Relevant bibliographies by topics / Molecular probes Diagnostic use

Academic literature on the topic 'Molecular probes Diagnostic use'

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Published: 10 December 2022
Last updated: 29 January 2023

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Journal articles on the topic "Molecular probes Diagnostic use"

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Courtney, Samantha, Zachary Stromberg, Adán Myers y Gutiérrez, Daniel Jacobsen, Loreen Stromberg, Kiersten Lenz, James Theiler, et al. "Optical Biosensor Platforms Display Varying Sensitivity for the Direct Detection of Influenza RNA." Biosensors 11, no. 10 (September 30, 2021): 367. http://dx.doi.org/10.3390/bios11100367.

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Detection methods that do not require nucleic acid amplification are advantageous for viral diagnostics due to their rapid results. These platforms could provide information for both accurate diagnoses and pandemic surveillance. Influenza virus is prone to pandemic-inducing genetic mutations, so there is a need to apply these detection platforms to influenza diagnostics. Here, we analyzed the Fast Evaluation of Viral Emerging Risks (FEVER) pipeline on ultrasensitive detection platforms, including a waveguide-based optical biosensor and a flow cytometry bead-based assay. The pipeline was also evaluated in silico for sequence coverage in comparison to the U.S. Centers for Disease Control and Prevention’s (CDC) influenza A and B diagnostic assays. The influenza FEVER probe design had a higher tolerance for mismatched bases than the CDC’s probes, and the FEVER probes altogether had a higher detection rate for influenza isolate sequences from GenBank. When formatted for use as molecular beacons, the FEVER probes detected influenza RNA as low as 50 nM on the waveguide-based optical biosensor and 1 nM on the flow cytometer. In addition to molecular beacons, which have an inherently high background signal we also developed an exonuclease selection method that could detect 500 pM of RNA. The combination of high-coverage probes developed using the FEVER pipeline coupled with ultrasensitive optical biosensors is a promising approach for future influenza diagnostic and biosurveillance applications.
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Betson, Mark, Nigel Allanson, and Philip Wainwright. "A review of methods to synthesise 4′-substituted nucleosides." Org. Biomol. Chem. 12, no. 46 (2014): 9291–306. http://dx.doi.org/10.1039/c4ob01449a.

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Ma, Ying-Yu, Ke-Tao Jin, Shi-Bing Wang, Hui-Ju Wang, Xiang-Min Tong, Dong-Sheng Huang, and Xiao-Zhou Mou. "Molecular Imaging of Cancer with Nanoparticle-Based Theranostic Probes." Contrast Media & Molecular Imaging 2017 (2017): 1–11. http://dx.doi.org/10.1155/2017/1026270.

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Although advancements in medical technology supporting cancer diagnosis and treatment have improved survival, these technologies still have limitations. Recently, the application of noninvasive imaging for cancer diagnosis and therapy has become an indispensable component in clinical practice. However, current imaging contrasts and tracers, which are in widespread clinical use, have their intrinsic limitations and disadvantages. Nanotechnologies, which have improved in vivo detection and enhanced targeting efficiency for cancer, may overcome some of the limitations of cancer diagnosis and therapy. Theranostic nanoparticles have great potential as a therapeutic model, which possesses the ability of their nanoplatforms to load targeted molecule for both imaging and therapeutic functions. The resulting nanosystem will likely be critical with the growth of personalized medicine because of their diagnostic potential, effectiveness as a drug delivery vehicle, and ability to oversee patient response to therapy. In this review, we discuss the achievements of modern nanoparticles with the goal of accurate tumor imaging and effective treatment and discuss the future prospects.
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Sguizzato, Maddalena, Petra Martini, Lorenza Marvelli, Walter Pula, Markus Drechsler, Martina Capozza, Enzo Terreno, et al. "Synthetic and Nanotechnological Approaches for a Diagnostic Use of Manganese." Molecules 27, no. 10 (May 13, 2022): 3124. http://dx.doi.org/10.3390/molecules27103124.

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The development of multimodal imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) allows the contemporary obtaining of metabolic and morphological information. To fully exploit the complementarity of the two imaging modalities, the design of probes displaying radioactive and magnetic properties at the same time could be very beneficial. In this regard, transition metals offer appealing options, with manganese representing an ideal candidate. As nanosized imaging probes have demonstrated great value for designing advanced diagnostic/theranostic procedures, this work focuses on the potential of liposomal formulations loaded with a new synthesized paramagnetic Mn(II) chelates. Negatively charged liposomes were produced by thin-layer hydration method and extrusion. The obtained formulations were characterized in terms of size, surface charge, efficiency of encapsulation, stability over time, relaxivity, effective magnetic moment, and in vitro antiproliferative effect on human cells by means of the MTT assay. The negatively charged paramagnetic liposomes were monodisperse, with an average hydrodynamic diameter not exceeding 200 nm, and they displayed good stability and no cytotoxicity. As determined by optical emission spectroscopy, manganese complexes are loaded almost completely on liposomes maintaining their paramagnetic properties.
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Rangasamy, Geronimo, Ortín, Coderch, Zapico, Ramos, and de Pascual-Teresa. "Molecular Imaging Probes Based on Matrix Metalloproteinase Inhibitors (MMPIs)." Molecules 24, no. 16 (August 16, 2019): 2982. http://dx.doi.org/10.3390/molecules24162982.

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Matrix metalloproteinases (MMPs) are a family of zinc- and calcium-dependent endopeptidases which are secreted or anchored in the cell membrane and are capable of degrading the multiple components of the extracellular matrix (ECM). MMPs are frequently overexpressed or highly activated in numerous human diseases. Owing to the important role of MMPs in human diseases, many MMP inhibitors (MMPIs) have been developed as novel therapeutics, and some of them have entered clinical trials. However, so far, only one MMPI (doxycycline) has been approved by the FDA. Therefore, the evaluation of the activity of a specific subset of MMPs in human diseases using clinically relevant imaging techniques would be a powerful tool for the early diagnosis and assessment of the efficacy of therapy. In recent years, numerous MMPIs labeled imaging agents have emerged. This article begins by providing an overview of the MMP subfamily and its structure and function. The latest advances in the design of subtype selective MMPIs and their biological evaluation are then summarized. Subsequently, the potential use of MMPI-labeled diagnostic agents in clinical imaging techniques are discussed, including positron emission tomography (PET), single-photon emission computed tomography (SPECT) and optical imaging (OI). Finally, this article concludes with future perspectives and clinical utility.
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M S, Kumar. "Snake Venom Toxins: Clinical use and as Diagnostic Agents." Shanlax International Journal of Arts, Science and Humanities 8, S1-Feb (February 6, 2021): 1–5. http://dx.doi.org/10.34293/sijash.v8is1-feb.3923.

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Snakes are fascinating creatures and were inhabitants of this world well before the earth was populated by ancient humans. Snakes with a lethal secretion known as venom were endowed by nature. Snake venom is a very poisonous mixture consisting of a number of molecules such as carbohydrates, nucleosides, amino acids, lipids, proteins and peptides, making it a cocktail of diversified molecules. Snake envenomation is responsible for the disruption in the envenomed victim’s fundamental physiological processes contributing to serious health problems. Millions of snakebites are recorded annually, and due to snake venom poisoning, a significant number of individuals are injured and die. However, through technical developments, many fatal snake venom toxins have found potential applications as diagnostic agents, medicinal agents, or drug leads. From the development of Captopril, the first drug derived from Bothrops jarararaca’s bradykinin potentiating peptide, to the disintegrins that have potent activity against some forms of cancers. Therefore, components of snake venom have shown tremendous potential for the development of lead compounds for new drugs. Complementary tools and techniques are currently being used to isolate and characterize peptides and to study their potential uses as molecular probes and templates for drug development and design investigation models.
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Neveling, Kornelia, Arjen R. Mensenkamp, Ronny Derks, Michael Kwint, Hicham Ouchene, Marloes Steehouwer, Bart van Lier, et al. "BRCA Testing by Single-Molecule Molecular Inversion Probes." Clinical Chemistry 63, no. 2 (February 1, 2017): 503–12. http://dx.doi.org/10.1373/clinchem.2016.263897.

