Differentially Melting Amplicons (CADMA), which allows very sensitive and specific detection of all mutation types. The principle of the method is to amplify wild-type and mutated sequences simultaneously using a three-primer system. A mutation-specific primer is designed to introduce melting temperature decreasing mutations in the resulting mutated amplicon, while a second overlapping primer is designed to amplify both wild-type and mutated sequences. When combined with a third common primer very sensitive mutation detection becomes possible, when using high-resolution melting (HRM) as detection platform. The introduction of melting temperature decreasing mutations in the mutated amplicon also allows for further mutation enrichment by fast coamplification at lower denaturation temperature PCR (COLD-PCR). For proof-of-concept, we have designed CADMA assays for clinically relevant BRAF, EGFR, KRAS, and PIK3CA mutations, which are sensitive to, between 0.025% and 0.25%, mutated alleles in a wild-type background. In conclusion, CADMA enables highly sensitive and specific mutation detection by HRM analysis. © 2011 Wiley Periodicals, Inc.

Detection of Onchocerca volvulus in Skin Snips by Microscopy and Real-Time Polymerase Chain Reaction: Implications for Monitoring and Evaluation Activities.

PubMed

Thiele, Elizabeth A; Cama, Vitaliano A; Lakwo, Thomson; Mekasha, Sindeaw; Abanyie, Francisca; Sleshi, Markos; Kebede, Amha; Cantey, Paul T

2016-04-01

Microscopic evaluation of skin biopsies is the monitoring and evaluation (M and E) method currently used by multiple onchocerciasis elimination programs in Africa. However, as repeated mass drug administration suppresses microfilarial loads, the sensitivity and programmatic utility of skin snip microscopy is expected to decrease. Using a pan-filarial real-time polymerase chain reaction with melt curve analysis (qPCR-MCA), we evaluated 1) the use of a single-step molecular assay for detecting and identifying Onchocerca volvulus microfilariae in residual skin snips and 2) the sensitivity of skin snip microscopy relative to qPCR-MCA. Skin snips were collected and examined with routine microscopy in hyperendemic regions of Uganda and Ethiopia (N= 500 each) and "residual" skin snips (tissue remaining after induced microfilarial emergence) were tested with qPCR-MCA. qPCR-MCA detected Onchocerca DNA in 223 residual snips: 139 of 147 microscopy(+) and 84 among microscopy(-) snips, suggesting overall sensitivity of microscopy was 62.3% (139/223) relative to qPCR-MCA (75.6% in Uganda and 28.6% in Ethiopia). These findings demonstrate the insufficient sensitivity of skin snip microscopy for reliable programmatic monitoring. Molecular tools such as qPCR-MCA can augment sensitivity and provide diagnostic confirmation of skin biopsies and will be useful for evaluation or validation of new onchocerciasis M and E tools. © The American Society of Tropical Medicine and Hygiene.

Sensitive HIV-1 detection in a homogeneous solution based on an electrochemical molecular beacon coupled with a nafion-graphene composite film modified screen-printed carbon electrode.

PubMed

Li, Bo; Li, Zhengliang; Situ, Bo; Dai, Zong; Liu, Qinlan; Wang, Qian; Gu, Dayong; Zheng, Lei

2014-02-15

A novel electrochemical sensing assay for sensitive determination of HIV-1 in a homogeneous solution has been developed using an electrochemical molecular beacon combined with a nafion-graphene composite film modified screen-printed carbon electrode (nafion-graphene/SPCE). The electrochemical molecular beacon (CAs-MB), comprising a special recognition sequence for the conserved region of the HIV-1 gag gene and a pair of carminic acid molecules as a marker, can indicate the presence of the HIV-1 target by its on/off electrochemical signal behavior. It is suitable for direct, electrochemical determination of HIV-1, thereby simplifying the detection procedure and improving the signal-to-noise (S/N) ratio. To further improve the sensitivity, the nafion-graphene/SPCE was used to monitor changes in the CAs-MB, which has notable advantages, such as being ultrasensitive, inexpensive, and disposable. Under optimized conditions, the peak currents showed a linear relationship with the logarithm of target oligonucleotide concentrations ranging from 40 nM to 2.56 μM, with a detection limit of 5 nM (S/N=3). This sensing assay also displays a good stability, with a recovery of 88-106.8% and RSD<7% (n=5) in real serum samples. This work may lead to the development of an effective method for early point-of-care diagnosis of HIV-1 infection. © 2013 Elsevier B.V. All rights reserved.

Plasmonic gold nanostar for biomedical sensing

NASA Astrophysics Data System (ADS)

Liu, Yang; Yuan, Hsiangkuo; Fales, Andrew M.; Vo-Dinh, Tuan

2014-03-01

Cancer has become one of most significant death reasons and causes approximately 7.9 million human deaths worldwide each year. The challenge to detect cancer at an early stage makes cancer-related biomarkers sensing attract more and more research interest and efforts. Surface-enhanced Raman scattering (SERS) provides a promising method for various biomarkers (DNA, RNA, protein, et al.) detection due to its high sensitivity, specificity and capability for multiple analytes detection. Raman spectroscopy is a non-destructive photon-scattering technique, which provides molecule-specific information on molecular vibrational energy levels. SERS takes advantage of plasmonic effects and can enhance Raman signal up to 1015 at "hot spots". Due to its excellent sensitivity, SERS has been capable of achieving single-molecule detection limit. Local pH environment has been identified to be a potential biomarker for cancer diagnosis since solid cancer contains highly acidic environments. A near-infrared (NIR) SERS nanoprobe based on gold nanostars for pH sensing is developed for future cancer detection. Near-infrared (NIR) light is more suitable for in vivo applications because of its low attenuation rate and tissue auto fluorescence. SERS spectrum of pH reporter under various pH environments is monitored and used for pH sensing. Furthermore, density functional theory (DFT) calculation is performed to investigate Raman spectra changes with pH at the molecular level. The study demonstrates that SERS is a sensitive tool to monitor minor molecular structural changes due to local pH environment for cancer detection.

Development and first evaluation of a novel multiplex real-time PCR on whole blood samples for rapid pathogen identification in critically ill patients with sepsis.

PubMed

van de Groep, Kirsten; Bos, Martine P; Savelkoul, Paul H M; Rubenjan, Anna; Gazenbeek, Christel; Melchers, Willem J G; van der Poll, Tom; Juffermans, Nicole P; Ong, David S Y; Bonten, Marc J M; Cremer, Olaf L

2018-04-26

Molecular tests may enable early adjustment of antimicrobial therapy and be complementary to blood culture (BC) which has imperfect sensitivity in critically ill patients. We evaluated a novel multiplex real-time PCR assay to diagnose bloodstream pathogens directly in whole blood samples (BSI-PCR). BSI-PCR included 11 species- and four genus-specific PCRs, a molecular Gram-stain PCR, and two antibiotic resistance markers. We collected 5 mL blood from critically ill patients simultaneously with clinically indicated BC. Microbial DNA was isolated using the Polaris method followed by automated DNA extraction. Sensitivity and specificity were calculated using BC as reference. BSI-PCR was evaluated in 347 BC-positive samples (representing up to 50 instances of each pathogen covered by the test) and 200 BC-negative samples. Bacterial species-specific PCR sensitivities ranged from 65 to 100%. Sensitivity was 26% for the Gram-positive PCR, 32% for the Gram-negative PCR, and ranged 0 to 7% for yeast PCRs. Yeast detection was improved to 40% in a smaller set-up. There was no overall association between BSI-PCR sensitivity and time-to-positivity of BC (which was highly variable), yet Ct-values were lower for true-positive versus false-positive PCR results. False-positive results were observed in 84 (4%) of the 2200 species-specific PCRs in 200 culture-negative samples, and ranged from 0 to 6% for generic PCRs. Sensitivity of BSI-PCR was promising for individual bacterial pathogens, but still insufficient for yeasts and generic PCRs. Further development of BSI-PCR will focus on improving sensitivity by increasing input volumes and on subsequent implementation as a bedside test.

Microsatellite instability in prostate cancer by PCR or next-generation sequencing.

PubMed

Hempelmann, Jennifer A; Lockwood, Christina M; Konnick, Eric Q; Schweizer, Michael T; Antonarakis, Emmanuel S; Lotan, Tamara L; Montgomery, Bruce; Nelson, Peter S; Klemfuss, Nola; Salipante, Stephen J; Pritchard, Colin C

2018-04-17

Microsatellite instability (MSI) is now being used as a sole biomarker to guide immunotherapy treatment for men with advanced prostate cancer. Yet current molecular diagnostic tests for MSI have not been evaluated for use in prostate cancer. We evaluated two next-generation sequencing (NGS) MSI-detection methods, MSIplus (18 markers) and MSI by Large Panel NGS (> 60 markers), and compared the performance of each NGS method to the most widely used 5-marker MSI-PCR detection system. All methods were evaluated by comparison to targeted whole gene sequencing of DNA mismatch-repair genes, and immunohistochemistry for mismatch repair genes, where available. In a set of 91 prostate tumors with known mismatch repair status (29-deficient and 62-intact mismatch-repair) MSIplus had a sensitivity of 96.6% (28/29) and a specificity of 100% (62/62), MSI by Large Panel NGS had a sensitivity of 93.1% (27/29) and a specificity of 98.4% (61/62), and MSI-PCR had a sensitivity of 72.4% (21/29) and a specificity of 100% (62/62). We found that the widely used 5-marker MSI-PCR panel has inferior sensitivity when applied to prostate cancer and that NGS testing with an expanded panel of markers performs well. In addition, NGS methods offer advantages over MSI-PCR, including no requirement for matched non-tumor tissue and an automated analysis pipeline with quantitative interpretation of MSI-status.

Testing the limits of sensitivity in a solid-state structural investigation by combined X-ray powder diffraction, solid-state NMR, and molecular modelling.

PubMed

Filip, Xenia; Borodi, Gheorghe; Filip, Claudiu

2011-10-28

A solid state structural investigation of ethoxzolamide is performed on microcrystalline powder by using a multi-technique approach that combines X-ray powder diffraction (XRPD) data analysis based on direct space methods with information from (13)C((15)N) solid-state Nuclear Magnetic Resonance (SS-NMR) and molecular modeling. Quantum chemical computations of the crystal were employed for geometry optimization and chemical shift calculations based on the Gauge Including Projector Augmented-Wave (GIPAW) method, whereas a systematic search in the conformational space was performed on the isolated molecule using a molecular mechanics (MM) approach. The applied methodology proved useful for: (i) removing ambiguities in the XRPD crystal structure determination process and further refining the derived structure solutions, and (ii) getting important insights into the relationship between the complex network of non-covalent interactions and the induced supra-molecular architectures/crystal packing patterns. It was found that ethoxzolamide provides an ideal case study for testing the accuracy with which this methodology allows to distinguish between various structural features emerging from the analysis of the powder diffraction data. This journal is © the Owner Societies 2011

Scalable and fast heterogeneous molecular simulation with predictive parallelization schemes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guzman, Horacio V.; Junghans, Christoph; Kremer, Kurt

Multiscale and inhomogeneous molecular systems are challenging topics in the field of molecular simulation. In particular, modeling biological systems in the context of multiscale simulations and exploring material properties are driving a permanent development of new simulation methods and optimization algorithms. In computational terms, those methods require parallelization schemes that make a productive use of computational resources for each simulation and from its genesis. Here, we introduce the heterogeneous domain decomposition approach, which is a combination of an heterogeneity-sensitive spatial domain decomposition with an a priori rearrangement of subdomain walls. Within this approach and paper, the theoretical modeling and scalingmore » laws for the force computation time are proposed and studied as a function of the number of particles and the spatial resolution ratio. We also show the new approach capabilities, by comparing it to both static domain decomposition algorithms and dynamic load-balancing schemes. Specifically, two representative molecular systems have been simulated and compared to the heterogeneous domain decomposition proposed in this work. Finally, these two systems comprise an adaptive resolution simulation of a biomolecule solvated in water and a phase-separated binary Lennard-Jones fluid.« less

Scalable and fast heterogeneous molecular simulation with predictive parallelization schemes

DOE PAGES

Guzman, Horacio V.; Junghans, Christoph; Kremer, Kurt; ...