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Abstract BACKGROUND Despite advances in next generation DNA sequencing (NGS), NGS-based single gene tests for diagnostic purposes require improvements in terms of completeness, quality, speed, and cost. Single-molecule molecular inversion probes (smMIPs) are a technology with unrealized potential in the area of clinical genetic testing. In this proof-of-concept study, we selected 2 frequently requested gene tests, those for the breast cancer genes BRCA1 and BRCA2, and developed an automated work flow based on smMIPs. METHODS The BRCA1 and BRCA2 smMIPs were validated using 166 human genomic DNA samples with known variant status. A generic automated work flow was built to perform smMIP-based enrichment and sequencing for BRCA1, BRCA2, and the checkpoint kinase 2 (CHEK2) c.1100del variant. RESULTS Pathogenic and benign variants were analyzed in a subset of 152 previously BRCA-genotyped samples, yielding an analytical sensitivity and specificity of 100%. Following automation, blind analysis of 65 in-house samples and 267 Norwegian samples correctly identified all true-positive variants (>3000), with no false positives. Consequent to process optimization, turnaround times were reduced by 60% to currently 10–15 days. Copy number variants were detected with an analytical sensitivity of 100% and an analytical specificity of 88%. CONCLUSIONS smMIP-based genetic testing enables automated and reliable analysis of the coding sequences of BRCA1 and BRCA2. The use of single-molecule tags, double-tiled targeted enrichment, and capturing and sequencing in duplo, in combination with automated library preparation and data analysis, results in a robust process and reduces routine turnaround times. Furthermore, smMIP-based copy number variation analysis could make independent copy number variation tools like multiplex ligation-dependent probes amplification dispensable.
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Fuller, Skylar L., Elizabeth A. Savory, Alexandra J. Weisberg, Jessica Z. Buser, Michael I. Gordon, Melodie L. Putnam, and Jeff H. Chang. "Isothermal Amplification and Lateral-Flow Assay for Detecting Crown-Gall-Causing Agrobacterium spp." Phytopathology® 107, no. 9 (September 2017): 1062–68. http://dx.doi.org/10.1094/phyto-04-17-0144-r.

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Agrobacterium is a genus of soilborne gram-negative bacteria. Members carrying oncogenic plasmids can cause crown gall disease, which has significant economic costs, especially for the orchard and nursery industries. Early and rapid detection of pathogenic Agrobacterium spp. is key to the management of crown gall disease. To this end, we designed oligonucleotide primers and probes to target virD2 for use in a molecular diagnostic tool that relies on isothermal amplification and lateral-flow-based detection. The oligonucleotide tools were tested in the assay and evaluated for detection limit and specificity in detecting alleles of virD2. One set of primers that successfully amplified virD2 when used with an isothermal recombinase was selected. Both tested probes had detection limits in picogram amounts of DNA. Probe 1 could detect all tested pathogenic isolates that represented most of the diversity of virD2. Finally, the coupling of lateral-flow detection to the use of these oligonucleotide primers in isothermal amplification helped to reduce the onerousness of the process, and alleviated reliance on specialized tools necessary for molecular diagnostics. The assay is an advancement for the rapid molecular detection of pathogenic Agrobacterium spp.
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Bentivoglio, Valeria, Michela Varani, Chiara Lauri, Danilo Ranieri, and Alberto Signore. "Methods for Radiolabelling Nanoparticles: PET Use (Part 2)." Biomolecules 12, no. 10 (October 20, 2022): 1517. http://dx.doi.org/10.3390/biom12101517.

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The use of radiolabelled nanoparticles (NPs) is a promising nuclear medicine tool for diagnostic and therapeutic purposes. Thanks to the heterogeneity of their material (organic or inorganic) and their unique physical and chemical characteristics, they are highly versatile for their use in several medical applications. In particular, they have shown interesting results as radiolabelled probes for positron emission tomography (PET) imaging. The high variability of NP types and the possibility to use several isotopes in the radiolabelling process implies different radiolabelling methods that have been applied over the previous years. In this review, we compare and summarize the different methods for NP radiolabelling with the most frequently used PET isotopes.
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Zenteno-Cuevas, Roberto, Betzaida Cuevas-Cordoba, Antonio Enciso, Leonor Enciso, and Aremy Cuellar. "Assessing the utility of three TaqMan probes for the diagnosis of tuberculosis and resistance to rifampin and isoniazid in Veracruz, México." Canadian Journal of Microbiology 58, no. 3 (March 2012): 318–25. http://dx.doi.org/10.1139/w11-127.

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Mutations at codons 526 and 531 in the rpoB gene and at 315 in the katG gene are considered diagnostic markers for resistance to rifampin and isoniazid in tuberculosis. The aim of this study was to design and evaluate three TaqMan probes for the identification of these mutations in 138 respiratory samples positive for acid-fast bacilli, and 32 clinical isolates from a region with considerable levels of drug resistance. The specificities of the probes for the diagnosis of resistance to both drugs were 100%; however, the sensitivities were calculated to be 50% for isoniazid and 56% for rifampin. DNA sequencing of rpoB and katG; and the spoligotyping assay of the clinical isolates, confirmed the diversity of the mutations and the presence of 11 spoligotypes with a shared international type and eight unique spoligotypes. Analysis of the respiratory samples identified 22 (16%) as drug-resistant and 4 (3%) as multidrug-resistant tuberculosis. The diagnostic value of the TaqMan probes was compromised by the diversity of mutations found in the clinical isolates. This highlights the need for better understanding of the molecular mechanisms responsible for drug resistance prior to the use of molecular probes, especially in regions with significant levels of drug-resistant tuberculosis.
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Dissertations / Theses on the topic "Molecular probes Diagnostic use"

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Wang, Lei. "Molecular Probes for Pancreatic Cancer Imaging." PDXScholar, 2016. http://pdxscholar.library.pdx.edu/open_access_etds/3108.

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Pancreatic ductal adenocarcinoma (PDAC) has the poorest five-year survival rate of any cancer. Currently, there are no effective diagnostics or chemotherapeutics. Surgical resection is the only curative therapy. However, most patients experience recurrence due largely to challenges in assessing tumor margin status in the operating room. Molecular probes that selectively highlight pancreatic cancer tissue, having the potential to improve PDAC margin assessment intraoperatively, are urgently needed. In this work, a series of red and near-infrared fluorescent probes is reported. Two were found to distribute to normal pancreas following systemic administration. One selectively accumulates in genetically modified mouse models of PDAC, providing cancer-specific fluorescence. In contrast to the small molecule probes reported previously, it possesses inherent affinity for PDAC cells and tissue, and thus does not require conjugation to targeting agents. Moreover, the probe exhibits intracellular accumulation and enables visualization of four levels of structure including the whole organ, tissue, individual cells and subcellular organelles. It can thus promote new strategies for precision image-guided surgery, pancreatic cancer detection, the monitoring of therapeutic outcomes and basic research.
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Vaccaro, Carlos. "Use of luminescence energy transfer probes to detect genetic variants." Thesis, University of North Texas, 2004. https://digital.library.unt.edu/ark:/67531/metadc4566/.

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The purpose of this research was to study the hybridization of molecular beacons under different conditions and designs. Data collected suggest that the inconsistency found in the emission intensity of several of these probes may be caused by 3 important factors: length of the probe, nucleotide sequence and, the formation of an alternative complex structure such as a dimer. Of all three factors, dimer formation is the most troublesome, since it reduces the emission of the reporter molecules. A new probe design was used to reduce dimer formation. The emission signal of the improved probe was several folds stronger than those probes with the early design. In this research, dimer formation is detected, furthermore a new probe with a different design was tested. If dimer formation can be reduced molecular beacons can be integrated into more complex hybridization systems providing an important tool in research and diagnosis of genetic disorders.
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Cheung, Lori. "Production of labeled DNA probes for the rapid diagnosis of disseminated candidiasis in immunocompromised patients." Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/26188.