2017-11-27

Multiscale and inhomogeneous molecular systems are challenging topics in the field of molecular simulation. In particular, modeling biological systems in the context of multiscale simulations and exploring material properties are driving a permanent development of new simulation methods and optimization algorithms. In computational terms, those methods require parallelization schemes that make a productive use of computational resources for each simulation and from its genesis. Here, we introduce the heterogeneous domain decomposition approach, which is a combination of an heterogeneity-sensitive spatial domain decomposition with an a priori rearrangement of subdomain walls. Within this approach and paper, the theoretical modeling and scalingmore » laws for the force computation time are proposed and studied as a function of the number of particles and the spatial resolution ratio. We also show the new approach capabilities, by comparing it to both static domain decomposition algorithms and dynamic load-balancing schemes. Specifically, two representative molecular systems have been simulated and compared to the heterogeneous domain decomposition proposed in this work. Finally, these two systems comprise an adaptive resolution simulation of a biomolecule solvated in water and a phase-separated binary Lennard-Jones fluid.« less

MEMS cantilever sensor for THz photoacoustic chemical sensing and pectroscopy

NASA Astrophysics Data System (ADS)

Glauvitz, Nathan E.

Sensitive Microelectromechanical System (MEMS) cantilever designs were modeled, fabricated, and tested to measure the photoacoustic (PA) response of gasses to terahertz (THz) radiation. Surface and bulk micromachining technologies were employed to create the extremely sensitive devices that could detect very small changes in pressure. Fabricated devices were then tested in a custom made THz PA vacuum test chamber where the cantilever deflections caused by the photoacoustic effect were measured with a laser interferometer and iris beam clipped methods. The sensitive cantilever designs achieved a normalized noise equivalent absorption coefficient of 2.83x10-10 cm-1 W Hz-½ using a 25 microW radiation source power and a 1 s sampling time. Traditional gas phase molecular spectroscopy absorption cells are large and bulky. The outcome of this research resulted was a photoacoustic detection method that was virtually independent of the absorption path-length, which allowed the chamber dimensions to be greatly reduced, leading to the possibility of a compact, portable chemical detection and spectroscopy system

Detection of early primary colorectal cancer with upconversion luminescent NP-based molecular probes

NASA Astrophysics Data System (ADS)

Liu, Chunyan; Qi, Yifei; Qiao, Ruirui; Hou, Yi; Chan, Kaying; Li, Ziqian; Huang, Jiayi; Jing, Lihong; Du, Jun; Gao, Mingyuan

2016-06-01

Early detection and diagnosis of cancers is extremely beneficial for improving the survival rate of cancer patients and molecular imaging techniques are believed to be relevant for offering clinical solutions. Towards early cancer detection, we developed a primary animal colorectal cancer model and constructed a tumor-specific imaging probe by using biocompatible NaGdF4:Yb,Er@NaGdF4 upconversion luminescent NPs for establishing a sensitive early tumor imaging method. The primary animal tumor model, which can better mimic the human colorectal cancer, was built upon continual administration of 1,2-dimethylhydrazine in Kunming mice and the tumor development was carefully monitored through histopathological and immunohistochemical analyses to reveal the pathophysiological processes and molecular features of the cancer microenvironment. The upconversion imaging probe was constructed through covalent coupling of PEGylated core-shell NPs with folic acid whose receptor is highly expressed in the primary tumors. Upon 980 nm laser excitation, the primary colorectal tumors in the complex abdominal environment were sensitively imaged owing to the ultralow background of the upconversion luminescence and the high tumor-targeting specificity of the nanoprobe. We believe that the current studies provide a highly effective and potential approach for early colorectal cancer diagnosis and tumor surgical navigation.Early detection and diagnosis of cancers is extremely beneficial for improving the survival rate of cancer patients and molecular imaging techniques are believed to be relevant for offering clinical solutions. Towards early cancer detection, we developed a primary animal colorectal cancer model and constructed a tumor-specific imaging probe by using biocompatible NaGdF4:Yb,Er@NaGdF4 upconversion luminescent NPs for establishing a sensitive early tumor imaging method. The primary animal tumor model, which can better mimic the human colorectal cancer, was built upon continual administration of 1,2-dimethylhydrazine in Kunming mice and the tumor development was carefully monitored through histopathological and immunohistochemical analyses to reveal the pathophysiological processes and molecular features of the cancer microenvironment. The upconversion imaging probe was constructed through covalent coupling of PEGylated core-shell NPs with folic acid whose receptor is highly expressed in the primary tumors. Upon 980 nm laser excitation, the primary colorectal tumors in the complex abdominal environment were sensitively imaged owing to the ultralow background of the upconversion luminescence and the high tumor-targeting specificity of the nanoprobe. We believe that the current studies provide a highly effective and potential approach for early colorectal cancer diagnosis and tumor surgical navigation. Electronic supplementary information (ESI) available: (1) Molecular structure of Jeffamine-modified FA; (2) immunohistochemical analysis of FR expression in the colorectal tissue derived from mice treated with NaCl at different weeks; (3) biodistributions of probes of NP-FA and NP-IgG in the main organs of mice. See DOI: 10.1039/c5nr07858j

Comparison of methods of DNA extraction for real-time PCR in a model of pleural tuberculosis.

PubMed

Santos, Ana; Cremades, Rosa; Rodríguez, Juan Carlos; García-Pachón, Eduardo; Ruiz, Montserrat; Royo, Gloria

2010-01-01

Molecular methods have been reported to have different sensitivities in the diagnosis of pleural tuberculosis and this may in part be caused by the use of different methods of DNA extraction. Our study compares nine DNA extraction systems in an experimental model of pleural tuberculosis. An inoculum of Mycobacterium tuberculosis was added to 23 pleural liquid samples with different characteristics. DNA was subsequently extracted using nine different methods (seven manual and two automatic) for analysis with real-time PCR. Only two methods were able to detect the presence of M. tuberculosis DNA in all the samples: extraction using columns (Qiagen) and automated extraction with the TNAI system (Roche). The automatic method is more expensive, but requires less time. Almost all the false negatives were because of the difficulty involved in extracting M. tuberculosis DNA, as in general, all the methods studied are capable of eliminating inhibitory substances that block the amplification reaction. The method of M. tuberculosis DNA extraction used affects the results of the diagnosis of pleural tuberculosis by molecular methods. DNA extraction systems that have been shown to be effective in pleural liquid should be used.

Metabolic primers for detection of (Per)chlorate-reducing bacteria in the environment and phylogenetic analysis of cld gene sequences.

PubMed

Bender, Kelly S; Rice, Melissa R; Fugate, William H; Coates, John D; Achenbach, Laurie A

2004-09-01

Natural attenuation of the environmental contaminant perchlorate is a cost-effective alternative to current removal methods. The success of natural perchlorate remediation is dependent on the presence and activity of dissimilatory (per)chlorate-reducing bacteria (DPRB) within a target site. To detect DPRB in the environment, two degenerate primer sets targeting the chlorite dismutase (cld) gene were developed and optimized. A nested PCR approach was used in conjunction with these primer sets to increase the sensitivity of the molecular detection method. Screening of environmental samples indicated that all products amplified by this method were cld gene sequences. These sequences were obtained from pristine sites as well as contaminated sites from which DPRB were isolated. More than one cld phylotype was also identified from some samples, indicating the presence of more than one DPRB strain at those sites. The use of these primer sets represents a direct and sensitive molecular method for the qualitative detection of (per)chlorate-reducing bacteria in the environment, thus offering another tool for monitoring natural attenuation. Sequences of cld genes isolated in the course of this project were also generated from various DPRB and provided the first opportunity for a phylogenetic treatment of this metabolic gene. Comparisons of the cld and 16S ribosomal DNA (rDNA) gene trees indicated that the cld gene does not track 16S rDNA phylogeny, further implicating the possible role of horizontal transfer in the evolution of (per)chlorate respiration.

Molecular engineering of cyanine dyes to design a panchromatic response in Co-sensitized dye-sensitized solar cells

DOE PAGES

Pepe, Giulio; Cole, Jacqueline M.; Waddell, Paul G.; ...

2016-04-05

Cyanines are optically tunable dyes with high molar extinction coefficients, suitable for applications in co-sensitized dye-sensitized solar cells (DSCs); yet, barely thus applied. This might be due to the lack of a rational molecular design strategy that efficiently exploits cyanine properties. This study computationally re-designs these dyes, to broaden their optical absorption spectrum and create dye···TiO 2 binding and co-sensitization functionality. This is achieved via a stepwise molecular engineering approach. Firstly, the structural and optical properties of four parent dyes are experimentally and computationally investigated: 3,3’-diethyloxacarbocyanine iodide, 3,3’-diethylthiacarbocyanine iodide, 3,3’-diethylthiadicarbocyanine iodide and 3,3’-diethylthiatricarbocyanine iodide. Secondly, the molecules are theoretically modifiedmore » and their energetics are analyzed and compared to the parent dyes. A dye···TiO 2 anchoring group (carboxylic or cyanoacrylic acid), absent from the parent dyes, is chemically substituted at different molecular positions to investigate changes in optical absorption. We find that cyanoacrylic acid substitution at the para-quinoidal position affects the absorption wavelength of all parent dyes, with an optimal bathochromic shift of ca. 40 nm. The theoretical lengthening of the polymethine chain is also shown to effect dye absorption. Two molecularly engineered dyes are proposed as promising co-sensitizers. Finally, corresponding dye···TiO 2 adsorption energy calculations corroborate their applicability, demonstrating the potential of cyanine dyes in DSC research.« less

Multi-Component Profiling of Trace Volatiles in Blood by Gas Chromatography/Mass Spectrometry with Dynamic Headspace Extraction

PubMed Central

Kakuta, Shoji; Yamashita, Toshiyuki; Nishiumi, Shin; Yoshida, Masaru; Fukusaki, Eiichiro; Bamba, Takeshi

2015-01-01

A dynamic headspace extraction method (DHS) with high-pressure injection is described. This dynamic extraction method has superior sensitivity to solid phase micro extraction, SPME and is capable of extracting the entire gas phase by purging the headspace of a vial. Optimization of the DHS parameters resulted in a highly sensitive volatile profiling system with the ability to detect various volatile components including alcohols at nanogram levels. The average LOD for a standard volatile mixture was 0.50 ng mL−1, and the average LOD for alcohols was 0.66 ng mL−1. This method was used for the analysis of volatile components from biological samples and compared with acute and chronic inflammation models. The method permitted the identification of volatiles with the same profile pattern as in vitro oxidized lipid-derived volatiles. In addition, the concentration of alcohols and aldehydes from the acute inflammation model samples were significantly higher than that for the chronic inflammation model samples. The different profiles between these samples could also be identified by this method. Finally, it was possible to analyze alcohols and low-molecular-weight volatiles that are difficult to analyze by SPME in high sensitivity and to show volatile profiling based on multi-volatile simultaneous analysis. PMID:26819905

Multi-Component Profiling of Trace Volatiles in Blood by Gas Chromatography/Mass Spectrometry with Dynamic Headspace Extraction.

PubMed

Kakuta, Shoji; Yamashita, Toshiyuki; Nishiumi, Shin; Yoshida, Masaru; Fukusaki, Eiichiro; Bamba, Takeshi

2015-01-01

A dynamic headspace extraction method (DHS) with high-pressure injection is described. This dynamic extraction method has superior sensitivity to solid phase micro extraction, SPME and is capable of extracting the entire gas phase by purging the headspace of a vial. Optimization of the DHS parameters resulted in a highly sensitive volatile profiling system with the ability to detect various volatile components including alcohols at nanogram levels. The average LOD for a standard volatile mixture was 0.50 ng mL(-1), and the average LOD for alcohols was 0.66 ng mL(-1). This method was used for the analysis of volatile components from biological samples and compared with acute and chronic inflammation models. The method permitted the identification of volatiles with the same profile pattern as in vitro oxidized lipid-derived volatiles. In addition, the concentration of alcohols and aldehydes from the acute inflammation model samples were significantly higher than that for the chronic inflammation model samples. The different profiles between these samples could also be identified by this method. Finally, it was possible to analyze alcohols and low-molecular-weight volatiles that are difficult to analyze by SPME in high sensitivity and to show volatile profiling based on multi-volatile simultaneous analysis.

An insight into the isolation, enumeration, and molecular detection of Listeria monocytogenes in food

PubMed Central

Law, Jodi Woan-Fei; Ab Mutalib, Nurul-Syakima; Chan, Kok-Gan; Lee, Learn-Han

2015-01-01

Listeria monocytogenes, a foodborne pathogen that can cause listeriosis through the consumption of food contaminated with this pathogen. The ability of L. monocytogenes to survive in extreme conditions and cause food contaminations have become a major concern. Hence, routine microbiological food testing is necessary to prevent food contamination and outbreaks of foodborne illness. This review provides insight into the methods for cultural detection, enumeration, and molecular identification of L. monocytogenes in various food samples. There are a number of enrichment and plating media that can be used for the isolation of L. monocytogenes from food samples. Enrichment media such as buffered Listeria enrichment broth, Fraser broth, and University of Vermont Medium (UVM) Listeria enrichment broth are recommended by regulatory agencies such as Food and Drug Administration-bacteriological and analytical method (FDA-BAM), US Department of Agriculture-Food and Safety (USDA-FSIS), and International Organization for Standardization (ISO). Many plating media are available for the isolation of L. monocytogenes, for instance, polymyxin acriflavin lithium-chloride ceftazidime aesculin mannitol, Oxford, and other chromogenic media. Besides, reference methods like FDA-BAM, ISO 11290 method, and USDA-FSIS method are usually applied for the cultural detection or enumeration of L. monocytogenes. most probable number technique is applied for the enumeration of L. monocytogenes in the case of low level contamination. Molecular methods including polymerase chain reaction, multiplex polymerase chain reaction, real-time/quantitative polymerase chain reaction, nucleic acid sequence-based amplification, loop-mediated isothermal amplification, DNA microarray, and next generation sequencing technology for the detection and identification of L. monocytogenes are discussed in this review. Overall, molecular methods are rapid, sensitive, specific, time- and labor-saving. In future, there are chances for the development of new techniques for the detection and identification of foodborne with improved features. PMID:26579116

An insight into the isolation, enumeration, and molecular detection of Listeria monocytogenes in food.

PubMed

Law, Jodi Woan-Fei; Ab Mutalib, Nurul-Syakima; Chan, Kok-Gan; Lee, Learn-Han

2015-01-01

Listeria monocytogenes, a foodborne pathogen that can cause listeriosis through the consumption of food contaminated with this pathogen. The ability of L. monocytogenes to survive in extreme conditions and cause food contaminations have become a major concern. Hence, routine microbiological food testing is necessary to prevent food contamination and outbreaks of foodborne illness. This review provides insight into the methods for cultural detection, enumeration, and molecular identification of L. monocytogenes in various food samples. There are a number of enrichment and plating media that can be used for the isolation of L. monocytogenes from food samples. Enrichment media such as buffered Listeria enrichment broth, Fraser broth, and University of Vermont Medium (UVM) Listeria enrichment broth are recommended by regulatory agencies such as Food and Drug Administration-bacteriological and analytical method (FDA-BAM), US Department of Agriculture-Food and Safety (USDA-FSIS), and International Organization for Standardization (ISO). Many plating media are available for the isolation of L. monocytogenes, for instance, polymyxin acriflavin lithium-chloride ceftazidime aesculin mannitol, Oxford, and other chromogenic media. Besides, reference methods like FDA-BAM, ISO 11290 method, and USDA-FSIS method are usually applied for the cultural detection or enumeration of L. monocytogenes. most probable number technique is applied for the enumeration of L. monocytogenes in the case of low level contamination. Molecular methods including polymerase chain reaction, multiplex polymerase chain reaction, real-time/quantitative polymerase chain reaction, nucleic acid sequence-based amplification, loop-mediated isothermal amplification, DNA microarray, and next generation sequencing technology for the detection and identification of L. monocytogenes are discussed in this review. Overall, molecular methods are rapid, sensitive, specific, time- and labor-saving. In future, there are chances for the development of new techniques for the detection and identification of foodborne with improved features.

A multivariate relationship for the impact sensitivities of energetic N-nitrocompounds based on bond dissociation energy.

PubMed

Li, Jinshan

2010-02-15

The ZPE-corrected N-NO(2) bond dissociation energies (BDEs(ZPE)) of a series of model N-nitrocompounds and typical energetic N-nitrocompounds have been calculated using density functional theory methods. Computed results show that using the 6-31G** basis set the UB3LYP calculated BDE(ZPE) is similar to the B3PW91 but is less than the UB3P86 and that for both UB3P86 and UB3PW91 methods the 6-31G(**) calculated BDE(ZPE) is close to the 6-31++G(**). For the series of model N-nitrocompounds it is drawn from the NBO analysis that at the UB3LYP/6-31G(**) level the order of BDE(ZPE) is not only in line with that of bond order but also with that of the energy gap between N-NO(2) bond and antibond orbitals. For the typical energetic N-nitrocompounds the impact sensitivity is strongly related to the BDE(ZPE) indeed, and based on the BDEs(ZPE) calculated at different density functional theory levels this work has established a good multivariate correlation of impact sensitivity with molecular parameters, which provides a method to address the sensitivity problem.

In Situ Determination of Trace Elements in Fish Otoliths by Laser Ablation Double Focusing Sector Field Inductively Coupled Plasma Mass Spectrometry Using a Solution Standard Addition Calibration Method

NASA Astrophysics Data System (ADS)

Chen, Z.; Jones, C. M.

2002-05-01

Microchemistry of fish otoliths (fish ear bones) is a very useful tool for monitoring aquatic environments and fish migration. However, determination of the elemental composition in fish otolith by ICP-MS has been limited to either analysis of dissolved sample solution or measurement of limited number of trace elements by laser ablation (LA)- ICP-MS due to low sensitivity, lack of available calibration standards, and complexity of polyatomic molecular interference. In this study, a method was developed for in situ determination of trace elements in fish otoliths by laser ablation double focusing sector field ultra high sensitivity Finnigan Element 2 ICP-MS using a solution standard addition calibration method. Due to the lack of matrix-match solid calibration standards, sixteen trace elements (Na, Mg, P, Cr, Mn, Fe, Ni, Cu, Rb, Sr, Y, Cd, La, Ba, Pb and U) were determined using a solution standard calibration with Ca as an internal standard. Flexibility, easy preparation and stable signals are the advantages of using solution calibration standards. In order to resolve polyatomic molecular interferences, medium resolution (M/delta M > 4000) was used for some elements (Na, Mg, P, Cr, Mn, Fe, Ni, and Cu). Both external calibration and standard addition quantification strategies are compared and discussed. Precision, accuracy, and limits of detection are presented.

Biomimetic ELISA detection of malachite green based on magnetic molecularly imprinted polymers.

PubMed

Li, Lu; Lin, Zheng-Zhong; Peng, Ai-Hong; Zhong, Hui-Ping; Chen, Xiao-Mei; Huang, Zhi-Yong

2016-11-01

A direct competitive enzyme-linked immunosorbent assay (ELISA) method was used for the detection of malachite green (MG) with a high sensitivity and selectivity using magnetic molecularly imprinted polymers (MMIPs) as a bionic antibody. MMIPs were prepared through emulsion polymerization using Fe 3 O 4 nanoparticles as magnetic nuclei, MG as a template, methacrylic acid (MAA) as a functional monomer, ethylene glycol dimethacrylate (EGDMA) as a crosslinking agent and span-80/tween-80 as mixed emulsifiers. The MMIPs were characterized by scanning electron micrographs (SEM), thermal-gravimetric analyzer (TGA), Fourier transform infrared spectrometer (FT-IR) and vibrating sample magnetometer (VSM), respectively. A high magnetic saturation value of 54.1emug -1 was obtained, resulting in rapid magnetic separation of MMIPs with an external magnet. The IC 50 of the established ELISA method was 20.1μgL -1 and the detection limit (based on IC 85 ) was 0.1μgL -1 . The MMIPs exhibited high selective binding capacity for MG with cross-reactivities less than 3.9% for MG structural analogues. The MG spiking recoveries were 85.0%-106% with the relative standard deviations less than 4.7%. The results showed that the biomimetic ELISA method by using MMIPs as bionic antibody could be used to detect MG rapidly in fish samples with a high sensitivity and accuracy. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantum Dot Platform for Single-Cell Molecular Profiling

NASA Astrophysics Data System (ADS)

Zrazhevskiy, Pavel S.

In-depth understanding of the nature of cell physiology and ability to diagnose and control the progression of pathological processes heavily rely on untangling the complexity of intracellular molecular mechanisms and pathways. Therefore, comprehensive molecular profiling of individual cells within the context of their natural tissue or cell culture microenvironment is essential. In principle, this goal can be achieved by tagging each molecular target with a unique reporter probe and detecting its localization with high sensitivity at sub-cellular resolution, primarily via microscopy-based imaging. Yet, neither widely used conventional methods nor more advanced nanoparticle-based techniques have been able to address this task up to date. High multiplexing potential of fluorescent probes is heavily restrained by the inability to uniquely match probes with corresponding molecular targets. This issue is especially relevant for quantum dot probes---while simultaneous spectral imaging of up to 10 different probes is possible, only few can be used concurrently for staining with existing methods. To fully utilize multiplexing potential of quantum dots, it is necessary to design a new staining platform featuring unique assignment of each target to a corresponding quantum dot probe. This dissertation presents two complementary versatile approaches towards achieving comprehensive single-cell molecular profiling and describes engineering of quantum dot probes specifically tailored for each staining method. Analysis of expanded molecular profiles is achieved through augmenting parallel multiplexing capacity with performing several staining cycles on the same specimen in sequential manner. In contrast to other methods utilizing quantum dots or other nanoparticles, which often involve sophisticated probe synthesis, the platform technology presented here takes advantage of simple covalent bioconjugation and non-covalent self-assembly mechanisms for straightforward probe preparation and specimen labeling, requiring no advanced technical skills and being directly applicable for a wide range of molecular profiling studies. Utilization of quantum dot platform for single-cell molecular profiling promises to greatly benefit both biomedical research and clinical diagnostics by providing a tool for addressing phenotypic heterogeneity within large cell populations, opening access to studying low-abundance events often masked or completely erased by batch processing, and elucidating biomarker signatures of diseases critical for accurate diagnostics and targeted therapy.

High-throughput analysis of sulfatides in cerebrospinal fluid using automated extraction and UPLC-MS/MS.

PubMed

Blomqvist, Maria; Borén, Jan; Zetterberg, Henrik; Blennow, Kaj; Månsson, Jan-Eric; Ståhlman, Marcus

2017-07-01

Sulfatides (STs) are a group of glycosphingolipids that are highly expressed in brain. Due to their importance for normal brain function and their potential involvement in neurological diseases, development of accurate and sensitive methods for their determination is needed. Here we describe a high-throughput oriented and quantitative method for the determination of STs in cerebrospinal fluid (CSF). The STs were extracted using a fully automated liquid/liquid extraction method and quantified using ultra-performance liquid chromatography coupled to tandem mass spectrometry. With the high sensitivity of the developed method, quantification of 20 ST species from only 100 μl of CSF was performed. Validation of the method showed that the STs were extracted with high recovery (90%) and could be determined with low inter- and intra-day variation. Our method was applied to a patient cohort of subjects with an Alzheimer's disease biomarker profile. Although the total ST levels were unaltered compared with an age-matched control group, we show that the ratio of hydroxylated/nonhydroxylated STs was increased in the patient cohort. In conclusion, we believe that the fast, sensitive, and accurate method described in this study is a powerful new tool for the determination of STs in clinical as well as preclinical settings. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

Specific detection of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella from single vegetative cells by a loop-mediated isothermal amplification method.

PubMed

Nagai, Satoshi; Itakura, Shigeru

2012-09-01

In this study, we succeeded in developing a loop-mediated isothermal amplification (LAMP) method that enables sensitive and specific detection of the toxic marine dinoflagellates Alexandrium tamarense and Alexandrium catenella from single cells of both laboratory cultures and naturally blooming cells within 25 min, by monitoring the turbidimeter from the start of the LAMP reaction. The fluorescence intensity was strong enough to allow discrimination between positive and negative results by naked eye under a UV lamp, even in amplified samples from a single cell, by using the LAMP method. Unambiguous detection by naked eye was possible even in half the volume of LAMP cocktail recommended by the manufacturer, suggesting the potential to significantly reduce the cost of Alexandrium monitoring. Therefore, we can conclude that this method is one of the most convenient, sensitive, and cost-effective molecular tools for Alexandrium monitoring. Copyright © 2012 Elsevier B.V. All rights reserved.

Iron oxide nanoparticle-micelles (ION-micelles) for sensitive (molecular) magnetic particle imaging and magnetic resonance imaging.