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The increasing incidence of disseminated (invasive) candidiasis is probably attributable to iatrogenic factors and to improved pre and postmortem evaluation. Premortem diagnosis of such infections have seldom been made early enough for successful treatment. In order to increase the likelihood of successful antifungal chemotherapy, rapid diagnosis of such infections is vital. However, present diagnostic procedures for invasive candidiasis are insensitive and often do not reliably differentiate superficial from invasive infections. This study was undertaken to produce DNA probes and to optimize conditions for rapid and efficient detection of Candida DNA. Seven random Candida albicans DNA fragments (2-7 kbp) were cloned into plasmid pACYC 184. These recombinant plasmids were labeled with either ³²p or biotin and used as probes. Two of the four recombinant plasmids tested were genus specific. The other two were slightly cross reactive with other yeasts (Saccharomyces cerevisiae and Hansenula anomala). Probes labeled with ³²p were twice as sensitive as the biotin probes. One ³²p labelled recombinant (#66) detected 7 Pg of target DNA , which corresponds to approximately 2 X 10⁵ C.albicans cells. With refined simple DNA extraction procedures for C.albicans (in serum), these recombinant probes could possibly be suitable for clinical application.
Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate
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Goetz, Joan. "Biocompatible luminescent probes for imaging and inhibition of cancers." HKBU Institutional Repository, 2018. https://repository.hkbu.edu.hk/etd_oa/532.

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This joint PhD program is part of a collaboration between Hong Kong Baptist University (Dr. Gary K-L Wong) and Laboratoire d'Ingénierie Moléculaire Appliquée à l'Analyse (LIMAA - Dr. Loïc Charbonnière) funded by the Alsace region to synthesize new nanoprobes for sensing, imaging, and inhibiting cancer diseases. The first work was to synthesize new hybrid ultrabright nanoparticles. They have been obtained from a La0.9Tb0.1F3 core and coated by different ligands. Thanks to a mechanism of antenna effect, the brightness of the nanoparticles has been significantly improved. The second work was to synthesize a new ligand to photosensitize water-soluble La0.90Eu0.1F3 nanoparticles in order to improve the emission of europium. A second ligand and new heterometallic nanoparticles have been synthesized with the aim to promote the energy transfer from Tb(III) ions on the surface of the NPs to Eu(III) ions in the core of the nanoparticles and to get a very long excited-state lifetime and an exceptional quantum yield in aqueous solution. The last work was to functionalize water-soluble graphitic-carbon nitride (g-C3N4) nanoparticles by porphyrins. The porphyrins have been synthesized to generate singlet oxygen (1O2), to host a Ga3+ ion inside their cavity and with two different linkers to be coupled to nanoparticles. This system aims to be a pH sensor, and a PDT and PET theranostic agent.
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Turetsky, Anna. "Companion Imaging Probes and Diagnostic Devices for B-Cell Lymphoma." Thesis, Harvard University, 2014. http://nrs.harvard.edu/urn-3:HUL.InstRepos:13094356.

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As new therapeutic targets and drugs are discovered for B-cell lymphoma and other cancers, companion diagnostics are also needed to determine target engagement, therapeutic efficacy, and patient segmentation for clinical trials. We first employed synthetic chemistry to build a platform for modifying small molecule drugs into imaging probes, using the poly(ADP-ribose) polymerase 1 (PARP1) inhibitor AZD2281 (Olaparib) as a model for technology development. Our results showed that small-molecule companion imaging drugs can be used for fluorescence imaging in cells, as well as for pharmacokinetic studies and positron emission tomography (PET) imaging in vivo, without significantly perturbing their target binding properties or cellular uptake. To apply this approach to B-cell lymphoma drugs currently in clinical trials, we modified an irreversible inhibitor of Bruton's Tyrosine Kinase (BTK), PCI-32765 (Ibrutinib), with the fluorophore Bodipy FL (BFL), and used it for imaging in cells and in a mouse window-chamber xenograft model. The excellent co-localization of our probe (Ibrutinib-BFL) with BTK demonstrated its utility for studying additional BTK inhibitors and as a companion imaging probe. In parallel, we hypothesized that central nervous system (CNS) lymphoma diagnosis from paucicellular cerebrospinal fluid (CSF) samples could be improved with molecular profiling of putative lymphoma cells trapped in a customized microfluidic chip. Following fabrication and characterization of a polydimethylsiloxane (PDMS) diagnostic device containing an array of affinity-free single-cell capture sites, we were able to efficiently recover >90% of lymphocytes, perform immunostaining on chip, and apply an image-processing algorithm to group cells based on their molecular marker expression, such as kappa/lambda light chain restriction. Additionally, in combination with Ibrutinib-BFL or other imaging drugs, we demonstrated the potential for on-chip drug imaging for use in conjunction with drug development. Finally, we applied bioorthogonal conjugation chemistries on cellulose paper for potential applications in lowering the cost of drug screening. We anticipate that these approaches will enable direct, molecular information for personalized treatment decisions in B-cell lymphomas, as well as provide a roadmap for the development of companion diagnostic probes and devices for additional indications.
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Rautiola, Davin. "Detection of Homocysteine with Bridged Viologen Chemical Probes." PDXScholar, 2014. https://pdxscholar.library.pdx.edu/open_access_etds/1541.

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Increased blood plasma concentrations of the aminothiol homocysteine (Hcy) are associated with a variety of disease states including those which cause impaired renal function, many forms of cardiovascular disease, and neurodegenerative diseases such as Alzheimer's. Therefore, Hcy has the potential to be a significant diagnostic biomarker. Routine monitoring of Hcy plasma concentration is encumbered by the time and resources required to quantify Hcy using currently accepted instrumental analysis methods. As part of the continuing effort to develop a quick, reliable, inexpensive, and user-friendly test to quantify Hcy at the point of care, we have designed a series of novel colorimetric and fluorescent chemical probes based on bridged viologen structures. The absorbance at 540 nm for the para-bridged bis-nitrile viologen probe (pCN) was found to be proportional to the concentration of Hcy analyte, with LOD = 2.17 μM and LOQ = 6.10 μM where unhealthy Hcy plasma concentrations are > 15 μM. The mechanism of reactivity between pCN and Hcy encompasses a dynamic set of reactions which involve pimerization of radical probe species and thioether adduct formation of pCN with Hcy. Preliminary results with fluorometric analogs of the bridged viologen probes are also presented.
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Nitin, Nitin. "Optical and MR Molecular Imaging Probes and Peptide-based Cellular Delivery for RNA Detection in Living Cells." Diss., Available online, Georgia Institute of Technology, 2005, 2005. http://etd.gatech.edu/theses/available/etd-08102005-120350/.

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Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2006.
Dr. X. Hu, Committee Member ; Dr. Al Merrill, Committee Member ; Dr. Niren Murthy, Committee Member ; Dr. Gang Bao, Committee Chair ; Dr. Nicholas Hud, Committee Member. Includes bibliographical references.
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Glick, Cindy Jennifer. "Development of androgen receptor messenger RNA targeted molecular beacons for use in the study of prostate cancer progression." Thesis, Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/26630.

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Thesis (M.S.)--Biomedical Engineering, Georgia Institute of Technology, 2009.
Committee Chair: Bao, Gang; Committee Member: Merrill, Alfred; Committee Member: Santangelo, Philip. Part of the SMARTech Electronic Thesis and Dissertation Collection.
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Hakuna, Lovemore. "Selective Indicators for Optical Determination of Disease Biomarkers." PDXScholar, 2014. http://pdxscholar.library.pdx.edu/open_access_etds/2053.