PubMed

Starmans, Lucas W E; Burdinski, Dirk; Haex, Nicole P M; Moonen, Rik P M; Strijkers, Gustav J; Nicolay, Klaas; Grüll, Holger

2013-01-01

Iron oxide nanoparticles (IONs) are a promising nanoplatform for contrast-enhanced MRI. Recently, magnetic particle imaging (MPI) was introduced as a new imaging modality, which is able to directly visualize magnetic particles and could serve as a more sensitive and quantitative alternative to MRI. However, MPI requires magnetic particles with specific magnetic properties for optimal use. Current commercially available iron oxide formulations perform suboptimal in MPI, which is triggering research into optimized synthesis strategies. Most synthesis procedures aim at size control of iron oxide nanoparticles rather than control over the magnetic properties. In this study, we report on the synthesis, characterization and application of a novel ION platform for sensitive MPI and MRI. IONs were synthesized using a thermal-decomposition method and subsequently phase-transferred by encapsulation into lipidic micelles (ION-Micelles). Next, the material and magnetic properties of the ION-Micelles were analyzed. Most notably, vibrating sample magnetometry measurements showed that the effective magnetic core size of the IONs is 16 nm. In addition, magnetic particle spectrometry (MPS) measurements were performed. MPS is essentially zero-dimensional MPI and therefore allows to probe the potential of iron oxide formulations for MPI. ION-Micelles induced up to 200 times higher signal in MPS measurements than commercially available iron oxide formulations (Endorem, Resovist and Sinerem) and thus likely allow for significantly more sensitive MPI. In addition, the potential of the ION-Micelle platform for molecular MPI and MRI was showcased by MPS and MRI measurements of fibrin-binding peptide functionalized ION-Micelles (FibPep-ION-Micelles) bound to blood clots. The presented data underlines the potential of the ION-Micelle nanoplatform for sensitive (molecular) MPI and warrants further investigation of the FibPep-ION-Micelle platform for in vivo, non-invasive imaging of fibrin in preclinical disease models of thrombus-related pathologies and atherosclerosis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Belinsky, Steven A; Palmisano, William A

A molecular marker-based method for monitoring and detecting cancer in humans. Aberrant methylation of gene promoters is a marker for cancer risk in humans. A two-stage, or "nested" polymerase chain reaction method is disclosed for detecting methylated DNA sequences at sufficiently high levels of sensitivity to permit cancer screening in biological fluid samples, such as sputum, obtained non-invasively. The method is for detecting the aberrant methylation of the p16 gene, O 6-methylguanine-DNA methyltransferase gene, Death-associated protein kinase gene, RAS-associated family 1 gene, or other gene promoters. The method offers a potentially powerful approach to population-based screening for the detection ofmore » lung and other cancers.« less

Increasing role of arthropod bites in tularaemia transmission in Poland - case reports and diagnostic methods.

PubMed

Formińska, Kamila; Zasada, Aleksandra A; Rastawicki, Waldemar; Śmietańska, Karolina; Bander, Dorota; Wawrzynowicz-Syczewska, Marta; Yanushevych, Mariya; Niścigórska-Olsen, Jolanta; Wawszczak, Marek

2015-01-01

The study describes four cases of tularaemia - one developed after contact with rabbits and three developed after an arthropod bite. Due to non-specific clinical symptoms, accurate diagnosis of tularaemia may be difficult. The increasing contribution of the arthropod vectors in the transmission of the disease indicates that special effort should be made to apply sensitive and specific diagnostic methods for tularaemia, and to remind health-care workers about this route of Francisella tularensis infections. The advantages and disadvantages of various diagnostic methods - molecular, serological and microbiological culture - are discussed. The PCR as a rapid and proper diagnostic method for ulceroglandular tularaemia is presented.

A Novel High-Throughput Method for Molecular Detection of Human Pathogenic Viruses Using a Nanofluidic Real-Time PCR System

PubMed Central

Coudray-Meunier, Coralie; Fraisse, Audrey; Martin-Latil, Sandra; Delannoy, Sabine; Fach, Patrick; Perelle, Sylvie

2016-01-01

Human enteric viruses are recognized as the main causes of food- and waterborne diseases worldwide. Sensitive and quantitative detection of human enteric viruses is typically achieved through quantitative RT-PCR (RT-qPCR). A nanofluidic real-time PCR system was used to develop novel high-throughput methods for qualitative molecular detection (RT-qPCR array) and quantification of human pathogenic viruses by digital RT-PCR (RT-dPCR). The performance of high-throughput PCR methods was investigated for detecting 19 human pathogenic viruses and two main process controls used in food virology. The conventional real-time PCR system was compared to the RT-dPCR and RT-qPCR array. Based on the number of genome copies calculated by spectrophotometry, sensitivity was found to be slightly better with RT-qPCR than with RT-dPCR for 14 viruses by a factor range of from 0.3 to 1.6 log10. Conversely, sensitivity was better with RT-dPCR than with RT-qPCR for seven viruses by a factor range of from 0.10 to 1.40 log10. Interestingly, the number of genome copies determined by RT-dPCR was always from 1 to 2 log10 lower than the expected copy number calculated by RT-qPCR standard curve. The sensitivity of the RT-qPCR and RT-qPCR array assays was found to be similar for two viruses, and better with RT-qPCR than with RT-qPCR array for eighteen viruses by a factor range of from 0.7 to 3.0 log10. Conversely, sensitivity was only 0.30 log10 better with the RT-qPCR array than with conventional RT-qPCR assays for norovirus GIV detection. Finally, the RT-qPCR array and RT-dPCR assays were successfully used together to screen clinical samples and quantify pathogenic viruses. Additionally, this method made it possible to identify co-infection in clinical samples. In conclusion, given the rapidity and potential for large numbers of viral targets, this nanofluidic RT-qPCR assay should have a major impact on human pathogenic virus surveillance and outbreak investigations and is likely to be of benefit to public health. PMID:26824897

Molecular dissection of colorectal cancer in pre-clinical models identifies biomarkers predicting sensitivity to EGFR inhibitors

PubMed Central

Schütte, Moritz; Risch, Thomas; Abdavi-Azar, Nilofar; Boehnke, Karsten; Schumacher, Dirk; Keil, Marlen; Yildiriman, Reha; Jandrasits, Christine; Borodina, Tatiana; Amstislavskiy, Vyacheslav; Worth, Catherine L.; Schweiger, Caroline; Liebs, Sandra; Lange, Martin; Warnatz, Hans- Jörg; Butcher, Lee M.; Barrett, James E.; Sultan, Marc; Wierling, Christoph; Golob-Schwarzl, Nicole; Lax, Sigurd; Uranitsch, Stefan; Becker, Michael; Welte, Yvonne; Regan, Joseph Lewis; Silvestrov, Maxine; Kehler, Inge; Fusi, Alberto; Kessler, Thomas; Herwig, Ralf; Landegren, Ulf; Wienke, Dirk; Nilsson, Mats; Velasco, Juan A.; Garin-Chesa, Pilar; Reinhard, Christoph; Beck, Stephan; Schäfer, Reinhold; Regenbrecht, Christian R. A.; Henderson, David; Lange, Bodo; Haybaeck, Johannes; Keilholz, Ulrich; Hoffmann, Jens; Lehrach, Hans; Yaspo, Marie-Laure

2017-01-01

Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I–IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling >4,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab. PMID:28186126

High Resolution Melt analysis for mutation screening in PKD1 and PKD2

PubMed Central

2011-01-01

Background Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disorder. It is characterized by focal development and progressive enlargement of renal cysts leading to end-stage renal disease. PKD1 and PKD2 have been implicated in ADPKD pathogenesis but genetic features and the size of PKD1 make genetic diagnosis tedious. Methods We aim to prove that high resolution melt analysis (HRM), a recent technique in molecular biology, can facilitate molecular diagnosis of ADPKD. We screened for mutations in PKD1 and PKD2 with HRM in 37 unrelated patients with ADPKD. Results We identified 440 sequence variants in the 37 patients. One hundred and thirty eight were different. We found 28 pathogenic mutations (25 in PKD1 and 3 in PKD2 ) within 28 different patients, which is a diagnosis rate of 75% consistent with literature mean direct sequencing diagnosis rate. We describe 52 new sequence variants in PKD1 and two in PKD2. Conclusion HRM analysis is a sensitive and specific method for molecular diagnosis of ADPKD. HRM analysis is also costless and time sparing. Thus, this method is efficient and might be used for mutation pre-screening in ADPKD genes. PMID:22008521

Update on hepatitis B and C virus diagnosis

PubMed Central

Villar, Livia Melo; Cruz, Helena Medina; Barbosa, Jakeline Ribeiro; Bezerra, Cristianne Sousa; Portilho, Moyra Machado; Scalioni, Letícia de Paula

2015-01-01

Viral hepatitis B and C virus (HBV and HCV) are responsible for the most of chronic liver disease worldwide and are transmitted by parenteral route, sexual and vertical transmission. One important measure to reduce the burden of these infections is the diagnosis of acute and chronic cases of HBV and HCV. In order to provide an effective diagnosis and monitoring of antiviral treatment, it is important to choose sensitive, rapid, inexpensive, and robust analytical methods. Primary diagnosis of HBV and HCV infection is made by using serological tests for detecting antigens and antibodies against these viruses. In order to confirm primary diagnosis, to quantify viral load, to determine genotypes and resistance mutants for antiviral treatment, qualitative and quantitative molecular tests are used. In this manuscript, we review the current serological and molecular methods for the diagnosis of hepatitis B and C. PMID:26568915

Extreme sensitivity biosensing platform based on hyperbolic metamaterials

NASA Astrophysics Data System (ADS)

Sreekanth, Kandammathe Valiyaveedu; Alapan, Yunus; Elkabbash, Mohamed; Ilker, Efe; Hinczewski, Michael; Gurkan, Umut A.; de Luca, Antonio; Strangi, Giuseppe

2016-06-01

Optical sensor technology offers significant opportunities in the field of medical research and clinical diagnostics, particularly for the detection of small numbers of molecules in highly diluted solutions. Several methods have been developed for this purpose, including label-free plasmonic biosensors based on metamaterials. However, the detection of lower-molecular-weight (<500 Da) biomolecules in highly diluted solutions is still a challenging issue owing to their lower polarizability. In this context, we have developed a miniaturized plasmonic biosensor platform based on a hyperbolic metamaterial that can support highly confined bulk plasmon guided modes over a broad wavelength range from visible to near infrared. By exciting these modes using a grating-coupling technique, we achieved different extreme sensitivity modes with a maximum of 30,000 nm per refractive index unit (RIU) and a record figure of merit (FOM) of 590. We report the ability of the metamaterial platform to detect ultralow-molecular-weight (244 Da) biomolecules at picomolar concentrations using a standard affinity model streptavidin-biotin.

Critical comparison of the on-line and off-line molecularly imprinted solid-phase extraction of patulin coupled with liquid chromatography.

PubMed

Lhotská, Ivona; Holznerová, Anežka; Solich, Petr; Šatínský, Dalibor

2017-12-01

Reaching trace amounts of mycotoxin contamination requires sensitive and selective analytical tools for their determination. Improving the selectivity of sample pretreatment steps covering new and modern extraction techniques is one way to achieve it. Molecularly imprinted polymers as selective sorbent for extraction undoubtedly meet these criteria. The presented work is focused on the hyphenation of on-line molecularly imprinted solid-phase extraction with a chromatography system using a column-switching approach. Making a critical comparison with a simultaneously developed off-line extraction procedure, evaluation of pros and cons of each method, and determining the reliability of both methods on a real sample analysis were carried out. Both high-performance liquid chromatography methods, using off-line extraction on molecularly imprinted polymer and an on-line column-switching approach, were validated, and the validation results were compared against each other. Although automation leads to significant time savings, fewer human errors, and required no handling of toxic solvents, it reached worse detection limits (15 versus 6 μg/L), worse recovery values (68.3-123.5 versus 81.2-109.9%), and worse efficiency throughout the entire clean-up process in comparison with the off-line extraction method. The difficulties encountered, the compromises made during the optimization of on-line coupling and their critical evaluation are presented in detail. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Magnetic molecularly imprinted polymers for the determination of β-agonist residues in milk by ultra high performance liquid chromatography with tandem mass spectrometry.

PubMed

Liu, Hongcheng; Lin, Xin; Lin, Tao; Zhang, Yulong; Luo, Yinglan; Li, Qiwan

2016-09-01

A simple, accurate, and highly sensitive analytical method was developed in this study for the determination of nine β-agonists in milk. In this method, a new magnetic adsorbent of molecularly imprinted polymers/magnetic nanoparticles prepared by simple physical blending was adopted, which enabled magnetic solid-phase extraction. Thus, the resultant material can be separated from the solvent rapidly and conveniently by a magnet. Two kinds of molecularly imprinted polymer/magnetic nanoparticles materials were fabricated, and the characteristics of materials such as the ratio, pH, amount, desorption, and regeneration were investigated. The analytes were quantified by ultra high performance liquid chromatography coupled to an electrospray ionization tandem mass spectrometer operating in multiple reaction monitoring modes. The detection limit of the method was 0.003-0.3 μg/L, and the detection capability was 0.01-0.3 μg/L. The recoveries of these compounds were 65.7-114% at three spiked levels. Reproducibility represented by relative standard deviation was 11.2% or less. The method was successfully applied to the screening of real samples obtained from local markets and confirmation of the suspected target analytes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

External quality assessment studies for laboratory performance of molecular and serological diagnosis of Chikungunya virus infection.