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The most abundant biological thiols, homocysteine (Hcy), cysteine (Cys) and glutathione (GSH) have been the subject of intense research due to their association with a wide range of diseases. They play a key role in maintaining the redox status of biological systems. Selective detection methods for these thiols are challenging due to their similar structures and properties. Current commercially available detection methods use separations, fragile and expensive enzymatic or immunogenic materials and complex instrumentation. This has led to a global effort towards developing simple and inexpensive optical probes and indicators selective for specific biological thiols. Highly selective chemical probes and simple methods for detection and potential quantification of Hcy and GSH in their natural biological media have been developed. These indicators and methods are relatively simple and inexpensive for potential application at point of care. The selective detection of Hcy using novel asymmetric viologen chemical probes at room temperature is described as well as the use of commercially available materials under photochemical conditions. These probes respond linearly proportional to increasing Hcy concentrations, potentially enabling the monitoring of Hcy levels in human plasma. Additionally, new methods for the selective determination of GSH in human plasma, as well as its quantification in whole blood deposited on filter paper (dried blood spots), is also presented herein.
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Abbaszadegan, Morteza 1955. "Detection of Giardia cysts by cDNA probe and application to water samples." Diss., The University of Arizona, 1991. http://hdl.handle.net/10150/191163.

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Giardia is the most common human parasite infection in the United States causing a lengthy diarrhea. Transmission of Giardia is by the fecal-oral route and numerous waterborne outbreaks have been documented. The Environmental Protection Agency has regulated Giardia in drinking water through the "Surface Water Treatment Rule." Current methods for detection of Giardia in water rely primarily on microscopic observation of water concentrates by immunofluorescent techniques. We evaluated the efficacy of using a gene-specific probe for the detection of Giardia species in water. A cDNA probe, 265 base pairs long, from the small subunit of rRNA of Giardia lamblia was used for detection of cysts. The replicative form of M13 vector with insert was isolated from lysed host E. coli XL1- Blue and used for production of the cDNA probe by nick translation with ³²P-labeled nucleotides. Seven different protocols were tested for extracting nucleic acids from the cysts. Using the most efficient procedure, disrupting Giardia cysts with glass beads in the presence of proteinase K, as few as 1 to 5 cysts per ml can be detected in water sample concentrates by dot-blot hybridization assays. Environmental concentrates from secondary and tertiary treated sewage or surface waters were screened for Giardia cysts by immunofluorescent and the genespecific probe. Positive signals were observed in sewage and surface water samples without floatation at ten fold greater dilutions than after floatation. It appeared that gene probe detection was slightly more sensitive than microscopic detection of Giardia cysts for wastewater samples. In six surface water samples and two sewage sample no positive results were found either by the cDNA probe or immunofluorescent. Usually, DNA probes are radiolabeled and the most commonly used is ³²P. ³²P is expensive, hazardous and has an extremely short half-life of 14.3 days, necessitating frequent preparation of the nucleic acid probes. Three non-radioactive labeling methods, chemiluminescence, enzyme-linked immunoassay and enhanced chemiluminescence were evaluated. The cDNA probe was labeled by nick translation for chemiluminescence method. Biotinylated deoxyuridine triphosphate was used in place of deoxythymidine triphosphate to produce biotinylated DNA strands. The result of hybridization was visualized by chemiluminescenct detection of DNA. The sensitivity of the chemiluminescent method and the 32P labeled probe was 0.1 pg of DNA in a slot-blot hybridization assay.
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Books on the topic "Molecular probes Diagnostic use"

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Alberto, Albertini, Paoletti Rodolfo, Reisfeld Ralph A, and International Symposium BIOTECH RIA '88, "Molecular Probes: Technology and Medical Applications" (1988 : Florence, Italy), eds. Molecular probes: Technology and medical applications. New York: Raven Press, 1989.

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R, Walker Matthew, and Rapley Ralph, eds. Molecular and antibody probes in diagnosis. Chichester: Wiley, 1993.

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S, Herrington C., and McGee J. O'D, eds. Diagnostic molecular pathology: A practical approach. Oxford: IRL Press at Oxford University Press, 1992.

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Xiaoyuan, Chen, ed. Nanoplatform-based molecular imaging. Hoboken, N.J: Wiley, 2010.

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Lin, Charles P., and Vasilis Ntziachristos. Molecular imaging III: 22-23 May, 2011, Munich, Germany. Bellingham, Wash: SPIE, 2011.

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Raghavachari, Ramesh, and Samuel Achilefu. Reporters, markers, dyes, nanoparticles, and molecular probes for biomedical applications IV: 23-25 January 2012, San Francisco, California, United States. Edited by SPIE (Society). Bellingham, Wash: SPIE, 2012.

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Raghavachari, Ramesh, and Samuel Achilefu. Reporters, markers, dyes, nanoparticles, and molecular probes for biomedical applications V: 4-6 February 2013, San Francisco, Calififornia, United States. Edited by SPIE (Society), SPIE Photonics West (Conference) (2013 : San Francisco, Calif.), and Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications (Conference) (5th : 2013 : San Francisco, Calif.). Bellingham, Washington: SPIE, 2013.

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Comparative diagnostic pharmacology: Clinical and research applications in living-system models. Ames, Iowa: Blackwell Pub. Professional, 2006.

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(Society), SPIE, ed. Reporters, markers, dyes, nanoparticles, and molecular probes for biomedical applications: 26-29 January 2009, San Jose, California, United States. Bellingham, Wash: SPIE, 2009.

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Achilefu, Samuel. Reporters, markers, dyes, nanoparticles, and molecular probes for biomedical applications: 26-29 January 2009, San Jose, California, United States. Bellingham, Wash: SPIE, 2009.

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Book chapters on the topic "Molecular probes Diagnostic use"

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Choi, Jung-Suk, and Anthony Berdis. "Artificial Nucleosides as Diagnostic Probes to Measure Translesion DNA Synthesis." In Methods in Molecular Biology, 237–49. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9216-4_15.

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Gao, Tang, Anyao Bi, Shuiqi Yang, Yi Liu, Xiangqi Kong, and Wenbin Zeng. "Applications of Nanoparticles Probes for Prostate Cancer Imaging and Therapy." In Molecular & Diagnostic Imaging in Prostate Cancer, 99–115. Cham: Springer International Publishing, 2018. http://dx.doi.org/10.1007/978-3-319-99286-0_6.

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Rydberg, Ulf. "Clinical aspects on molecular probes, markers and metabolism." In Toward a Molecular Basis of Alcohol Use and Abuse, 115–20. Basel: Birkhäuser Basel, 1994. http://dx.doi.org/10.1007/978-3-0348-7330-7_12.

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Sharma, Sulbha K., and Michael R. Hamblin. "The Use of Fluorescent Probes to Detect ROS in Photodynamic Therapy." In Methods in Molecular Biology, 215–29. New York, NY: Springer US, 2020. http://dx.doi.org/10.1007/978-1-0716-0896-8_17.

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Metcalfe, Paul. "Validation of Genotyping Protocols for Diagnostic Use." In Molecular Typing of Blood Cell Antigens, 1–3. New York, NY: Springer New York, 2015. http://dx.doi.org/10.1007/978-1-4939-2690-9_1.

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Minet, O., J. Beuthan, K. Licha, and C. Mahnke. "The Biomedical Use of Rescaling Procedures in Optical Biopsy and Optical Molecular Imaging." In Fluorescence Spectroscopy, Imaging and Probes, 349–60. Berlin, Heidelberg: Springer Berlin Heidelberg, 2002. http://dx.doi.org/10.1007/978-3-642-56067-5_21.