PubMed

Jacobsen, Sonja; Patel, Pranav; Schmidt-Chanasit, Jonas; Leparc-Goffart, Isabelle; Teichmann, Anette; Zeller, Herve; Niedrig, Matthias

2016-03-01

Since the re-emergence of Chikungunya virus (CHIKV) in Reunion in 2005 and the recent outbreak in the Caribbean islands with an expansion to the Americas the CHIK diagnostic became very important. We evaluate the performance of laboratories regarding molecular and serological diagnostic of CHIK worldwide. A panel of 12 samples for molecular and 13 samples for serology were provided to 60 laboratories in 40 countries for evaluating the sensitivity and specificity of molecular and serology testing. The panel for molecular diagnostic testing was analysed by 56 laboratories returning 60 data sets of results whereas the 56 and 60 data sets were returned for IgG and IgM diagnostic from the participating laboratories. Twenty-three from 60 data sets performed optimal, 7 acceptable and 30 sets of results require improvement. From 50 data sets only one laboratory shows an optimal performance for IgM detection, followed by 9 data sets with acceptable and the rest need for improvement. From 46 IgG serology data sets 20 provide an optimal, 2 an acceptable and 24 require improvement performance. The evaluation of some of the diagnostic performances allows linking the quality of results to the in-house methods or commercial assays used. The external quality assurance for CHIK diagnostics provides a good overview on the laboratory performance regarding sensitivity and specificity for the molecular and serology diagnostic required for the quick and reliable analysis of suspected CHIK patients. Nearly half of the laboratories have to improve their diagnostic profile to achieve a better performance. Copyright © 2016 Z. Published by Elsevier B.V. All rights reserved.

Synthesis of molecularly imprinted dye-silica nanocomposites with high selectivity and sensitivity: Fluorescent imprinted sensor for rapid and efficient detection of τ-fluvalinate in vodka.

PubMed

Wang, Yunyun; Wang, Jixiang; Cheng, Rujia; Sun, Lin; Dai, Xiaohui; Yan, Yongsheng

2018-04-01

An imprinted fluorescent sensor was fabricated based on SiO 2 nanoparticles encapsulated with a molecularly imprinted polymer containing allyl fluorescein. High fluorine cypermethirin as template molecules, methyl methacrylate as functional monomer, and allyl fluorescein as optical materials synthesized a core-shell fluorescent molecular imprinted sensor, which showed a high and rapid sensitivity and selectivity for the detection of τ-fluvalinate. The sensor presented appreciable sensitivity with a limit of 13.251 nM, rapid detection that reached to equilibrium within 3 min, great linear relationship in the relevant concentration range from 0 to 150 nM, and excellent selectivity over structural analogues. In addition, the fluorescent sensor demonstrated desirable regeneration ability (eight cycling operations). The molecularly imprinted polymers ensured specificity, while the fluorescent dyes provided the stabile sensitivity. Finally, an effective application of the sensor was implemented by the detection of τ-fluvalinate in real samples from vodka. The molecularly imprinted fluorescent sensor showed a promising potential in environmental monitoring and food safety. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Surface similarity-based molecular query-retrieval

PubMed Central

Singh, Rahul

2007-01-01

Background Discerning the similarity between molecules is a challenging problem in drug discovery as well as in molecular biology. The importance of this problem is due to the fact that the biochemical characteristics of a molecule are closely related to its structure. Therefore molecular similarity is a key notion in investigations targeting exploration of molecular structural space, query-retrieval in molecular databases, and structure-activity modelling. Determining molecular similarity is related to the choice of molecular representation. Currently, representations with high descriptive power and physical relevance like 3D surface-based descriptors are available. Information from such representations is both surface-based and volumetric. However, most techniques for determining molecular similarity tend to focus on idealized 2D graph-based descriptors due to the complexity that accompanies reasoning with more elaborate representations. Results This paper addresses the problem of determining similarity when molecules are described using complex surface-based representations. It proposes an intrinsic, spherical representation that systematically maps points on a molecular surface to points on a standard coordinate system (a sphere). Molecular surface properties such as shape, field strengths, and effects due to field super-positioningcan then be captured as distributions on the surface of the sphere. Surface-based molecular similarity is subsequently determined by computing the similarity of the surface-property distributions using a novel formulation of histogram-intersection. The similarity formulation is not only sensitive to the 3D distribution of the surface properties, but is also highly efficient to compute. Conclusion The proposed method obviates the computationally expensive step of molecular pose-optimisation, can incorporate conformational variations, and facilitates highly efficient determination of similarity by directly comparing molecular surfaces and surface-based properties. Retrieval performance, applications in structure-activity modeling of complex biological properties, and comparisons with existing research and commercial methods demonstrate the validity and effectiveness of the approach. PMID:17634096

A combined EPR and MD simulation study of a nitroxyl spin label with restricted internal mobility sensitive to protein dynamics.

PubMed

Oganesyan, Vasily S; Chami, Fatima; White, Gaye F; Thomson, Andrew J

2017-01-01

EPR studies combined with fully atomistic Molecular Dynamics (MD) simulations and an MD-EPR simulation method provide evidence for intrinsic low rotameric mobility of a nitroxyl spin label, Rn, compared to the more widely employed label MTSL (R1). Both experimental and modelling results using two structurally different sites of attachment to Myoglobin show that the EPR spectra of Rn are more sensitive to the local protein environment than that of MTSL. This study reveals the potential of using the Rn spin label as a reporter of protein motions. Copyright © 2016 Elsevier Inc. All rights reserved.

High sensitivity contrast enhanced optical coherence tomography for functional in vivo imaging

NASA Astrophysics Data System (ADS)

Liba, Orly; SoRelle, Elliott D.; Sen, Debasish; de la Zerda, Adam

2017-02-01

In this study, we developed and applied highly-scattering large gold nanorods (LGNRs) and custom spectral detection algorithms for high sensitivity contrast-enhanced optical coherence tomography (OCT). We were able to detect LGNRs at a concentration as low as 50 pM in blood. We used this approach for noninvasive 3D imaging of blood vessels deep in solid tumors in living mice. Additionally, we demonstrated multiplexed imaging of spectrally-distinct LGNRs that enabled observations of functional drainage in lymphatic networks. This method, which we call MOZART, provides a platform for molecular imaging and characterization of tissue noninvasively at cellular resolution.

236U measurement with accelerator mass spectrometry at CIAE

NASA Astrophysics Data System (ADS)

Wang, Xianggao; Jiang, Shan; He, Ming; Dong, Kejun; Wang, Wei; Li, Chaoli; He, Guozhu; Li, Shizhuo; Gong, Jie; Lu, Liyuan; Wu, Shaoyong

2010-07-01

236U is a long-lived radioactive isotope which is produced principally by thermal neutron capture on 235U. 236U may be potentially applied in geological research and nuclear safeguards. Accelerator mass spectrometry is presently the most sensitive technique for the measurement of 236U and a measurement method for long-lived heavy ion 236U has been developed. The set-up uses a dedicated injector and the newly proposed 208Pb 16O2- molecular ions for the simulation of 236U ion transport. A sensitivity of lower than 10 -10 has been achieved for the isotopic ratio 236U/ 238U in present work.

Sensitivity, Specificity, PPV, and NPV for Predictive Biomarkers.

PubMed

Simon, Richard

2015-08-01

Molecularly targeted cancer drugs are often developed with companion diagnostics that attempt to identify which patients will have better outcome on the new drug than the control regimen. Such predictive biomarkers are playing an increasingly important role in precision oncology. For diagnostic tests, sensitivity, specificity, positive predictive value, and negative predictive are usually used as performance measures. This paper discusses these indices for predictive biomarkers, provides methods for their calculation with survival or response endpoints, and describes assumptions involved in their use. Published by Oxford University Press 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Prediction of Chemical Respiratory Sensitizers Using GARD, a Novel In Vitro Assay Based on a Genomic Biomarker Signature

PubMed Central

Albrekt, Ann-Sofie; Borrebaeck, Carl A. K.; Lindstedt, Malin

2015-01-01

Background Repeated exposure to certain low molecular weight (LMW) chemical compounds may result in development of allergic reactions in the skin or in the respiratory tract. In most cases, a certain LMW compound selectively sensitize the skin, giving rise to allergic contact dermatitis (ACD), or the respiratory tract, giving rise to occupational asthma (OA). To limit occurrence of allergic diseases, efforts are currently being made to develop predictive assays that accurately identify chemicals capable of inducing such reactions. However, while a few promising methods for prediction of skin sensitization have been described, to date no validated method, in vitro or in vivo, exists that is able to accurately classify chemicals as respiratory sensitizers. Results Recently, we presented the in vitro based Genomic Allergen Rapid Detection (GARD) assay as a novel testing strategy for classification of skin sensitizing chemicals based on measurement of a genomic biomarker signature. We have expanded the applicability domain of the GARD assay to classify also respiratory sensitizers by identifying a separate biomarker signature containing 389 differentially regulated genes for respiratory sensitizers in comparison to non-respiratory sensitizers. By using an independent data set in combination with supervised machine learning, we validated the assay, showing that the identified genomic biomarker is able to accurately classify respiratory sensitizers. Conclusions We have identified a genomic biomarker signature for classification of respiratory sensitizers. Combining this newly identified biomarker signature with our previously identified biomarker signature for classification of skin sensitizers, we have developed a novel in vitro testing strategy with a potent ability to predict both skin and respiratory sensitization in the same sample. PMID:25760038

Microwave-assisted on-spot derivatization for gas chromatography-mass spectrometry based determination of polar low molecular weight compounds in dried blood spots.

PubMed

Sadones, Nele; Van Bever, Elien; Archer, John R H; Wood, David M; Dargan, Paul I; Van Bortel, Luc; Lambert, Willy E; Stove, Christophe P

2016-09-23

Dried blood spot (DBS) sampling and analysis is increasingly being applied in bioanalysis. Although the use of DBS has many advantages, it is also associated with some challenges. E.g. given the limited amount of available material, highly sensitive detection techniques are often required to attain sufficient sensitivity. In gas chromatography coupled to mass spectrometry (GC-MS), derivatization can be helpful to achieve adequate sensitivity. Because this additional sample preparation step is considered as time-consuming, we introduce a new derivatization procedure, i.e. "microwave-assisted on-spot derivatization", to minimize sample preparation of DBS. In this approach the derivatization reagents are directly applied onto the DBS and derivatization takes place in a microwave instead of via conventional heating. In this manuscript we evaluated the applicability of this new concept of derivatization for the determination of two polar low molecular weight molecules, gamma-hydroxybutyric acid (GHB) and gabapentin, in DBS using a standard GC-MS configuration. The method was successfully validated for both compounds, with imprecision and bias values within acceptance criteria (<20% at LLOQ, <15% at 3 other QC levels). Calibration lines were linear over the 10-100μg/mL and 1-30μg/mL range for GHB and gabapentin, respectively. Stability studies revealed no significant decrease of gabapentin and GHB in DBS upon storage at room temperature for at least 84 days. Furthermore, DBS-specific parameters, including hematocrit and volume spotted, were evaluated. As demonstrated by the analysis of GHB and gabapentin positive samples, "microwave-assisted on-spot derivatization" proved to be reliable, fast and applicable in routine toxicology. Moreover, other polar low molecular weight compounds of interest in clinical and/or forensic toxicology, including vigabatrin, beta-hydroxybutyric acid, propylene glycol, diethylene glycol, 1,4-butanediol and 1,2-butanediol, can also be detected using this method. Copyright © 2016 Elsevier B.V. All rights reserved.

Genomic, transcriptomic, and proteomic approaches towards understanding the molecular mechanisms of salt tolerance in Frankia strains isolated from Casuarina trees.