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McCann, Thomas E., Nobuyuki Kosaka, Peter L. Choyke, and Hisataka Kobayashi. "The Use of Fluorescent Proteins for Developing Cancer-Specific Target Imaging Probes." In Methods in Molecular Biology, 191–204. Totowa, NJ: Humana Press, 2012. http://dx.doi.org/10.1007/978-1-61779-797-2_13.

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Bursell, Sven-Erik, Peter C. Magnante, and Leo T. Chylack. "In Vivo Uses of Quasi-Elastic Light Scattering Spectroscopy as a Molecular Probe in the Anterior Segment of the Eye." In Noninvasive Diagnostic Techniques in Ophthalmology, 342–65. New York, NY: Springer New York, 1990. http://dx.doi.org/10.1007/978-1-4613-8896-8_18.

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Crampton, Julian M., and Susannah M. Hill. "Generation and use of species-specific DNA probes for insect vector identification." In The Molecular Biology of Insect Disease Vectors, 384–98. Dordrecht: Springer Netherlands, 1997. http://dx.doi.org/10.1007/978-94-009-1535-0_33.

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Pudney, Christopher R., Sam Hay, and Nigel S. Scrutton. "Practical Aspects on the Use of Kinetic Isotope Effects as Probes of Flavoprotein Enzyme Mechanisms." In Methods in Molecular Biology, 161–75. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-0452-5_8.

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Conference papers on the topic "Molecular probes Diagnostic use"

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Bárcena, Carlos, Chalermchai Khemtong, Girija S. Chaubey, Chase W. Kessinger, J. Ping Liu, and Jinming Gao. "Zinc Superparamagnetic Iron Oxide Nanoparticles for Use as MRI Contrast Agents." In ASME 2007 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2007. http://dx.doi.org/10.1115/sbc2007-176240.

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Molecular imaging has become a rapidly evolving field used in various applications to target macromolecules and biological process [1,2]. Various imaging systems, such as single photon emission computed tomography (SPECT), positron emission tomography (PET), computerized tomography (CT), and magnetic resonance imaging (MRI), use non-invasive techniques that provide disease-specific information through diagnostic imaging. Early detection of disease demonstrates the potential benefit of these systems.
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Giannelli, B. F. "MOLECULAR GENETICS OF HAEMOPHILIA." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643981.

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Haemophilia B, an X-linked recessive disease with an incidence of 1/30,000 newborn males, is due to defects in the gene for coagulation factor IX, which is on the long am of the X chromosome at band Xq27.1. This gene consists of approximately 34 Kb and contains 8 exons which specify a mRtfc of 2803 residues coding for a protein of 415 aa preceded by a prepro signal peptide of 46 aa. Coripanson of the functional domains of the factor IX protein with the exon structure of the gene supports the exon/protein domain hypothesis of gene evolution. The factor IX gene seems to be formed by a number of functionally and evolutionally independent modules. The signal peptide and the gla (γcarboxy-glutamic) region encoded in the first three exons are homologous to those of factor X, protein C and prothrombin. Thevfourth and fifth exons which code for the connecting peptide are homologous to one another and to the epidermal growth factor, a module that has been used in the construction of a great variety of proteins including different members of the coagulation and fibrinolytic pathways. The sixth exon encodes the activation peptide region, while the catalytic region of factor IX is coded by the seventh and eighth exon. This is at variance with other serine protease genes that have different exons for the segments containing the cardinal ami no-acids of the active centre (histidine, aspartic acid and serine).Natural selection acts against detrimental mutations of the factor IX gene and at each generation a proportion of haemophilia B genes is eliminated, as a significant number of patients does not reproduce. There appears to be no selective advantage to the heterozygote and therefore haemophilia B is maintained in the population by new mutations. Consequently, a significant proportion of patients should be born to non-carrier mothers, and unrelated patients should carry different gene defects, as recently verified by detailed analysis of individual haemophilia B genes.The defects of factor IX described so far comprise both point mutations and gene deletions. The latter affect either part or the whole of the gene and are often associated with the development of antibodies against therapeutically adninistered factor IX (the inhibitor complication). Since gene deletions may result in the complete absenceof factor IX synthesis or in the production of an extremely abnormal product, it has been suggested that mutationspreventing the synthesis of a factor IX gene product capable of inducing immune tolerance to normal factor IX is important in predisposing to the inhibitor complication.Among the point mutations described so far, those affecting the signal peptide are of particular interest. Substitutions of the arginine at positions -4 and -1 cause failure of propeptide cleavage. Thus they indicate that the propeptide consists of 18 aa an(lthat lts excision is necessary for factor IX function. It appears also that the propeptide contains a signal for γcarboxylation which has been conserved during the evolution of different γcarboxylated proteins.In spite of coagulant treatment, haemophilia B is a serious disease and one for which genetic counselling is required. Paramount for this is the detection of carriers and the diagnosis ofaffected male fetuses. DNA probes derived from the cloned factor IX gene have been used for this purpose. Carrier and first or second trimester prenatal diagnoses have been done using factors IX gene markers to follow the transmission of haemophilia B genes. Six sequence variations causing restriction fragment length polymorphisms (RFLP) in the factor IX gene have been detected and used as markers for such indirect diagnoses The efficiency of the above markers is reduced by linkage disequilibrium but, nevertheless, they offer definite carrier and nremtal diagnoses in 75-80% of the relatives of familial cases of haemophilia B.The indirect detection of gene defects is of modest help in the counselling of individuals from the families of isolated patients, but new methods for the direct detection of gene mutations promise better results in such families and also the attainment of % diagnostic success in relatives of familial cases.Finally the successful expression of recombinant factor IX genes in tissue culture and transgenic mammals raises hopes of therapeutic advances.
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DeLeo, Michael J., Matthew J. Gounis, Ajay K. Wakhloo, and Alexei A. Bogdanov. "Validation of Di-5-HT-Gd-DTPA, an Enzyme-Specific MR Contrast Agent for Myeloperoxidase, in the Rabbit Elastase Model of Cerebrovascular Aneurysm." In ASME 2009 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2009. http://dx.doi.org/10.1115/sbc2009-206346.

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Characterization of molecular imaging probes in multiple animal models of disease is essential to increase their diagnostic potential. For example, we recently demonstrated visualization of active inflammation in a rabbit model saccular aneurysm using clinical field strength MRI and the paramagnetic MR contrast agent di-5-HT-GdDTPA, which has been shown in vitro to be sensitive and specific for the enzyme myeloperoxidase (MPO). While the use of transgenic mice (MPO−/−) has demonstrated specificity of di-5-HT-GdDTPA for MPO in a model of myocardial infarction [1], MPO-deficient rabbits are not available. Therefore, in this study, we sought to validate di-5-HT-GdDTPA MPO specificity in the New Zealand white rabbit by comparing serial enhancement ratios of di-5-HT-GdDTPA to a structurally similar MR contrast agent, di-Tyr-GdDTPA, which is activated by peroxidases but not by MPO. Structural diagrams of the synthesis of the two agents are demonstrated in Figure 1 [2].
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Schweitzer, Dietrich, Matthias Klemm, Stefan Schenke, Silvio Quick, Lydia Deutsch, Susanne Jentsch, and Martin Hammer. "FLIM in Ophthalmology - a Diagnostic Tool for Metabolic Mapping." In Optical Molecular Probes, Imaging and Drug Delivery. Washington, D.C.: OSA, 2011. http://dx.doi.org/10.1364/omp.2011.otub2.

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Wang, Cheng, Margaret Folaron, Eunice Chen, P. Jack Hoopes, Kenneth Tichauer, and Kimberley Samkoe. "Paired-agent imaging demonstrates improved diagnostic ability compared to single targeted agents for guiding head and neck squamous cell carcinoma resection." In Optical Molecular Probes, Imaging and Drug Delivery. Washington, D.C.: OSA, 2019. http://dx.doi.org/10.1364/omp.2019.ow1d.5.