PubMed

Oshone, Rediet; Ngom, Mariama; Chu, Feixia; Mansour, Samira; Sy, Mame Ourèye; Champion, Antony; Tisa, Louis S

2017-08-18

Soil salinization is a worldwide problem that is intensifying because of the effects of climate change. An effective method for the reclamation of salt-affected soils involves initiating plant succession using fast growing, nitrogen fixing actinorhizal trees such as the Casuarina. The salt tolerance of Casuarina is enhanced by the nitrogen-fixing symbiosis that they form with the actinobacterium Frankia. Identification and molecular characterization of salt-tolerant Casuarina species and associated Frankia is imperative for the successful utilization of Casuarina trees in saline soil reclamation efforts. In this study, salt-tolerant and salt-sensitive Casuarina associated Frankia strains were identified and comparative genomics, transcriptome profiling, and proteomics were employed to elucidate the molecular mechanisms of salt and osmotic stress tolerance. Salt-tolerant Frankia strains (CcI6 and Allo2) that could withstand up to 1000 mM NaCl and a salt-sensitive Frankia strain (CcI3) which could withstand only up to 475 mM NaCl were identified. The remaining isolates had intermediate levels of salt tolerance with MIC values ranging from 650 mM to 750 mM. Comparative genomic analysis showed that all of the Frankia isolates from Casuarina belonged to the same species (Frankia casuarinae). Pangenome analysis revealed a high abundance of singletons among all Casuarina isolates. The two salt-tolerant strains contained 153 shared single copy genes (most of which code for hypothetical proteins) that were not found in the salt-sensitive(CcI3) and moderately salt-tolerant (CeD) strains. RNA-seq analysis of one of the two salt-tolerant strains (Frankia sp. strain CcI6) revealed hundreds of genes differentially expressed under salt and/or osmotic stress. Among the 153 genes, 7 and 7 were responsive to salt and osmotic stress, respectively. Proteomic profiling confirmed the transcriptome results and identified 19 and 8 salt and/or osmotic stress-responsive proteins in the salt-tolerant (CcI6) and the salt-sensitive (CcI3) strains, respectively. Genetic differences between salt-tolerant and salt-sensitive Frankia strains isolated from Casuarina were identified. Transcriptome and proteome profiling of a salt-tolerant strain was used to determine molecular differences correlated with differential salt-tolerance and several candidate genes were identified. Mechanisms involving transcriptional and translational regulation, cell envelop remodeling, and previously uncharacterized proteins appear to be important for salt tolerance. Physiological and mutational analyses will further shed light on the molecular mechanism of salt tolerance in Casuarina associated Frankia isolates.

Molecular weight distribution of polysaccharides from edible seaweeds by high-performance size-exclusion chromatography (HPSEC).

PubMed

Gómez-Ordóñez, Eva; Jiménez-Escrig, Antonio; Rupérez, Pilar

2012-05-15

Biological properties of polysaccharides from seaweeds are related to their composition and structure. Many factors such as the kind of sugar, type of linkage or sulfate content of algal biopolymers exert an influence in the relationship between structure and function. Besides, the molecular weight (MW) also plays an important role. Thus, a simple, reliable and fast HPSEC method with refractive index detection was developed and optimized for the MW estimation of soluble algal polysaccharides. Chromatogram shape and repeatability of retention time was considerably improved when sodium nitrate was used instead of ultrapure water as mobile phase. Pullulan and dextran standards of different MW were used for method calibration and validation. Also, main polysaccharide standards from brown (alginate, fucoidan, laminaran) and red seaweeds (kappa- and iota-carrageenan) were used for quantification and method precision and accuracy. Relative standard deviation (RSD) of repeatability for retention time, peak areas and inter-day precision was below 0.7%, 2.5% and 2.6%, respectively, which indicated good repeatability and precision. Recoveries (96.3-109.8%) also showed its fairly good accuracy. Regarding linearity, main polysaccharide standards from brown or red seaweeds showed a highly satisfactory correlation coefficient (r>0.999). Moreover, a good sensitivity was shown, with corresponding limits of detection and quantitation in mg/mL of 0.05-0.21 and 0.16-0.31, respectively. The method was applied to the MW estimation of standard algal polysaccharides, as well as to the soluble polysaccharide fractions from the brown seaweed Saccharina latissima and the red Mastocarpus stellatus, respectively. Although distribution of molecular weight was broad, the good repeatability for retention time provided a good precision in MW estimation of polysaccharides. Water- and alkali-soluble fractions from S. latissima ranged from very high (>2400 kDa) to low MW compounds (<6 kDa); this high heterogeneity could be attributable to the complex polysaccharide composition of brown algae. Regarding M. stellatus, sulfated galactans followed a descending order of MW (>1400 kDa to <10 kDa), related to the different solubility of carrageenans in red seaweeds. In summary, the method developed allows for the molecular weight analysis of seaweed polysaccharides with very good precision, accuracy, linearity and sensitivity within a short time. Copyright © 2012 Elsevier B.V. All rights reserved.

Nested methylation-specific polymerase chain reaction cancer detection method

DOEpatents

Belinsky, Steven A [Albuquerque, NM; Palmisano, William A [Edgewood, NM

2007-05-08

A molecular marker-based method for monitoring and detecting cancer in humans. Aberrant methylation of gene promoters is a marker for cancer risk in humans. A two-stage, or "nested" polymerase chain reaction method is disclosed for detecting methylated DNA sequences at sufficiently high levels of sensitivity to permit cancer screening in biological fluid samples, such as sputum, obtained non-invasively. The method is for detecting the aberrant methylation of the p16 gene, O 6-methylguanine-DNA methyltransferase gene, Death-associated protein kinase gene, RAS-associated family 1 gene, or other gene promoters. The method offers a potentially powerful approach to population-based screening for the detection of lung and other cancers.

Automated Online Solid-Phase Derivatization for Sensitive Quantification of Endogenous S-Nitrosoglutathione and Rapid Capture of Other Low-Molecular-Mass S-Nitrosothiols.

PubMed

Wang, Xin; Garcia, Carlos T; Gong, Guanyu; Wishnok, John S; Tannenbaum, Steven R

2018-02-06

S-Nitrosothiols (RSNOs) constitute a circulating endogenous reservoir of nitric oxide and have important biological activities. In this study, an online coupling of solid-phase derivatization (SPD) with liquid chromatography-mass spectrometry (LC-MS) was developed and applied in the analysis of low-molecular-mass RSNOs. A derivatizing-reagent-modified polymer monolithic column was prepared and adapted for online SPD-LC-MS. Analytes from the LC autosampler flowed through the monolithic column for derivatization and then directly into the LC-MS for analysis. This integration of the online derivatization, LC separation, and MS detection facilitated system automation, allowing rapid, laborsaving, and sensitive detection of RSNOs. S-Nitrosoglutathione (GSNO) was quantified using this automated online method with good linearity (R 2 = 0.9994); the limit of detection was 0.015 nM. The online SPD-LC-MS method has been used to determine GSNO levels in mouse samples, 138 ± 13.2 nM of endogenous GSNO was detected in mouse plasma. Besides, the GSNO concentrations in liver (64.8 ± 11.3 pmol/mg protein), kidney (47.2 ± 6.1 pmol/mg protein), heart (8.9 ± 1.8 pmol/mg protein), muscle (1.9 ± 0.3 pmol/mg protein), hippocampus (5.3 ± 0.9 pmol/mg protein), striatum (6.7 ± 0.6 pmol/mg protein), cerebellum (31.4 ± 6.5 pmol/mg protein), and cortex (47.9 ± 4.6 pmol/mg protein) were also successfully quantified. When the derivatization was performed within 8 min, followed by LC-MS detection, samples could be rapidly analyzed compared with the offline manual method. Other low-molecular-mass RSNOs, such as S-nitrosocysteine and S-nitrosocysteinylglycine, were captured by rapid precursor-ion scanning, showing that the proposed method is a potentially powerful tool for capture, identification, and quantification of RSNOs in biological samples.

Comparison of diagnostic methods to detect Histoplasma capsulatum in serum and blood samples from AIDS patients

PubMed Central

da Silva, Marcos Vinicius; Criado, Paulo Ricardo; Luiz, Olinda do Carmo; Vicentini, Adriana Pardini

2018-01-01

Background Although early and rapid detection of histoplasmosis is essential to prevent morbidity and mortality, few diagnostic tools are available in resource-limited areas, especially where it is endemic and HIV/AIDS is also epidemic. Thus, we compared conventional and molecular methods to detect Histoplasma capsulatum in sera and blood from HIV/AIDS patients. Methodology We collected a total of 40 samples from control volunteers and patients suspected of histoplasmosis, some of whom were also infected with other pathogens. Samples were then analyzed by mycological, serological, and molecular methods, and stratified as histoplasmostic with (group I) or without AIDS (group II), uninfected (group III), and infected with HIV and other pathogens only (group IV). All patients were receiving treatment for histoplasmosis and other infections at the time of sample collection. Results Comparison of conventional methods with nested PCR using primers against H. capsulatum 18S rRNA (HC18S), 5.8S rRNA ITS (HC5.8S-ITS), and a 100 kDa protein (HC100) revealed that sensitivity against sera was highest for PCR with HC5.8S-ITS, followed by immunoblotting, double immunodiffusion, PCR with HC18S, and PCR with HC100. Specificity was equally high for double immunodiffusion, immunoblotting and PCR with HC100, followed for PCR with HC18S and HC5.8-ITS. Against blood, sensitivity was highest for PCR with HC5.8S-ITS, followed by PCR with HC18S, Giemsa staining, and PCR with HC100. Specificity was highest for Giemsa staining and PCR with HC100, followed by PCR with HC18S and HC5.8S-ITS. PCR was less efficient in patients with immunodeficiency due to HIV/AIDS and/or related diseases. Conclusion Molecular techniques may detect histoplasmosis even in cases with negative serology and mycology, potentially enabling early diagnosis. PMID:29342162

Scrambled eggs: A highly sensitive molecular diagnostic workflow for Fasciola species specific detection from faecal samples

PubMed Central

Calvani, Nichola Eliza Davies; Windsor, Peter Andrew; Bush, Russell David

2017-01-01

Background Fasciolosis, due to Fasciola hepatica and Fasciola gigantica, is a re-emerging zoonotic parasitic disease of worldwide importance. Human and animal infections are commonly diagnosed by the traditional sedimentation and faecal egg-counting technique. However, this technique is time-consuming and prone to sensitivity errors when a large number of samples must be processed or if the operator lacks sufficient experience. Additionally, diagnosis can only be made once the 12-week pre-patent period has passed. Recently, a commercially available coprological antigen ELISA has enabled detection of F. hepatica prior to the completion of the pre-patent period, providing earlier diagnosis and increased throughput, although species differentiation is not possible in areas of parasite sympatry. Real-time PCR offers the combined benefits of highly sensitive species differentiation for medium to large sample sizes. However, no molecular diagnostic workflow currently exists for the identification of Fasciola spp. in faecal samples. Methodology/Principal findings A new molecular diagnostic workflow for the highly-sensitive detection and quantification of Fasciola spp. in faecal samples was developed. The technique involves sedimenting and pelleting the samples prior to DNA isolation in order to concentrate the eggs, followed by disruption by bead-beating in a benchtop homogeniser to ensure access to DNA. Although both the new molecular workflow and the traditional sedimentation technique were sensitive and specific, the new molecular workflow enabled faster sample throughput in medium to large epidemiological studies, and provided the additional benefit of speciation. Further, good correlation (R2 = 0.74–0.76) was observed between the real-time PCR values and the faecal egg count (FEC) using the new molecular workflow for all herds and sampling periods. Finally, no effect of storage in 70% ethanol was detected on sedimentation and DNA isolation outcomes; enabling transport of samples from endemic to non-endemic countries without the requirement of a complete cold chain. The commercially-available ELISA displayed poorer sensitivity, even after adjustment of the positive threshold (65–88%), compared to the sensitivity (91–100%) of the new molecular diagnostic workflow. Conclusions/Significance Species-specific assays for sensitive detection of Fasciola spp. enable ante-mortem diagnosis in both human and animal settings. This includes Southeast Asia where there are potentially many undocumented human cases and where post-mortem examination of production animals can be difficult. The new molecular workflow provides a sensitive and quantitative diagnostic approach for the rapid testing of medium to large sample sizes, potentially superseding the traditional sedimentation and FEC technique and enabling surveillance programs in locations where animal and human health funding is limited. PMID:28915255

Traditional and Modern Cell Culture in Virus Diagnosis.

PubMed

Hematian, Ali; Sadeghifard, Nourkhoda; Mohebi, Reza; Taherikalani, Morovat; Nasrolahi, Abbas; Amraei, Mansour; Ghafourian, Sobhan

2016-04-01

Cell cultures are developed from tissue samples and then disaggregated by mechanical, chemical, and enzymatic methods to extract cells suitable for isolation of viruses. With the recent advances in technology, cell culture is considered a gold standard for virus isolation. This paper reviews the evolution of cell culture methods and demonstrates why cell culture is a preferred method for identification of viruses. In addition, the advantages and disadvantages of both traditional and modern cell culture methods for diagnosis of each type of virus are discussed. Detection of viruses by the novel cell culture methods is considered more accurate and sensitive. However, there is a need to include some more accurate methods such as molecular methods in cell culture for precise identification of viruses.

Paper-Based MicroRNA Expression Profiling from Plasma and Circulating Tumor Cells.

PubMed

Leong, Sai Mun; Tan, Karen Mei-Ling; Chua, Hui Wen; Huang, Mo-Chao; Cheong, Wai Chye; Li, Mo-Huang; Tucker, Steven; Koay, Evelyn Siew-Chuan

2017-03-01

Molecular characterization of circulating tumor cells (CTCs) holds great promise for monitoring metastatic progression and characterizing metastatic disease. However, leukocyte and red blood cell contamination of routinely isolated CTCs makes CTC-specific molecular characterization extremely challenging. Here we report the use of a paper-based medium for efficient extraction of microRNAs (miRNAs) from limited amounts of biological samples such as rare CTCs harvested from cancer patient blood. Specifically, we devised a workflow involving the use of Flinders Technology Associates (FTA) ® Elute Card with a digital PCR-inspired "partitioning" method to extract and purify miRNAs from plasma and CTCs. We demonstrated the sensitivity of this method to detect miRNA expression from as few as 3 cancer cells spiked into human blood. Using this method, background miRNA expression was excluded from contaminating blood cells, and CTC-specific miRNA expression profiles were derived from breast and colorectal cancer patients. Plasma separated out during purification of CTCs could likewise be processed using the same paper-based method for miRNA detection, thereby maximizing the amount of patient-specific information that can be derived from a single blood draw. Overall, this paper-based extraction method enables an efficient, cost-effective workflow for maximized recovery of small RNAs from limited biological samples for downstream molecular analyses. © 2016 American Association for Clinical Chemistry.

[Detection of rubella virus RNA in clinical material by real time polymerase chain reaction method].

PubMed

Domonova, É A; Shipulina, O Iu; Kuevda, D A; Larichev, V F; Safonova, A P; Burchik, M A; Butenko, A M; Shipulin, G A

2012-01-01

Development of a reagent kit for detection of rubella virus RNA in clinical material by PCR-RT. During development and determination of analytical specificity and sensitivity DNA and RNA of 33 different microorganisms including 4 rubella strains were used. Comparison of analytical sensitivity of virological and molecular-biological methods was performed by using rubella virus strains Wistar RA 27/3, M-33, "Orlov", Judith. Evaluation of diagnostic informativity of rubella virus RNAisolation in various clinical material by PCR-RT method was performed in comparison with determination of virus specific serum antibodies by enzyme immunoassay. A reagent kit for the detection of rubella virus RNA in clinical material by PCR-RT was developed. Analytical specificity was 100%, analytical sensitivity - 400 virus RNA copies per ml. Analytical sensitivity of the developed technique exceeds analytical sensitivity of the Vero E6 cell culture infection method in studies of rubella virus strains Wistar RA 27/3 and "Orlov" by 11g and 31g, and for M-33 and Judith strains is analogous. Diagnostic specificity is 100%. Diagnostic specificity for testing samples obtained within 5 days of rash onset: for peripheral blood sera - 20.9%, saliva - 92.5%, nasopharyngeal swabs - 70.1%, saliva and nasopharyngeal swabs - 97%. Positive and negative predictive values of the results were shown depending on the type of clinical material tested. Application of reagent kit will allow to increase rubella diagnostics effectiveness at the early stages of infectious process development, timely and qualitatively perform differential diagnostics of exanthema diseases, support tactics of anti-epidemic regime.

Overview of hybridization and detection techniques.

PubMed

Hilario, Elena

2007-01-01

A misconception regarding the sensitivity of nonradioactive methods for screening genomic DNA libraries often hinders the establishment of these environmentally friendly techniques in molecular biology laboratories. Nonradioactive probes, properly prepared and quantified, can detect DNA target molecules to the femtomole range. However, appropriate hybridization techniques and detection methods should also be adopted for an efficient use of nonradioactive techniques. Detailed descriptions of genomic library handling before and during the nonradioactive hybridization and detection are often omitted from publications. This chapter aims to fill this void by providing a collection of technical tips on hybridization and detection techniques.

Advances in biological dosimetry

NASA Astrophysics Data System (ADS)

Ivashkevich, A.; Ohnesorg, T.; Sparbier, C. E.; Elsaleh, H.

2017-01-01

Rapid retrospective biodosimetry methods are essential for the fast triage of persons occupationally or accidentally exposed to ionizing radiation. Identification and detection of a radiation specific molecular ‘footprint’ should provide a sensitive and reliable measurement of radiation exposure. Here we discuss conventional (cytogenetic) methods of detection and assessment of radiation exposure in comparison to emerging approaches such as gene expression signatures and DNA damage markers. Furthermore, we provide an overview of technical and logistic details such as type of sample required, time for sample preparation and analysis, ease of use and potential for a high throughput analysis.

Molecular mimicry between cockroach and helminth glutathione S-transferases promotes cross-reactivity and cross-sensitization

USDA-ARS?s Scientific Manuscript database

The extensive similarities between helminth proteins and allergens are thought to contribute to helminth-driven allergic sensitization. We investigated the molecular and structural similarities between Bla g 5, a major glutathione-S transferase (GST) allergen of cockroaches, and the GST of Wucherer...

Retrieving transient conformational molecular structure information from inner-shell photoionization of laser-aligned molecules

DOE PAGES

Wang, Xu; Le, Anh -Thu; Yu, Chao; ...

2016-03-30

We discuss a scheme to retrieve transient conformational molecular structure information using photoelectron angular distributions (PADs) that have averaged over partial alignments of isolated molecules. The photoelectron is pulled out from a localized inner-shell molecular orbital by an X-ray photon. We show that a transient change in the atomic positions from their equilibrium will lead to a sensitive change in the alignment-averaged PADs, which can be measured and used to retrieve the former. Exploiting the experimental convenience of changing the photon polarization direction, we show that it is advantageous to use PADs obtained from multiple photon polarization directions. Lastly, amore » simple single-scattering model is proposed and benchmarked to describe the photoionization process and to do the retrieval using a multiple-parameter fitting method.« less

Temporal mapping of photochemical reactions and molecular excited states with carbon specificity

NASA Astrophysics Data System (ADS)

Wang, K.; Murahari, P.; Yokoyama, K.; Lord, J. S.; Pratt, F. L.; He, J.; Schulz, L.; Willis, M.; Anthony, J. E.; Morley, N. A.; Nuccio, L.; Misquitta, A.; Dunstan, D. J.; Shimomura, K.; Watanabe, I.; Zhang, S.; Heathcote, P.; Drew, A. J.

2017-04-01

Photochemical reactions are essential to a large number of important industrial and biological processes. A method for monitoring photochemical reaction kinetics and the dynamics of molecular excitations with spatial resolution within the active molecule would allow a rigorous exploration of the pathway and mechanism of photophysical and photochemical processes. Here we demonstrate that laser-excited muon pump-probe spin spectroscopy (photo-μSR) can temporally and spatially map these processes with a spatial resolution at the single-carbon level in a molecule with a pentacene backbone. The observed time-dependent light-induced changes of an avoided level crossing resonance demonstrate that the photochemical reactivity of a specific carbon atom is modified as a result of the presence of the excited state wavefunction. This demonstrates the sensitivity and potential of this technique in probing molecular excitations and photochemistry.

[Clinical application of testing methods on acid-fast bacteria].

PubMed

Ichiyama, Satoshi; Suzuki, Katsuhiro

2005-02-01

Clinical bacteriology pertaining to acid-fast bacteria has made marked advances over the past decade, initiated by the development of a DNA probe kit for identification of acid-fast bacteria. Wide-spread use of nucleic acid amplification for rapid detection of tubercle bacillus contributed more greatly than any other factor to such advances in this field. At present, 90% of all kits used for nucleic acid amplification in the world are consumed in Japan. Unfortunately, not a few clinicians in Japan have a false idea that the smear method and nucleic acid amplification are necessary but culture is not. In any event nucleic acid amplification has exerted significant impacts on the routine works at bacteriology laboratories. Among others, collecting bacteria by pretreatment with NALC-NaOH has simplified the introduction of the collective mode smear method and liquid media. Furthermore, as clinicians have become increasingly more experienced with various methods of molecular biology, it now seems possible to apply these techniques for detection of genes encoding drug resistance and for utilization of molecular epidemiology in routine laboratory works. Meanwhile, attempts to diagnose acid-fast bacteriosis by checking blood for antibody have also been made, primarily in Japan. At present, two kits for detecting antibodies to glycolipids (LAM, TDM, etc.) are covered by national health insurance in Japan. We have an impression that in Japan clinicians do not have adequate knowledge and skill to make full use of these new testing methods clinically. We, as the chairmen of this symposium, hope that this symposium will help clinicians increase their skill related to new testing methods, eventually leading to stimulation of advances in clinical practices related to acid-fast bacteria in Japan. 1. Smear microscopy by concentration method and broth culture system: Kazunari TSUYUGUCHI (Clinical Research Center, National Hospital Organization Kinki-chuo Chest Medical Center) Smear microscopy and culture still remain the cornerstone to diagnose tuberculosis. However, the classical methods in Japan using direct microscopy and Ogawa solid media were not sufficient for clinical use. In recent years substantial advance has been made in these fields. Concentration of clinical samples by centrifugation improves the sensitivity of smear microscopy with excellent reproducibility. The Mycobacteria Growth Indicator Tube (MGIT) system using liquid media yields high sensitivity and rapidity. Using these methods, more and more tuberculosis cases would be correctly diagnosed and treated adequately based on drug susceptibility testing. 2. New technologies for anti-tuberculosis drug susceptibility testing: Satoshi MITARAI (Bacteriology Division, Reference Centre for Mycobacterium, Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association) Several new technologies have been developed to obtain anti-tuberculosis drug susceptibility testing (AST) results rapidly, utilising liquid culture and molecular technologies. Mycobacterium Growth Indicator Tube (MGIT), as a popular liquid culturing and AST system, was evaluated for its accuracy and usefulness. As for isoniazid, MGIT showed 12.6% of discordant result comparing with standard method. These MGIT resistant and Ogawa susceptible strains had relatively high MICs ranging 0.13 to 2.0 microg/ml. The molecular detection of resistant gene mutation is also a useful method to estimate drug resistance rapidly. The rpoB mutation detection is reliable with high sensitivity and specificity. 3. Nucleic acid amplification and novel diagnostic methods: Shunji TAKAKURA (Department of Clinical Laboratory Medicine, Kyoto University Graduate School of Medicine) Sensitivities of nucleic acid amplification tests (NAATs) for the diagnosis of tuberculosis meet clinical requirement that patients with high-risk of transmission should be identified within a day. Comparison of the performance of various NAATs is difficult because of the difference in sample processing and in samples tested among methods and reports. Considering the limitations of NAATs (low sensitivity compared with culture, inability to differentiate dead bacilli from the living), further advances would be expected when novel technologies could confer additional information, such as drug susceptibility, quantity, viability, and genotype. 4. Serodiagnosis of Mycobacterium avium complex lung disease: Seigo KITADA (Department of Internal Medicine, National Hospital Organization Toneyama National Hospital) Mycobacterium avium complex (MAC) organisms are ubiquitous in environment and a contamination in respiratory tracts is sometimes observed, and that complex the diagnosis. We developed a serodiagnostic method for MAC disease using an enzyme immunoassay with the MAC-specific glycopeptidolipid (GPL) core as antigen. A significant increase in GPL core antibodies was detected in sera of patients with MAC pulmonary diseases compared to patients who were colonized with MAC, patients with M. kansasii disease and tuberculosis and healthy subjects. The serodiagnosis is useful for diagnosis of MAC lung disease. 5. Molecular epidemiologic tools for tuberculosis: IS6110 RFLP, Spoligotyping, and VNTR: Tomoshige MATSUMOTO, Hiromi ANO, Tetsuya TAKASHIMA, Izuo TSUYUGUCHI (Osaka Prefectural Medical Center for Respiratory and Allergic Diseases) We have performed molecular typing on about 1,300 culture positive clinical isolates that made up the majority of tuberculosis strains in part of southeast Osaka since 2001 until now. By spoligotyping, about 75% of entire strains belonged to the Beijing strain. Particular spoligotyping descriptions, which were not described in SpolDBIII, were found in the strains with lower than 6 copies of IS6110 RFLP. We described them as Osaka type. We could also show that direct typing from Tb PCR positive sputum of patients with tuberculosis was possible by VNTR and that VNTR with 16 loci was useful in tuberculosis typing in Osaka.

Biotechnical use of polymerase chain reaction for microbiological analysis of biological samples.