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Hu, Hui, and Manoochehr Koochesfahani. "Molecular Tagging Techniques for Micro-Flow and Micro-Scale Heat Transfer Studies." In ASME 2009 Fluids Engineering Division Summer Meeting. ASMEDC, 2009. http://dx.doi.org/10.1115/fedsm2009-78059.

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We report recent progresses made in development of novel molecule-based flow diagnostic techniques, named as Molecular Tagging techniques, to achieve simultaneous measurements of multiple important flow variables (such as flow velocity and temperature) for micro-flows and micro-scale heat transfer studies. Instead of using tiny particles, specially-designed phosphorescent molecules, which can be turned into long-lasting glowing molecules upon excitation by photons of appropriate wavelength, are used as tracers for both velocity and temperature measurements. A pulsed laser is used to “tag” the tracer molecules in the regions of interest, and the movements of the tagged molecules are imaged at two successive times within the photoluminescence lifetime of the tracer molecules. The measured Lagrangian displacement of the tagged molecules between the two image acquisitions provides the estimate of the fluid velocity vector. The simultaneous temperature measurement is achieved by taking advantage of the temperature dependence of phosphorescence lifetime, which is estimated from the intensity ratio of the tagged molecules in the two images. The implementation and application of the MTV&T technique are demonstrated by conducting simultaneous velocity and temperature measurements to qunatify the transient behavior of electroosmotic flow (EOF) inside a microchannel and to reveal the unsteady heat transfer, mass transfer and phase changing process inside micro-sized water droplets pertinent to wind turbine icing phenomena.
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Meunier, Michel. "Plasmonics nanoparticles for use in theranostics (Conference Presentation)." In Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications XII, edited by Samuel Achilefu and Ramesh Raghavachari. SPIE, 2020. http://dx.doi.org/10.1117/12.2556323.

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Obeidat, A. T., A. E. Kaplan, J. B. Khurgin, and M. D. Stem. "Single-Fiber Optical Probe for Two-Photon Induced Fluorescence of Biological Markers." In Biomedical Optical Spectroscopy and Diagnostics. Washington, D.C.: Optica Publishing Group, 2006. http://dx.doi.org/10.1364/bosd.1996.ft8.

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The use of fluorescent dyes which can be attached to specific cellular components as biological markers has become a well- established biomedical technique in recent years [1]. When optically stimulated, these dyes emit a characteristic fluorescence, enabling the structure and organization of the stained sample to be readily studied and visualized by means of fluorescence spectroscopy and confocal microscopy [2]. Moreover, some dyes exhibit enhanced fluorescence if attached to certain biological molecules. For instance, 4’,6- diamidino-2-phenylindole (DAPI) exhibits such enhanced fluorescence when attached to the AT base pairs on a DNA strand [3], Such properties are useful for mapping DNA structure or identifying the presence of particular molecules in a sample.
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Morla-Folch, Judit, Guillem Vargas Nadal, Antonio Ardizzone, Siarhei Kurhuzenkau, Silvia Illa-Tuset, Jordi Faraudo, Mykhailo Bondar, et al. "Quatsomes, novel fluorescent organic nanoparticles and their use as bioimaging probes (Conference Presentation)." In Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications XI, edited by Samuel Achilefu and Ramesh Raghavachari. SPIE, 2019. http://dx.doi.org/10.1117/12.2523535.

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de la Salle, C., M. J. Baas, L. Grunebaum, R. Gialeraki, T. Mandalaki, and J.-P. Cazenave. "MOLECULAR ANALYSIS OF COAGULATION FACTOR VIII AND IX GENES BY DNA PROBES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643873.

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About 250 individuals belonging to 44 families with hemophilia A or B were studied in our laboratory. The detection of carriers was first established by pedigree analysis of each family . and coagulation and immunological assays of factor VIII or IX. The availability of specific probes for the molecular study of these two genes makes possible a diagnosis with certainty in the case of informative families. 25 families of hemophilia A were studied. For each person, blood was collected into EDTA and leucocyte DNA was extracted, digested by restriction endonucleases, electrophoresed in 0.9 % agarose gels and transferred to nitrocellulose filters by Southern blotting. Two probes were used for the analysis of factor VIII gene. The St 14 probe (J.L. Mandel) located on the q28 region of the X chromosome and closely linked to the gene, determines a restriction fragment length polymorphism (RFLP) when the DNA is digested by the enzyme TaqI. The p114-12 genomic probe (Genentech) corresponding to the exons 17 and 18 of the factor VIII gene, reveals a RFLP in the DNA digested by the enzyme BclI. 19 families -of hemophilia B were studied. A total factor IX cDNA probe was used for the screening of potential deletions in the case of hemophiliacs with circulating antibodies. A genomic probe containing the exons II, III and IV of factor IX was used to detect the TaqI RFLP. For the study of factor VIII gene, the extragenic probe St 14 gives a very high percentage of informativity (about 90 %) but recombination can occur between the probe and the gene. The p 114-12 probe, which is used to confirm the results given by the St 14 probe, gives about 20 % informativity. In our study, we were able to diagnose carrier state with certainty in 92 % of the families. For hemophilia B, the genomic probe gives about 40 % informativity. A large deletion of the region of the factor IX gene has been found in one family and remains to be mapped. In conclusion, carrier detection and prenatal diagnosis can be established with certainty by molecular studies in most cases of hemophilia A using the St 14 probe, with a 5 % risk of recombination when the BclI RFLP cannot confirm. This diagnosis is possible in about 40 % of the cases of hemophilia B.
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Reports on the topic "Molecular probes Diagnostic use"

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Dawson, William O., Moshe Bar-Joseph, Charles L. Niblett, Ron Gafny, Richard F. Lee, and Munir Mawassi. Citrus Tristeza Virus: Molecular Approaches to Cross Protection. United States Department of Agriculture, January 1994. http://dx.doi.org/10.32747/1994.7570551.bard.

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Citrus tristeza virus (CTV) has the largest genomes among RNA viruses of plants. The 19,296-nt CTV genome codes for eleven open reading frames (ORFs) and can produce at least 19 protein products ranging in size from 6 to 401 kDa. The complex biology of CTV results in an unusual composition of CTV-specific RNAs in infected plants which includes multiple defective RNAs and mixed infections. The complex structure of CTV populations poses special problems for diagnosis, strain differentiation, and studies of pathogenesis. A manipulatable genetic system with the full-length cDNA copy of the CTV genome has been created which allows direct studies of various aspects of the CTV biology and pathology. This genetic system is being used to identify determinants of the decline and stem-pitting disease syndromes, as well as determinants responsible for aphid transmission.
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Splitter, Gary, and Menachem Banai. Microarray Analysis of Brucella melitensis Pathogenesis. United States Department of Agriculture, 2006. http://dx.doi.org/10.32747/2006.7709884.bard.