PubMed

Lantz, P G; Abu al-Soud, W; Knutsson, R; Hahn-Hägerdal, B; Rådström, P

2000-01-01

Since its introduction in the mid-80s, polymerase chain reaction (PCR) technology has been recognised as a rapid, sensitive and specific molecular diagnostic tool for the analysis of micro-organisms in clinical, environmental and food samples. Although this technique can be extremely effective with pure solutions of nucleic acids, it's sensitivity may be reduced dramatically when applied directly to biological samples. This review describes PCR technology as a microbial detection method, PCR inhibitors in biological samples and various sample preparation techniques that can be used to facilitate PCR detection, by either separating the micro-organisms from PCR inhibitors and/or by concentrating the micro-organisms to detectable concentrations. Parts of this review are updated and based on a doctoral thesis by Lantz [1] and on a review discussing methods to overcome PCR inhibition in foods [2].

Method and apparatus for enhancing laser absorption sensitivity

NASA Technical Reports Server (NTRS)

Webster, Christopher R. (Inventor)

1987-01-01

A simple optomechanical method and apparatus is described for substantially reducing the amplitude of unwanted multiple interference fringes which often limit the sensitivities of tunable laser absorption spectrometers. An exterior cavity is defined by partially transmissible surfaces such as a laser exit plate, a detector input, etc. That cavity is spoiled by placing an oscillating plate in the laser beam. For tunable diode laser spectroscopy in the mid-infrared region, a Brewster-plate spoiler allows the harmonic detection of absorptances of less than 10 to the -5 in a single laser scan. Improved operation is achieved without subtraction techniques, without complex laser frequency modulation, and without distortion of the molecular lineshape signal. The technique is applicable to tunable lasers operating from UV to IR wavelengths and in spectrometers which employ either short or long pathlengths, including the use of retroreflectors or multipass cells.

Human relevance of an in vitro gene signature in HaCaT for skin sensitization.

PubMed

van der Veen, Jochem W; Hodemaekers, Henny; Reus, Astrid A; Maas, Wilfred J M; van Loveren, Henk; Ezendam, Janine

2015-02-01

The skin sensitizing potential of chemicals is mainly assessed using animal methods, such as the murine local lymph node assay. Recently, an in vitro assay based on a gene expression signature in the HaCaT keratinocyte cell line was proposed as an alternative to these animal methods. Here, the human relevance of this gene signature is assessed through exposure of freshly isolated human skin to the chemical allergens dinitrochlorobenzene (DNCB) and diphenylcyclopropenone (DCP). In human skin, the gene signature shows similar direction of regulation as was previously observed in vitro, suggesting that the molecular processes that drive expression of these genes are similar between the HaCaT cell line and freshly isolated skin, providing evidence for the human relevance of the gene signature. Copyright © 2014 Elsevier Ltd. All rights reserved.

Plasmonic biosensor for label-free G-quadruplexes detection

NASA Astrophysics Data System (ADS)

Qiu, Suyan; Zhao, Fusheng; Santos, Greggy M.; Shih, Wei-Chuan

2016-03-01

G-quadruplex, readily formed by the G-rich sequence, potentially distributes in over 40 % of all human genes, such as the telomeric DNA with the G-rich sequence found at the end of the chromosome. The G-quadruplex structure is supposed to possess a diverse set of critical functions in the mammalian genome for transcriptional regulation, DNA replication and genome stability. However, most of the currently available methods for G-quadruplex identification are restricted to fluorescence techniques susceptible to poor sensitivity. It is essential to propose methods with higher sensitivity to specifically recognize the G-quadruplexes. In this study, we demonstrate a label-free plasmonic biosensor for G-quadruplex detection by relying on the advantages of nanoporous gold (NPG) disks that provide high-density plasmonic hot spots, suitable for molecular recognition capability without the requirement for labeling processes.

Analysis of Publically Available Skin Sensitization Data from REACH Registrations 2008–2014

PubMed Central

Luechtefeld, Thomas; Maertens, Alexandra; Russo, Daniel P.; Rovida, Costanza; Zhu, Hao; Hartung, Thomas

2017-01-01

Summary The public data on skin sensitization from REACH registrations already included 19,111 studies on skin sensitization in December 2014, making it the largest repository of such data so far (1,470 substances with mouse LLNA, 2,787 with GPMT, 762 with both in vivo and in vitro and 139 with only in vitro data). 21% were classified as sensitizers. The extracted skin sensitization data was analyzed to identify relationships in skin sensitization guidelines, visualize structural relationships of sensitizers, and build models to predict sensitization. A chemical with molecular weight > 500 Da is generally considered non-sensitizing owing to low bioavailability, but 49 sensitizing chemicals with a molecular weight > 500 Da were found. A chemical similarity map was produced using PubChem’s 2D Tanimoto similarity metric and Gephi force layout visualization. Nine clusters of chemicals were identified by Blondel’s module recognition algorithm revealing wide module-dependent variation. Approximately 31% of mapped chemicals are Michael’s acceptors but alone this does not imply skin sensitization. A simple sensitization model using molecular weight and five ToxTree structural alerts showed a balanced accuracy of 65.8% (specificity 80.4%, sensitivity 51.4%), demonstrating that structural alerts have information value. A simple variant of k-nearest neighbors outperformed the ToxTree approach even at 75% similarity threshold (82% balanced accuracy at 0.95 threshold). At higher thresholds, the balanced accuracy increased. Lower similarity thresholds decrease sensitivity faster than specificity. This analysis scopes the landscape of chemical skin sensitization, demonstrating the value of large public datasets for health hazard prediction. PMID:26863411

Current Progress of Nanomaterials in Molecularly Imprinted Electrochemical Sensing.

PubMed

Zhong, Chunju; Yang, Bin; Jiang, Xinxin; Li, Jianping

2018-01-02

Nanomaterials have received much attention during the past decade because of their excellent optical, electronic, and catalytic properties. Nanomaterials possess high chemical reactivity, also high surface energy. Thus, provide a stable immobilization platform for biomolecules, while preserving their reactivity. Due to the conductive and catalytic properties, nanomaterials can also enhance the sensitivity of molecularly imprinted electrochemical sensors by amplifying the electrode surface, increasing the electron transfer, and catalyzing the electrochemical reactions. Molecularly imprinted polymers that contain specific molecular recognition sites can be designed for a particular target analyte. Incorporating nanomaterials into molecularly imprinted polymers is important because nanomaterials can improve the response signal, increase the sensitivity, and decrease the detection limit of the sensors. This study describes the classification of nanomaterials in molecularly imprinted polymers, their analytical properties, and their applications in the electrochemical sensors. The progress of the research on nanomaterials in molecularly imprinted polymers and the application of nanomaterials in molecularly imprinted polymers is also reviewed.

Sensitive detection of microRNA in complex biological samples by using two stages DSN-assisted target recycling signal amplification method.

PubMed

Zhang, Kai; Wang, Ke; Zhu, Xue; Xu, Fei; Xie, Minhao

2017-01-15

MicroRNA (miRNA) has become an important biomarker candidate for cancer diagnosis, prognosis, and therapy. In this study, we have developed a novel fluorescence method for sensitive and specific miRNA detection via duplex specific nuclease (DSN) signal amplification and demonstrated its practical application in biological samples. Malachite green (MG) was employed as a "label-free" signal transducer since fluorescence of MG could be enhanced by 100-fold when MG were binding to a G-quadruplex structure formed within the d(G 2 T) 13 G sequence. The proposed signal amplification strategy is an integrated "biological circuit" designed to initiate a cascade of enzymatic reactions in order to detect, amplify, and measure a specific miRNA sequence by using the isothermal cleavage property of a DSN. The circuit is composed of two molecular switches operating in series: the amplification reaction activated by a specific miRNA and the strand-displacement polymerization reaction designed to initiate molecular beacon-assisted amplification and signal transduction by using MG/G-quadruplex complex. The hsa-miR-141 (miR141) was chosen as a target miRNA because its level specifically abnormal in a wide range of common human cancers including breast, lung, colon, and prostate cancer. The proposed method allowed quantitative sequence-specific detection of miR141 (with a detection limit of 1.03pM) in a dynamic range from 1pM to 10μM, with an excellent ability to discriminate differences in miRNAs. Moreover, the detection assay was applied to quantify miR141 in cancerous cell lysates. On the basis of these findings, we believe that this proposed sensitive and specific assay has great potential as a miRNA quantification method for use in biomedical research and clinical diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Hydroxyl as a Tracer of Dark Gas in a Diffuse Molecular Cloud

NASA Astrophysics Data System (ADS)

White, Josh; Donate, Emmanuel; Magnani, Loris A.

2017-06-01

In an attempt to determine the extent of dark molecular gas at high Galactic latitudes, we have conducted a survey of OH at 18 cm in a region containing the diffuse molecular cloud MBM 53. Dark molecular gas is a term that refers to molecular hydrogen that is either difficult or impossible to detect by conventional spectroscopic means. While models of photo-dissociation regions predict that some molecular hydrogen is found under conditions where other species are too low in abundance to be detected by radio spectroscopy, recent estimates have predicted that as much dark molecular gas exists as that normally detected by CO(1-0) surveys. However, more sensitive surveys either in the CO(1-0) line or other tracers should detect some of this gas. We observed 44 lines of sight at 18 cm to see if very sensitive OH observations could detect some of the dark molecular gas in the Pegasus-Pisces region. Our data were taken with the 305 m Arecibo radiotelescope and have typical rms values of 6-7 mK. We compared our OH observations with the Georgia/Harvard-Smithsonian CfA high-latitude CO(1-0) survey. Of 8 OH detections at 1667 MHz, 5 were not detected by the CO survey and indicate that at least some of the dark molecular gas may be traced by sensitive OH observations.

Learning contextual gene set interaction networks of cancer with condition specificity

PubMed Central

2013-01-01

Background Identifying similarities and differences in the molecular constitutions of various types of cancer is one of the key challenges in cancer research. The appearances of a cancer depend on complex molecular interactions, including gene regulatory networks and gene-environment interactions. This complexity makes it challenging to decipher the molecular origin of the cancer. In recent years, many studies reported methods to uncover heterogeneous depictions of complex cancers, which are often categorized into different subtypes. The challenge is to identify diverse molecular contexts within a cancer, to relate them to different subtypes, and to learn underlying molecular interactions specific to molecular contexts so that we can recommend context-specific treatment to patients. Results In this study, we describe a novel method to discern molecular interactions specific to certain molecular contexts. Unlike conventional approaches to build modular networks of individual genes, our focus is to identify cancer-generic and subtype-specific interactions between contextual gene sets, of which each gene set share coherent transcriptional patterns across a subset of samples, termed contextual gene set. We then apply a novel formulation for quantitating the effect of the samples from each subtype on the calculated strength of interactions observed. Two cancer data sets were analyzed to support the validity of condition-specificity of identified interactions. When compared to an existing approach, the proposed method was much more sensitive in identifying condition-specific interactions even in heterogeneous data set. The results also revealed that network components specific to different types of cancer are related to different biological functions than cancer-generic network components. We found not only the results that are consistent with previous studies, but also new hypotheses on the biological mechanisms specific to certain cancer types that warrant further investigations. Conclusions The analysis on the contextual gene sets and characterization of networks of interaction composed of these sets discovered distinct functional differences underlying various types of cancer. The results show that our method successfully reveals many subtype-specific regions in the identified maps of biological contexts, which well represent biological functions that can be connected to specific subtypes. PMID:23418942

Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules.

PubMed

Kühnemund, Malte; Hernández-Neuta, Iván; Sharif, Mohd Istiaq; Cornaglia, Matteo; Gijs, Martin A M; Nilsson, Mats

2017-05-05

Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Assessing direct analysis in real-time-mass spectrometry (DART-MS) for the rapid identification of additives in food packaging.

PubMed

Ackerman, L K; Noonan, G O; Begley, T H

2009-12-01

The ambient ionization technique direct analysis in real time (DART) was characterized and evaluated for the screening of food packaging for the presence of packaging additives using a benchtop mass spectrometer (MS). Approximate optimum conditions were determined for 13 common food-packaging additives, including plasticizers, anti-oxidants, colorants, grease-proofers, and ultraviolet light stabilizers. Method sensitivity and linearity were evaluated using solutions and characterized polymer samples. Additionally, the response of a model additive (di-ethyl-hexyl-phthalate) was examined across a range of sample positions, DART, and MS conditions (temperature, voltage and helium flow). Under optimal conditions, molecular ion (M+H+) was the major ion for most additives. Additive responses were highly sensitive to sample and DART source orientation, as well as to DART flow rates, temperatures, and MS inlet voltages, respectively. DART-MS response was neither consistently linear nor quantitative in this setting, and sensitivity varied by additive. All additives studied were rapidly identified in multiple food-packaging materials by DART-MS/MS, suggesting this technique can be used to screen food packaging rapidly. However, method sensitivity and quantitation requires further study and improvement.