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Original Objectives 1. To determine the Brucella genes that lead to chronic macrophage infection. 2. To identify Brucella genes that contribute to infection. 3. To confirm the importance of Brucella genes in macrophages and placental cells by mutational analysis. Background Brucella spp. is a Gram-negative facultative intracellular bacterium that infects ruminants causing abortion or birth of severely debilitated animals. Brucellosis continues in Israel, caused by B. melitensis despite an intensive eradication campaign. Problems with the Rev1 vaccine emphasize the need for a greater understanding of Brucella pathogenesis that could improve vaccine designs. Virulent Brucella has developed a successful strategy for survival in its host and transmission to other hosts. To invade the host, virulent Brucella establishes an intracellular niche within macrophages avoiding macrophage killing, ensuring its long-term survival. Then, to exit the host, Brucella uses placenta where it replicates to high numbers resulting in abortion. Also, Brucella traffics to the mammary gland where it is secreted in milk. Missing from our understanding of brucellosis is the surprisingly lillie basic information detailing the mechanisms that permit bacterial persistence in infected macrophages (chronic infection) and dissemination to other animals from infected placental cells and milk (acute infection). Microarray analysis is a powerful approach to determine global gene expression in bacteria. The close genomic similarities of Brucella species and our recent comparative genomic studies of Brucella species using our B. melitensis microarray, suqqests that the data obtained from studying B. melitensis 16M would enable understanding the pathogenicity of other Brucella organisms, particularly the diverse B. melitensis variants that confound Brucella eradication in Israel. Conclusions Results from our BARD studies have identified previously unknown mechanisms of Brucella melitensis pathogenesis- i.e., response to blue light, quorum sensing, second messenger signaling by cyclic di-GMP, the importance of genomic island 2 for lipopolysaccharide in the outer bacterial membrane, and the role of a TIR domain containing protein that mimics a host intracellular signaling molecule. Each one of these pathogenic mechanisms offers major steps in our understanding of Brucella pathogenesis. Strikingly, our molecular results have correlated well to the pathognomonic profile of the disease. We have shown that infected cattle do not elicit antibodies to the organisms at the onset of infection, in correlation to the stealth pathogenesis shown by a molecular approach. Moreover, our field studies have shown that Brucella exploit this time frame to transmit in nature by synchronizing their life cycle to the gestation cycle of their host succumbing to abortion in the last trimester of pregnancy that spreads massive numbers of organisms in the environment. Knowing the bacterial mechanisms that contribute to the virulence of Brucella in its host has initiated the agricultural opportunities for developing new vaccines and diagnostic assays as well as improving control and eradication campaigns based on herd management and linking diagnosis to the pregnancy status of the animals. Scientific and Agricultural Implications Our BARD funded studies have revealed important Brucella virulence mechanisms of pathogenesis. Our publication in Science has identified a highly novel concept where Brucella utilizes blue light to increase its virulence similar to some plant bacterial pathogens. Further, our studies have revealed bacterial second messengers that regulate virulence, quorum sensing mechanisms permitting bacteria to evaluate their environment, and a genomic island that controls synthesis of its lipopolysaccharide surface. Discussions are ongoing with a vaccine company for application of this genomic island knowledge in a Brucella vaccine by the U.S. lab. Also, our new technology of bioengineering bioluminescent Brucella has resulted in a spin-off application for diagnosis of Brucella infected animals by the Israeli lab by prioritizing bacterial diagnosis over serological diagnosis.
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Davis, Robert E., Edna Tanne, James P. Prince, and Meir Klein. Yellow Disease of Grapevines: Impact, Pathogen Molecular Detection and Identification, Epidemiology, and Potential for Control. United States Department of Agriculture, September 1994. http://dx.doi.org/10.32747/1994.7568792.bard.

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Grapevine yellows diseases characterized by similar symptoms have been reported in several countries including Israel, the United States, France, Italy, Spain, Germany and Australia. These diseases are among the most serious known in grapevine, but precise knowledge of the pathogens' identities and modes of their spread is needed to devise effective control stratgegies. The overall goals of this project were to develop improved molecular diagnostic procedures for detection and identification of the presumed mycoplasmalike organism (MLO) pathogens, now termed phytoplasmas, and to apply these procedures to investigate impact and spread and potential for controlling grapevine yellows diseases. In the course of this research project, increased incidence of grapevine yellows was found in Israel and the United States; the major grapevine yellows phytoplasma in Israel was identified and tis 16S rRNA gene characterized; leafhopper vectors of this grapevine yellows phytoplasma in Israel were identified; a second phytoplasma was discovered in diseased grapevines in Israel; the grapevine yellows disease in the U.S. was found to be distinct from that in Israel; grapevine yellows in Virginia, USA, was found to be caused by two different phytoplasmas; both phytoplasmas in Virginia grapevines were molecularly characterized and classified; commercial grapevines in Europe were discovered to host a phytoplasma associated with aster yellow disease in the USA, but this phytoplasma has not been found in grapevine in the USA; the Australian grapevine yellows phytoplasma was found to be distinct from the grapevine phytoplasmas in Israel, the United States and Europe and was described and named "Candidatus phytoplasma australiense", and weed host plants acting as potential reservoirs of the grapevine phytoplasmas were discovered. These and other findings from the project should aid in the design and development of strategies for managing the grapevine yellows disease problem.
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Banai, Menachem, and Gary Splitter. Molecular Characterization and Function of Brucella Immunodominant Proteins. United States Department of Agriculture, July 1993. http://dx.doi.org/10.32747/1993.7568100.bard.

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The BARD project was a continuation of a previous BARD funded research project. It was aimed at characterization of the 12kDa immunodominant protein and subsequently the cloning and expression of the gene in E. coli. Additional immunodominant proteins were sought among genomic B. abortus expression library clones using T-lymphocyte proliferation assay as a screening method. The 12kDa protein was identified as the L7/L12 ribosomal protein demonstrating in the first time the role a structural protein may play in the development of the host's immunity against the organism. The gene was cloned from B. abortus (USA) and B. melitensis (Israel) showing identity of the oligonucleotide sequence between the two species. Further subcloning allowed expression of the protein in E. coli. While the native protein was shown to have DTH antigenicity its recombinant analog lacked this activity. In contrast the two proteins elicited lymphocyte proliferation in experimental murine brucellosis. CD4+ cells of the Th1 subset predominantly responded to this protein demonstrating the development of protective immunity (g-IFN, and IL-2) in the host. Similar results were obtained with bovine Brucella primed lymphocytes. UvrA, GroE1 and GroEs were additional Brucella immunodominant proteins that demonstrated MHC class II antigenicity. The role cytotoxic cells are playing in the clearance of brucella cells was shown using knock out mice defective either in their CD4+ or CD8+ cells. CD4+ defective mice were able to clear brucella as fast as did normal mice. In contrast mice which were defective in their CD8+ cells could not clear the organisms effectively proving the importance of this subtype cell line in development of protective immunity. The understanding of the host's immune response and the expansion of the panel of Brucella immunodominant proteins opened new avenues in vaccine design. It is now feasible to selectively use immunodominant proteins either as subunit vaccine to fortify immunity of older animals or as diagnostic reagents for the serological survaillance.
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Bercovier, Herve, Raul Barletta, and Shlomo Sela. Characterization and Immunogenicity of Mycobacterium paratuberculosis Secreted and Cellular Proteins. United States Department of Agriculture, January 1996. http://dx.doi.org/10.32747/1996.7573078.bard.

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Our long-term goal is to develop an efficient acellular vaccine against paratuberculosis based on protein antigen(s). A prerequisite to achieve this goal is to analyze and characterize Mycobacterium paratuberculosis (Mpt) secreted and cellular proteins eliciting a protective immune response. In the context of this general objective, we proposed to identify, clone, produce, and characterize: the Mpt 85B antigen and other Mpt immunoreactive secreted proteins, the Mpt L7/L12 ribosomal protein and other immunoreactive cellular proteins, Mpt protein determinants involved in invasion of epithelial cells, and Mpt protein antigens specifically expressed in macrophages. Paratuberculosis is still a very serious problem in Israel and in the USA. In the USA, a recent survey evaluated that 21.6% of the dairy herd were infected with Mpt resulting in 200-250 million dollars in annual losses. Very little is known on the virulence factors and on protective antigens of Mpt. At present, the only means of controlling this disease are culling or vaccination. The current vaccines do not allow a clear differentiation between infected and vaccinated animals. Our long-term goal is to develop an efficient acellular paratuberculosis vaccine based on Mpt protein antigen(s) compatible with diagnostic tests. To achieve this goal it is necessary to analyze and characterize secreted and cellular proteins candidate for such a vaccine. Representative Mpt libraries (shuttle plasmid and phage) were constructed and used to study Mpt genes and gene products described below and will be made available to other research groups. In addition, two approaches were performed which did not yield the expected results. Mav or Mpt DNA genes that confer upon Msg or E. coli the ability to invade and/or survive within HEp-2 cells were not identified. Likewise, we were unable to characterize the 34-39 kDa induced secreted proteins induced by stress factors due to technical difficulties inherent to the complexity of the media needed to support substantial M. pt growth. We identified, isolated, sequenced five Mpt proteins and expressed four of them as recombinant proteins that allowed the study of their immunological properties in sensitized mice. The AphC protein, found to be up regulated by low iron environment, and the SOD protein are both involved in protecting mycobacteria against damage and killing by reactive oxygen (Sod) and nitrogen (AhpC) intermediates, the main bactericidal mechanisms of phagocytic cells. SOD and L7/L12 ribosomal proteins are structural proteins constitutively expressed. 85B and CFP20 are both secreted proteins. SOD, L7/L12, 85B and CFP20 were shown to induce a Th1 response in immunized mice whereas AphC was shown by others to have a similar activity. These proteins did not interfere with the DTH reaction of naturally infected cows. Cellular immunity provides protection in mycobacterial infections, therefore molecules inducing cellular immunity and preferentially a Th1 pathway will be the best candidate for the development of an acellular vaccine. The proteins characterized in this grant that induce a cell-mediated immunity and seem compatible with diagnostic tests, are good candidates for the construction of a future acellular vaccine.
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Levisohn, Sharon, Mark Jackwood, and Stanley Kleven. New Approaches for Detection of Mycoplasma iowae Infection in Turkeys. United States Department of Agriculture, February 1995. http://dx.doi.org/10.32747/1995.7612834.bard.

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Mycoplasma iowae (Mi) is a pathogenic avian mycoplasma which causes mortality in turkey embryos and as such has clinical and economic significance for the turkey breeder industry. Control of Mi infection is severely hampered by lack of adequate diagnostic tests, together with resistance to most antibiotics and resilience to environment. A markedly high degree of intra-species antigenic variation also contributes to difficulties in detection and control of infection. In this project we have designed an innovative gene-based diagnostic test based on specific amplification of the 16S rRNA gene of Mi. This reaction, designed Multi-species PCR-RFLP test, also amplifies the DNA of the pathogenic avian mycoplasmas M. gallisepticum (Mg) and M. synoviae (Ms). This test detects DNA equivalent to about 300 cfu Mi or either of the other two target mycoplasmas, individually or in mixed infection. It is a quick test, applicable to a wide variety of clinical samples, such as allantoic fluid or tracheal or cloacal swab suspensions. Differential diagnosis is carried out by gel electro-phoresis of the PCR amplicon digested with selected restriction enzymes (Restriction Fragment Length Polymorphism). This can also be readily accomplished by using a simple Dot-Blot hybridization assay with digoxigenin-labeled oligonucleotide probes reacting specifically with unique Mi, Mg or Ms sequences in the PCR amplicon. The PCR/OLIGO test increased sensitivity by at least 10-fold with a capacity for rapid testing of large numbers of samples. Experimental infection trials were carried out to evaluate the diagnostic tools and to study pathogenesis of Mi infection. Field studies and experimental infection of embryonated eggs indicated both synergistic and competitive interaction of mycoplasma pathogens in mixed infection. The value of the PCR diagnostic tests for following the time course of egg transmission was shown. A workable serological test (Dot Immunobinding Assay) was also developed but there was no clear-cut evidence that infected turkeys develop an immune response. Typing of a wide spectrum of Mi field isolates by a variety of gene-based molecular techniques indicated a higher degree of genetic homogeneity than predicted on the basis of the phenotypic variability. All known strains of Mi were detected by the method developed. Together with an M. meleagridis-PCR test based on the same gene, the Multi-species PCR test is a highly valuable tool for diagnosis of pathogenic mycoplasmas in single or mixed infection. The further application of this rapid and specific test as a part of Mi and overall mycoplasma control programs will be dependent on developments in the turkey industry.
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Epel, Bernard, and Roger Beachy. Mechanisms of intra- and intercellular targeting and movement of tobacco mosaic virus. United States Department of Agriculture, November 2005. http://dx.doi.org/10.32747/2005.7695874.bard.

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To cause disease, plant viruses must replicate and spread locally and systemically within the host. Cell-to-cell virus spread is mediated by virus-encoded movement proteins (MPs), which modify the structure and function of plasmodesmata (Pd), trans-wall co-axial membranous tunnels that interconnect the cytoplasm of neighboring cells. Tobacco mosaic virus (TMV) employ a single MP for cell- cell spread and for which CP is not required. The PIs, Beachy (USA) and Epel (Israel) and co-workers, developed new tools and approaches for study of the mechanism of spread of TMV that lead to a partial identification and molecular characterization of the cellular machinery involved in the trafficking process. Original research objectives: Based on our data and those of others, we proposed a working model of plant viral spread. Our model stated that MPᵀᴹⱽ, an integral ER membrane protein with its C-terminus exposed to the cytoplasm (Reichel and Beachy, 1998), alters the Pd SEL, causes the Pd cytoplasmic annulus to dilate (Wolf et al., 1989), allowing ER to glide through Pd and that this gliding is cytoskeleton mediated. The model claimed that in absence of MP, the ER in Pd (the desmotubule) is stationary, i.e. does not move through the Pd. Based on this model we designed a series of experiments to test the following questions: -Does MP potentiate ER movement through the Pd? - In the presence of MP, is there communication between adjacent cells via ER lumen? -Does MP potentiate the movement of cytoskeletal elements cell to cell? -Is MP required for cell-to-cell movement of ER membranes between cells in sink tissue? -Is the binding in situ of MP to RNA specific to vRNA sequences or is it nonspecific as measured in vitro? And if specific: -What sequences of RNA are involved in binding to MP? And finally, what host proteins are associated with MP during intracellular targeting to various subcellular targets and what if any post-translational modifications occur to MP, other than phosphorylation (Kawakami et al., 1999)? Major conclusions, solutions and achievements. A new quantitative tool was developed to measure the "coefficient of conductivity" of Pd to cytoplasmic soluble proteins. Employing this tool, we measured changes in Pd conductivity in epidermal cells of sink and source leaves of wild-type and transgenic Nicotiana benthamiana (N. benthamiana) plants expressing MPᵀᴹⱽ incubated both in dark and light and at 16 and 25 ᵒC (Liarzi and Epel, 2005 (appendix 1). To test our model we measured the effect of the presence of MP on cell-to-cell spread of a cytoplasmic fluorescent probe, of two ER intrinsic membrane protein-probes and two ER lumen protein-probes fused to GFP. The effect of a mutant virus that is incapable of cell-to-cell spread on the spread of these probes was also determined. Our data shows that MP reduces SEL for cytoplasmic molecules, dilates the desmotubule allowing cell-cell diffusion of proteins via the desmotubule lumen and reduces the rate of spread of the ER membrane probes. Replicase was shown to enhance cell-cell spread. The data are not in support of the proposed model and have led us to propose a new model for virus cell-cell spread: this model proposes that MP, an integral ER membrane protein, forms a MP:vRNAER complex and that this ER-membrane complex diffuses in the lipid milieu of the ER into the desmotubule (the ER within the Pd), and spreads cell to cell by simple diffusion in the ER/desmotubule membrane; the driving force for spread is the chemical potential gradient between an infected cell and contingent non-infected neighbors. Our data also suggests that the virus replicase has a function in altering the Pd conductivity. Transgenic plant lines that express the MP gene of the Cg tobamovirus fused to YFP under the control the ecdysone receptor and methoxyfenocide ligand were generated by the Beachy group and the expression pattern and the timing and targeting patterns were determined. A vector expressing this MPs was also developed for use by the Epel lab . The transgenic lines are being used to identify and isolate host genes that are required for cell-to-cell movement of TMV/tobamoviruses. This line is now being grown and to be employed in proteomic studies which will commence November 2005. T-DNA insertion mutagenesis is being developed to identify and isolate host genes required for cell-to-cell movement of TMV.
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